Evaluation of Eight Different Cephalosporins for Detection of Cephalosporin Resistance in Salmonella enterica and Escherichia coli.
Aarestrup FM, Hasman H, Veldman K, Mevius D.
1 WHO Collaborating Centre for Antimicrobial Resistance in Food borne Pathogens and EU Community Reference Laboratory for Antimicrobial Resistance, National Food Institute, Technical University of Denmark , Copenhagen, Denmark .
Abstract
This study evaluates the efficacy of eight different cephalosporins for detection of cephalosporin resistance mediated by extended spectrum beta-lactamases (ESBL) and plasmidic AmpC beta-lactamases in Salmonella and Escherichia coli. A total of 138 E. coli and 86 Salmonella isolates with known beta-lactamase genes were tested for susceptibility toward cefoperazone, cefotaxime, cefpodoxime, cefquinome, ceftazidime, ceftiofur, ceftriaxone, and cefuroxime using minimum inhibitory concentration determinations and disc diffusion. The collection consisted of 84 ampicillin-susceptible, 57 ampicillin-resistant but cephalosporin-susceptible, 56 ESBL isolates and 19 isolates with plasmidic AmpC, as well as 10 ampC hyper-producing E. coli. The minimum inhibitory concentration distributions and zone inhibitions varied with the tested compound. Ampicillin-resistant isolates showed reduced susceptibility to the cephalosporins compared to ampicillin-susceptible isolates. Cefoperazone, cefquinome, and cefuroxime were not useful in detecting isolates with ESBL or plasmidic AmpC. The best substances for detection were cefotaxime, cefpodoxime, and ceftriaxone, whereas ceftazidime and ceftiofur were not as efficient. Ceftriaxone may be the recommended substance for monitoring because of some ability in separating ampC hyper-producing E. coli from ESBL and plasmidic AmpC isolates.
PMID: 20624078 [PubMed - as supplied by publisher]
Monday, September 20, 2010
Thursday, September 2, 2010
NSAIDS for canine use
Pain Control In Dogs And Cats
Ron Hines DVM PhD
Dear Reader,
You should never give your pet the medications I write about without expert, hands-on advice from your local veterinarian. Do not give doses higher than recommended. Although mixing medications is sometimes helpful, it should not be done without expert counsel. This article is for your general knowledge only.
Those of us who have and work with pets know that they experience pain similar to human beings. Although we cannot prove this scientifically, we are so closely attuned to our four-legged friends that we know when they are uncomfortable and troubled by pain. You, as the pet’s owner, are more likely to notice signs of pain than your veterinarian because you are more attuned and bonded to your own pet.
Pain can be a temporary problem or a persistent one. Acute or sudden pain can be the result of surgery or sudden damage to any of the major organs, muscles or bones of the body. However, Pain can be a temporary problem or a persistent one. Some causes of chronic pain in older pets are hip dysplasia, arthritis of the spine and joints instability.
All pain-relieving drugs are called analgesics. Controlling pain in your pet actually improves the outcome of many diseases or surgery. Whenever possible, it is better to give pain-controlling medications early rather than waiting until the pain becomes severe.
It is hard to objectively judge the severity of pain in human beings and even more difficult to do so in animals. Our thresholds to pain differ between one person and another and from one animal to another. Some sources claim a five-fold difference between individual pets. Pain perception depends partially on species, breed, age, gender, time of day and your pet’s individual temperament. Young animals tend to have a lower threshold to pain. Older and debilitated pets are more stoic and may not show as much response to pain - but they feel it just the same. Hunting and working breeds of dogs are more resistant to expressing pain than toy or miniature breeds. Signs of pain are subtler in cats than in dogs. When you take your pet in to an animal hospital, your pet’s is usually worrying about the visit and strange environment will often ignore the pain that you noticed at home. This can be very frustrating for owners who tell me “but he limped at home!”
Cats in pain are more stoic than dogs and they hide pain more effectively. Cats will often hunch up when the pain is in their tummies and be reluctant to move. Some cats hiss if a painful site is touched or become unresponsive to affection and petting. Other cats become aggressive and belligerent when in pain. Often their food consumption goes down. Some cats in acute pain meow pathetically. They often carry their ears down. Many pain control medications are bitter. It can be extremely hard to get cats to accept them. For this reason, medications are best when they are prepared in tasteless capsules by a compounding pharmacy.
In both cats and dogs, pain may lead to over-grooming the area that is painful. This can lead to hair loss and self-mutilation of the area. Some pets tremble and move with their stomachs tensed up. Others tremble. Some will show lameness of an affected leg while others become aggressive, pant or grimace. Any sudden behavior change can be a symptom of pain. Excessive salivation, licking of the lips, dilation of the eyes, rapid breathing and increased heart rate may all be attributable to pain. Some dogs in pain also eat less. Some become restless and do not sleep well. Some stop grooming and appear dejected. Pain can cause an increase in body temperature (fever), respiration, heart rate and blood pressure.
When the pain of hip dysplasia or spinal arthritis in dogs is severe, the dog may be unwilling to rear up on its back legs for a treat. I will often pinch the toe of a pet that appears to be in pain to judge the severity of the pain. If the cat or dog reacts to the toe pinch then I assume the severity of its other pain is less than that of the pinch.
Pain alone can actually change the results of blood chemistry analysis. Dogs and cats in pain may have elevated blood sugar. Their blood cortisol (steroid) and white cell levels often increase. Pain can also interfere with the immune system, increase the risk of infections and slowing the healing of wounds and surgical incisions.
You must understand that complete elimination of long-term pain may be impossible or even undesirable in your pet. But it can be minimized with a number of medications. There are five major classes of medicine that can be used to control pain in dogs and cats. Cats do not metabolize many drugs as well as other animals, so your options are fewer. All of the following medications must be used with extreme caution in cats. You and your veterinarian must weigh the advantages of pain medications in cats against the possible damage that might occur.
There are some general rules in using pain control medications in dogs and cats. The first is to try to give the medication early before the pain becomes too intense. The second is that it is usually safer and more effective to give lower doses of two or more pain control medications that have different modes of action rather than a higher dose of a single medication. Doses should always be kept to the bare minimum needed to give relief. Older patients should receive lower doses less frequently than younger more robust pets. It is also wise to check kidney and liver function before and during the use of pain control medications.
Pain control medications used in pets
The Non-steroidal Anti-inflammatory Agents (NSAIDs):
In 1989 scientists discovered the first of the NSAIDs. Pain sensation and inflammation rely on messenger chemicals called prostaglandins. There are two types of prostaglandins, the “good ones” called COX 1 and the “bad” ones called COX 2. Older NSAIDs prevent the formation both good and bad prostaglandins. “Good prostaglandins protect the lining of the stomach and small intestine and also assist clotting and kidney blood flow. “Bad” prostaglandins cause inflammation, swelling and pain. They have become the most frequently used pain medications used in pets. They are widely used in humans as well. There are basically two kinds of NSAIDs. I call the newer ones (COX 2 blockers)and the older ones (Cox 1 and Cox2 blockers). They both work by blocking an enzyme called cyclooxygenase (COX) which is necessary for both prostaglandin formation. The older NSAIDs cause more problems because they block both the pain-producing prostaglandins and the good, protective, prostaglandins. We suspect that NSAIDs also work directly on the brain to block pain sensation.
Now, the enzyme, Cyclooxygenase occurs in 2 forms: The ”good” COX 1 which controls the formation of “good” prostaglandins that protect the lining of the stomach and intestine, the blood clotting process and blood flow to the kidneys. The “bad” one, COX 2, encourages pain and inflammation. This is a simplified explanation but it should be sufficient for readers. Although all the newer NSAIDs work in the same way, some seem more effective in blocking COX 2 in a particular pet. So don’t give up if the first medication you use doesn’t appear to work.
All NSAIDs are well absorbed when given orally. They are eliminated from the body by the liver and kidneys.
When your pet is in acute (sudden) pain, your veterinarian will often use injectable forms of NSAIDs for fast relief.
The Special Problem Of Cats and NSAIDs
Cats are very special animals. They have a unique body biochemistry and liver function that make the NSAIDs more dangerous to them. Your cat’s liver does not have enough of a specific enzyme (bilirubin-glucuronide enzyme). Because of this, NSAIDs tend to linger in the cat’s blood stream. So NSAIDs must be given in very limited doses and they must be given less frequently.
It appears that two of the few NSAIDs that cats tolerate fairly well are meloxicam and ketoprofen. These medications come in flavored syrup as well as tablets. The volume of syrup needed is small cats so they usually accept it. Both compounds are registered for use in cats in Canada and Europe. There, they appear to be relatively safe especially when given for short periods of time. But neither medication is licensed by the FDA for use in cats in the United States.
Because there is a great deal of variation between individual cats as to the effects of these drugs, your veterinarian should monitor your cat closely for any side effects. This is especially true if either drug is given for extended periods of time.
Possible Side Effects in Pets
Because the liver plays an important roll in eliminating NSAIDs from the body, It is best not to use NSAIDs in the face of known liver disease, because animals with liver disease do not remove these drugs from their bodies normally. Also, on occasion, NSAIDs may cause sudden liver failure. According to the FDA’s records on the toxicity of approved products for dogs, Carprofen (Rimadyl) had had the most problems. However, this may be because it is the most widely prescribed.
From time to time, all of the NSAIDs will cause kidney damage in cats and dogs. This is because they can limit blood flow to the kidneys. This is not a problem if your pet has normal kidneys. But if your pet already has some kidney blood flow damage, more might result. This is especially true with the older NSAIDs. COX-2 enzyme increases blood circulation in the kidneys. When COX-2 is inhibited by the older NSAID versions blood flow through the kidneys can drop to dangerous levels.
Another common side effect is stomach and intestinal bleeding. Again, this is more of a problem with the older NSAIDs. When this occurs, your pet will experience vomiting and diarrhea. If this occurs the medication must be stopped or the dose decreased. This side effect occurs more in pets than in humans that receive NSAIDs. It is especially true of the older NSAIDs, which I listed below. An early warning sign of bleeding is a decreased number of circulating red blood cells. Your veterinarian can check for this periodically.
If I must use the older NSAIDs, I often suggest over-the-counter medications that limit stomach acidity (cimetidine, ranitidine, famotidine) Pantoprazole or omeprazole may also have this potential but I have not used them. These medications may minimize NSAID side effects. Some veterinarians give a synthetic prostaglandin, mistoprostol, with NSAIDs to coat and protect the pet’s stomach and intestines. Omeprazole may also be beneficial but I have found no data on its use in pets.
When administering NSAIDs, do not use two different ones at the same time. They should also not be given in combination with corticosteroids (prednisolone, prednisone, dexamethasone, etc).
Also, pets receiving diuretics such as Lasix (furosemide) are also more susceptible to NSAID side effect.
The Newer And Safer NSAIDs:
Most of this group were originally developed for use in humans. They are safer than the “old” NSAIDs. Because the newer ones are COX-2 selective, they are less likely to cause stomach or intestinal bleeding. This is the reason they were very popular in people until the Vioxx (rofecoxib) scare occurred. Luckily, dogs and cats do not share this risk because they do not commonly have heart attacks. Only Meloxicam injection is approved for short term use in cats.
These are dog or human products. None are approved in the United States for use in cats. But because veterinarians want to relieve suffering, we sometimes give them anyway. There are much fewer options in treating pain in cats. When we do use them in cats, it is called an “off label use”.
Before using these drugs, your veterinarian may suggest bloodwork to determine how well the liver and kidneys are functioning in your pet. If it is for long-term use, I would definitely suggest that. It is generally unwise to give this group of medications at the same time corticosteroid-type medications are given. The exception is in end-of-life situations.
Carprofen (Rimadyl, Pfizer):
Introduced in the USA in 1997 by Pfizer Animal Health Co for use in dogs, this NSAID is very similar to meloxicam. Carprofen is often given to dogs before surgery to decrease post-operative pain.
The other approved use is to combat the pain of arthritis. It is available for dogs in two oral forms, caplets and chewable tablets. In Europe, Canada and other countries, carprofen is also registered for short-term therapy in cats.
It will, on rare occasions (2 per 1000 dogs) , cause liver damage in dogs. Particularly Labrador Retrievers.
Ketoprofen (Orudis, Oruvai, Actron, Oruvail, Orudis-KT, Ketofen) :
This medication is sold for human use, over-the-counter at most pharmacies and super-centers. It is only approved in the US for use in people and horses. In Europe and Canada it is approved for use in dogs and cats. There it is available in tablet or injectable form.
All the factors and precautions I mention in the introduction to NSAIDs apply to ketoprofen.
As with the other NSAIDs, ketoprofen is processed in the liver to inactive byproducts that are eliminated by the kidneys. Side effects can includine intestinal upset with vomiting and diarrhea similar to other NSAIDs. Word-of-mouth and published articles recommend this medication to relieve short-term pain (up to 5 days) in dogs and cats. However, this is not an approved use in the United States. Once given by mouth, ketoprofen is rapidly absorbed. After 2-3 hours, blood levels of this medication are only half their original levels.
Side effects, including liver damage and kidney disease, have been reported in pets. Because ketoprofen can adversely affect blood clotting, I do not suggest it be given before or after surgery.
Etodolac (Etogesic, Wyeth Co):
Etodolac is approved for use in dogs in the United States. It is also quite similar to carprofen. Etodolac is given once a day to manage arthritis. It can be given with or without food.
The most commonly reported side effects to etodolac are diarrhea, vomiting, or mopyness and inactivity. When given at the recommended dose, side effects are rare. But if the dose is trebled, vomiting, intestinal bleeding, and weight loss often occur. Etodolac is eliminated by the liver and with the stool. All the cautions I mentioned in the introduction apply to etodolac.
Meloxicam (Metacam, Mobic, Borringer-Ingelheim, Merial Co):
This potent inhibitor of prostaglandin synthesis is used for the treatment of the acute and chronic pain associated with muscle disease and arthritis. It is also used in the management of surgical pain. Meloxicam is a favorite of mine in this group. This is probably because occasionally I myself have take the human brand of meloxicam called Mobic. It is marketed for use in dogs in the United States. It is available in oral tablet, suspension and injectable forms.
Meloxicam is approved for use in dogs. A larger, loading dose is given on the first day. On succeeding days, the dose should be lowered to the lowest possible amount that keeps the dog pain free or nearly so. Intestinal safety seems to be greater for meloxicam than for many other NSAIDs.
The injectable form of Metacam is approved for cats as a one-time, subcutaneous injection for post-surgical pain. If given more than one time or if other NSAIDs are given, kidney and liver toxicity may occur in cats. Both their kidney and liver function must be monitored frequently. If your veterinarian elects to use it in cats, it should not be given more than two or three days per week. Because of the nature of NSAIDs in cats, you must weight the potential risks against the benefits and make your own decision along with your veterinarian.
Deracoxib (Deramaxx, Novartis):
Deracoxib was first approved for controlling post-operative pain. In 2003, it won approval for the prevention of chronic arthritis pain. It is available in beef-flavored chewable tablets. When used to control the pain of surgery, it should be given by injection about two hours before the surgery. It can then be given for up to six days following the surgery. The dose of deracoxib is lowered when the drug is intended for long-term use in the treatment of arthritis.
Deracoxib is related to a class of antibiotic drugs called "sulfonamides" which means they contains a contain sulfur in their structure. However deracoxib is not an antibiotic. It should not be used in pets that have a history of problems taking sulfas. It should not be given after long periods of anesthesia. Do not use deracoxib in dogs that weigh less than 4 lbs. Do not administer it to pregnant dogs, nursing mothers or dogs under age 4 months of age. It is best to give this medication with food. It should never be given with corticosteroid medications. Do not use deracoxib in dogs that are dehydrated, or taking diuretics, or dogs that have preexisting kidney, liver, heart or circulatory problems. Symptoms of overdosage may include diarrhea, vomiting, and bloody stools.
Duracoxib is unique among the newer NSAIDs in the long length of time it controls pain. It persists in the blood stream longer than other NSAIDs. As with the other NSAIDs, the medication will occasionally cause life-threatening stomach punctures so dogs on this medication need to be monitored closely. It should not be used in dogs with an elevated BUN or Creatinine – signs of kidney disease. In such dogs it can exacerbate kidney failure and uremia. A recent study found that the risk of intestinal perforation was quite high when this product was given at doses exceeding the manufacturer's recomendations or when the dogs received corticosteroids while on the medication.
Studies, using deracoxib in cats have been run. A specially made liquid formula was accepted readily by the cats, and no adverse effects were observed. But several more scientific studies are needed before we know the effectiveness and safety of deracoxib in cats. Until then, you must consider the use of deracoxib in cats as an experiment with unknown risks and benefits.
Tepoxalin (Zubrin):
First marketed for dogs in 2003 by the Schering-Plough Animal Health Corporation,
This medication has properties similar to both carprofen and ketoprofen. This medication not only inhibits “bad” prostaglandin formation but also acts through different pathways to block pain.
Firocoxib (Prevacox):
Firocoxib is similar to dericoxib. It was recently approved in the United States and Europe for the control of pain and inflammation associated with arthritis in dogs. It is available in a chewable tablets.
Tolmetin (Tolectin, McNeil Co, Janssen-Ortho):
I have no experience with this medication. Tolmetin was approved for human use in 1997. It is used to treat rheumatoid arthritis. It is usually taken three times a day. I am not aquatinted with its use in pets. Studies in animals have shown that tolmetin to possess anti-inflammatory, analgesic, and anti-fever activity. In rats, tolmetin prevents the development of arthritis and also decreases inflammation. Tolmetin appears to reduce prostaglandin synthesis similar to other NSAIDs.
In humans it may cause headache, dizziness, nervousness, upset stomach, stomach pain or cramps, vomiting, diarrhea or constipation and gas.
Meclofenamic acid (Arquel):
Meclofenamic acid is FDA-approved for use in dogs This NSAID is available as an oral tablet. Meclofenamic acid has a therapeutic index that is lower than that of other NSAID, possibly due to the way it circulates in the liver. This means that the necessary dose for pain relief is quite close to the dose that can cause side effects. It is sold for the treatment of pain and inflammation - especially that associated with arthritis. It may take three or four days for pain relief to be seen in your dog. Side effects that can occur include vomiting, diarrhea, lack of appetite, bloody stool, black tarry stool, or ulcers in the stomach or small intestines. Less common side effects are depression, fever, behavior changes, fast breathing, edema inability to control urine, or irreversible anemia.
Do not use Meclofenamic acid in dogs that are hypersensitive (allergic) to this drug or any other NSAIDs. Do not use the medication at full dosage for more than 5-6 days. After that period, the dose should be decrease to the minimum dose and most infrequent administration that controls your pet’s pain. That is, give it only a few days per week if possible.
All the earlier warning I have given concerning NSAIDs apply to meclofenamic acid. Do not use this drug in dogs that have kidney or liver problems or heart disease. Tests must be run by your veterinarian to rule these conditions out. Give with a full meal. Do not give meclofenamic acid for a week prior to surgery or the week after surgery. Do not give to dehydrated pets or those taking diuretics for heart or lung problems. Do not use this product in dogs with clotting deficiencies such as Von Willebrand’s disease. Do not give the medication to pregnant or nursing mothers. Do not give to pets under eight months of age. This product is not approved for use in cats. Do not give with other NSAIDs or sulfa antibiotics, glipizide, or valproic acid or oral anticoagulants. In epileptic dogs, Meclofenamic acid may increase blood concentrations of phenytoin. If you exceed the recommended dosage you may see evidence of stomach ulcers and kidney damage.
Vedaprofen (Quadrisol-1 and Quadrisol-5, Intervet Co):
I have no personal experience with vedaprofen. However, reports out of the Netherlands, Portugal and Asia suggest to me that this medication may have significant potential uses in dogs and possibly cats. But because all NSAIDs have similar effects on the body, I do not expect it to be free of the side effects present with all NSAIs.
Intervet, on its European site, states that Quadrisol “has been tested in numerous field trials and has been proven to be safe, effective and well tolerated by dogs” and is said to be safe for use in nursing mothers. However, the possible side-effects are the same as the other newer NSAIDs. It is marketed for dogs for the relief of pain and control of inflammation associated with short-term injury or long-term for arthritic problems. It comes in gel form in a dosing syringe. It should be given with food. It is sold in two formulations: Quadrisol-1 is for use in dogs weighing under 5 pounds and Quadrisol-5 for dogs weighing over 5 pounds. In some countries Quadrisol-1 is also marketed for the management of fever and post-operative pain in cats. In studies in the Netherlands it was found to be a bit more effective than meloxicam.
Celecoxib (Celebrex, Pfizer Inc):
This drug is not approved for use in pets. In one study in beagles, there was dangerous variation in the length of time the drug stayed in the body as well as great variation in blood levels between dogs. I do not recommend its use until we understand it better.
Valdecoxib (Bextra):
This medication was introduced to the US market in 2001 as an arthritis remedy for humans. Valdecoxib is a potent and specific inhibitor of cyclooxygenase-2 (COX-2). However, side effects in humans led to it’s being banned in several countries. There is no data on its use in animals.
The Older, Less Safe NSAIDs:
These medications are much less specific than the newer NSAIDs. These medications reduce both the “good” and the bad prostaglandins. Bleeding is the most common side effect of these drugs. Despite this common side effects, the older NSAIDs are still used today in veterinary medicine because they are so much cheaper than the newer ones. Do not give these older medications after major surgery because they can lengthen the time that wounds bleed. Most dogs receiving these medications eventually develop gastro-intestinal problems and must stop taking the medications.
Aspirin (acetylsalicylic acid):
In 1899, the German company, Bayer, began marketing the new drug "Aspirin". Over the succeeding hundred years, aspirin became the most widely used anti-inflammatory drug in humans and dogs. It can be purchased in various forms including plain, buffered, and enteric-coated formulations as well as topical creams and rectal suppositories. It begins reducing pain in 1-2 hours after it is swallowed. Aspirin like drugs are called salicylates.
None of them, including aspirin, were ever approved by the FDA for use in pets. Aspirin is metabolized and eliminated by the kidneys after being processed in the liver. Before the newer NSAIDs were available, it was commonly used in dogs. It is more dangerous in cats, because they lack a liver enzyme, glucuronyl transferase. Because of this, cats have difficulty processing and eliminating aspirin. Aspirin lingers very long in the blood stream of cats (40hrs). Because of this, I would never give aspirin to cats - but some veterinarians do give the buffered form to cats. No human pill-form of aspirin should be given whole, to small pets. In dogs aspirin is eliminated within 7.5 hrs. Veterinarians used aspirin for the relief of pain associated with muscle or bone inflammation or arthritis. Aspirin should never be used in pets suffering from kidney disease or high blood pressure. I rarely give it because of the high rate of side effects from prolonged use. In cats, it has been used every two days to prevent and dissolve blood clots. Misoprostol helps in reducing stomach and intestinal ulceration associated with aspirin. Aspirin overdose in dogs or cats will result in salicylate poisoning. This is characterized by hemorrhage, severe blood acid-base abnormalities, coma, seizures, and death.
Ibuprofen (Advil, Motrin, Nuprin, Medipren):
Ibuprofen is an arylpropionic acid derivative that has been used in dogs as an anti-inflammatory agent. It is not approved for use in dogs or cats. Dogs are much more likely to develop gastro-intestinal side effects from ibuprofen administration than are humans. For this reason, I never give this medication to pets and don’t advise you doing so. At therapeutic doses, adverse effects observed in dogs include vomiting, diarrhea, gastro-intestinal bleeding, and kidney infection.
Phenylbutazone (Butazolodine, “bute”):
Phenylbutazone is another older NSAID agents that has been used in veterinary medicine for over thirty years to treat arthritis. It is primarily used in horses but was also administered to dogs. Veterinarians rarely administer it today. It is approved by the FDA for pain control in dogs but not cats. Its side effects in dogs can include ulceration and bleeding of the stomach and intestines as well as anemia. In dogs, phenylbutazone has been associated with bleeding disorders, liver damage, kidney damage, and rare cases of irreversible bone marrow suppression leading to death.
Piroxicam (Feldene, Pfizer):
Piroxicam is an older NSAID that is a member of the oxicam group of drugs. It is approved for use in humans only. Although it works well in humans I have found it to cause stomach distress in most dogs that I have tried this medication on. Piroxicam undergoes extensive recycling and processing in the liver of dogs This results in a prolonged presence in the plasma of dogs. Gastric and intestinal ulceration and bleeding and kidney damage have been observed in dogs receiving piroxicam.
Naproxen (Naprosyn. Aleve, Roche Co):
This NSAID is not approved for use in pets and I have never given it. One dose is said to lasts 45-92 hours in dogs. Dogs are extremely sensitive to its toxic effects. So, wjem it is used, it should only be given every second or third day. I am not familiar enough with this drug to suggest a safe dose. Because of the bad side effects that often occur, I do not recommend using naproxen in pets.
Flunixin meglumine (Banamine, Shering-Plough):
Flunixin meglumine is a potent injectable NSAID, which is particularly good for intestinal pain. In the United States, it is approved only for use in horses. Veterinarians have use it frequently in treating the pain associated with parvovirus intestinal disease in dogs and for treating post-surgical stomach pain. However, leading textbooks suggest it not be used at all in dogs. When given, it should not be given for more than two days. Recent research has shown that it can retard healing in dogs – especially when the intestines have been cut. The pain reducing effect of an injection of flunixin meglumine only lasts for a few hours. Long term administration of flunixin meglumine to dogs results in severe gastro-intestinal ulceration and kidney damage. It should not be given more than once or twice.
Ibuprofen (Advil, Motrin, Nuprin, Medipren):
This is a great anti-inflammatory drug in humans but it consistently causes ulcers in dogs after 2-6 weeks of use. In dogs, it will eventually cause ulcers of the stomach as well as vomiting. At a dose low enough to not have these side effects, the drug probably does not relieve pain. I do not recommend the use of ibuprofen in pets.
Indomethacin (Indocin, Indocin-SR):
When it is given to pets at doses high enough to relieve pain, Indomethacin is highly toxic to the gastro-intestinal tract of dogs. It often results in severe ulceration, bleeding, and dark, bloody stools. Do not use it in pets.
Pain Relievers Unrelated To NSAIDs :
Acetaminophen (Tylenol):
Acetaminophen is a para-aminophenol derivative with anti-fever and pain control activity activity, but very little anti-inflammatory effect. Acetaminophen does not produce stomach ulcers or retard blood clotting. Acetaminophen is more effective in inhibiting COX enzymes in the brain rather than in the body. I have never found it to be effective in reducing pain in dogs. In dogs, dose-dependent bad side effects include depression, vomiting, and destruction of blood hemoglobin. It should never be use in cats due to their lack of liver glucuronosyl transferase and the potential for hemolytic anemia and liver destruction. I have read that one extra-strength Tylenol will kill a cat.
Methocarbamol (Robaxin):
Methocarbamol is a muscle relaxant that exerts its effect by acting on the central nervous system (the nerves that control the muscles) rather than on the muscles themselves. It is a rather save drug. Although it does not directly lessen pain, it may relieve muscle tension associated with arthritis in pets. It has weak sedative properties and may make the urine appear darker.
Dissociative Anethetic Agents:
Ketamine (Ketaset, Vetalar):
This medication is commonly used as a general anesthetic in cats. However, studies have found that it reduces pain when it is applied to the skin as a gel or paste. A specialty compounding pharmacy can provide it. There have been some studies that found that ketamine, given by mouth, also alleviates pain. Of course, these studies work best in humans who can tell you if they are feeling less pain. It is also an NMDA receptor antagonist. NMDA is involved in the sensation of pain.
Alpha-2 agonists or Blockers:
Xylazine (Rompun):
Xylazine is an injectable sedative and anesthetic that is approved for use in dogs. It belongs to a class called alpha-2 agonists. When given by intramuscular injection at less than anesthetic doses, it is a very effective pain reliever. Anesthetic doses commonly cause dogs to vomit, but at low dosages, vomiting
and decreased breathing are rare.
I like to incorporate xylazine into my anesthetic protocols for dogs, because pain relief persists long after the surgery is completed. I have not personally used this xylazine in cats for pain relief but several sources suggest dosages that can be used in cats. I, personally, would not give xylazine to cats.
Medetomidine hydrochloride (Domitor, Novartis Co):
Medetomidine hydrochloride is approved as an anesthetic and pain reliever for use in dogs recovering from surgery. It also belongs to a class of drugs called alpha-2 adrenoreceptor agonists. At anesthetic doses this medication slows heart rate.
It is metabolized but the liver and excreted through the kidneys so it should not be given to dogs that have liver or kidney disease. It should also not be given to dogs at times that they are agitated and fearful at the animal hospital or dogs that might go into shock due to coexisting diseases.
NMDA Receptor Antagonists or Blockers:
NMDA receptor antagonists block pain by binding to the N-methyl-D-aspartate (NMDA) receptor. NMDA blocking medications include ketamine, dextromethorphan, memantine, and amantadine. The opioids methadone, dextropropoxyphene, and ketobemidone are also antagonists at the NMDA receptor.
Amantadine (Symmetrel):
Amantadine is the most commonly used oral drug in this class. It was originally developed as an antiviral compound, and has also been used to treat drug reactions that affect coordination (extrapyramidal reactions) and Parkinson's disease in humans. It appears to effectively block pain in dogs. There is positive information on its use in cats but I have never used it in cats. The dose of amantadine must be reduced in pets with poorly functioning kidneys. It has been given on a daily basis, but in most cases, it is given for one to two weeks and then discontinued until pain worsens. Side effects are uncommon, but may include agitation or diarrhea.
Dextromethorphan (Robitussin, Dexalone, Vicks Formula 44, etc):
This is a common cough suppressant used in humans and pets. But it is also an NMDA inhibitor. This is a rather safe medication when given at recommended dosage. It may have some potential in allieving pain in pets but I have never seen reference to this use.
Gabapentin (Neurontin):
Gabapentin has been used for many years to treat chronic pain in humans. It may offer some potential for use in dogs to lessen chronic pain. I have never personally used this medication in pets.
Tricyclic antidepressants (Elavil):
From studies in humans, we know that the tricyclic antidepressant, (Elavil), is sometimes effective in reducing chronic pain. I have administered it to dogs and cats without serious problems.
Joint Protective (Chondro-protective) agents (Cosequin, Arthroflex, etc.):
These nutritional supplements can be given safely to dogs and cats and many veterinarians dispense them. They supply the body with the building blocks of joint cartilage. In theory at least, they should be helpful in relieving some of the joint pain of arthritis. Similar products are sold over-the-counter at human pharmacies. They all seem to be non-toxic. They include polysulfated glycosoaminoglycans, glucosamine and chondroitin sulfate. However, I am a guinea pig in the Harvard Lifetime Study of Professionals. In a recent newsletter I received, glucosamine and chondroitin were found to be worthless in treating joint pain in humans. So I feel that the drugs effectiveness in dogs is now also in doubt.
Corticosteroids:
All of the corticosteroids mimic the effects of the body's own cortisone which is produced in the two adrenal gland. The are all the most effective blockers of inflammation and resulting pain. However, they all have major side effects when given over extended periods of time. These changes include, weight gain, fluid gain, increased thirst and urination, thinning of the skin, liver changes, decreased resistance to infectious disease, mood swings, and increased blood sugar. When they must be used, they should be given in the minimal amount that will control and inflammation and should not be given more than two or three times a week. A few corticosteroids used in humans for nasal allergies and one, beclomethasone, that is used on dogs topically can be applied every day. But they have very limited or no effects within the body.
Prednisone/Prednisolone (generic):
Prednisone and prednisolone are members of the glucocorticoid class of steroid hormones. They mimic the effects of natural cortisol, which is produced by the adrenal gland. They break down stored resources (fats, sugars and proteins) so that they may be used as fuels in times of stress. We do not use the glucocorticoids for their influences on glucose and protein metabolism; we use them because they are the second most effective anti-inflammatory, anti-pain medications other than Narcotic Opiate drugs. However, they have severe non-painful side effects and must only be given occasionally in measured doses or reserved for dog and cats in the twilight of their lives.
Dexamethasone (Azium, Voren):
Dexamethasone is another member of the cortisone class of hormones. This means they are steroids but, unlike the anabolic steroids that we hear about in sports which build muscle mass and strength, dexamethasone causes break-down of stored resources (fats, sugars and proteins). Dexamethazone is also a powerful anti-inflammation, anti-pain medication. However, the same side effects that limit prednisone/prednisolone apply to this medication.
It should only be given to control pain when all other medications have failed.
The Government Regulated Opiate Narcotics:
Opiates or Opioids are the most powerful pain-relieving compounds available for pets and humans. They all mimic natural brain chemicals that limit our perception of pain. However, they are highly addictive and should be reserved for pain that will not respond to other medications or when pets are in terminal condition. Also, with time, doses have to be increased to obtain comparable pain relief. Side effects include euphoria (joyous feeling), depression of breathing, physical dependence, and slowed heart rate constipation and itching. They may also cause contraction of the pupils of the eyes, sedation and unusual taste in foods. In humans, they are generally given to alleviate the pain of terminal cancer or painful nerve conditions. In pets, they are given to stop the pain of arthritis or cancer, when all other medications fail. In dogs in late hip dysplasia, corticosteroids are more likely to be given. Injectable and oral forms of opiates are rarely dispensed for pets in the United States. In the US, the Drug Enforcement Agency (DEA) frowns on any long-term use of opioids, fearing they might their diversion to illegal human use. The DEA calls all the opiates CII or class-2 narcotics. Veterinarians and physicians fear prosecution by the DEA and use them as little as possible. None of the controlled opioid class of drugs with the exception of fentanyl has FDA approval for use in pets. Because cats are deficient in glutathione liver enzyme, the half-life of some opioids in cats may be prolonged and doses must be smaller.
Fentanyl (Duragesic, Sublimaze, Ortho-McNeil Co):
Fentanyl patches are the most commonly used form of opiate narcotic used in pets. Fentanyl was first synthesized in the laboratory in the late 1950s to control pain in humans – particularly those suffering from cancer. It is about 90 times as potent as morphine. Unlike oral or injectable opioid narcotics, fentanyl patches slowly and continuously releases the drug giving smooth, continuous pain relief. Fentanyl passes through the pet’s skin and provides very steady pain relief. Veterinarians most often used fentanyl short-term, after surgery, in advanced cancer or subsequent to body injury. Fentanyl is sold as a transdermal adhesive patch which is placed on a hairless portion of the pet’s skin. In medium and larger dogs, complete patches can be given. In large cats, a whole patch can also be used. In smaller cats, only a portion of the plastic liner is folded back. Patches should never be cut. In dogs, one patch lasts for 72 hours but it takes 12-36 hours for enough of the drug to be absorbed. In cats, the effect of the patch can last up to 120 hours. Cats absorb enough of the drug to be effective within 5-8 hours. In dogs and cats, side effects include excitement or lethargy, poor appetite, and low body temperature. Fentanyl patches are sold in sizes that deliver 25, 50, 75 and 100 micrograms of the drug per hour. The 25 mcg/hr patches work well on 8-12 pound cats when only half the protective liner is removed. I find it best to cover the patch with an elastic bandage. Fur stubble interferes with delivery of the drug so that 50 mcg/hr human patches actually deliver about 37 mcg/hr in pets.
A common danger is the pet or a household member eating the patch or the patch being exposed to excessive heat, such as a heating pad, electric blanket, or a home heater vent. Should this occur, naloxone or Buprenorphine need to be given immediately. A suggested dose of naloxone is 0.2 mg/kg, repeated until breathing returns to normal (1). It should not be used in pets receiving Anipryl (selegiline hydrochloride) for Cushing’s disease or for Canine Cognitive Dysfunction Syndrome (doggy Altzheimers). Be very careful if you have small children. These patches can be fatal if swallowed.
Tramadol (Ultram):
This new synthetic opiate-like drug is a derivative of codeine. Because tramadol is not technically an opiate, it is not controlled by the Drug Enforcement Administration. It is one of the most promising drugs available to veterinarians for treating chronic pain in your pet. It has two modes of action: mu agonism and monoamine reuptake inhibition similar to the newer, human antidepressants. It appears to be comparable to meperidine or codeine in its pain-relieving affect. Administering tramadol with NSAIDs, or mu agonists increases the pain relief. . It tends not to cause vomiting as the opioids often do. The drug is inexpensive and available in generic form. Tramadol can be used for pain relief in both dogs and cats. Since most NSAIDs are dangerous in cats, tramadol provides a nice choice for cats with chronic pain issues). Because of it’s bitter taste, it should be prepared for cats in gelatin capsules. Side effects are uncommon but they include sleepiness and upset stomach and intestine. It must never be given with the newer, human, anti-depressants (SSRIs). In dogs, tramadol is usually given twice a day – but it can be given more frequently if required.
Morphine (Oramorph SR, Roxane):
Morphine sulfate (a CII regulated narcotic) is available in tablet, liquid and capsule preparations. Cats have been given the liquid formula - but most cats and some dog strongly dislike the bitter taste. I have never used this drug in my practice, although I saw it used when I was in veterinary school. Although it is a centuries-old drug, it is rarely used in animals in the United States. It is a very effective barrier to pain. Morphine was usually given to dogs by injection. It went into disfavor because clients occasionally took the pet’s medication themselves and because of strong government opposition to it’s use. This is because of the stigma of narcotics addiction in humans. Also, because the dog dose is so much higher than the human dose, owner fatalities occurred when the tablets were dispensed for dogs but taken by the pet owner.
Meperidine (Demerol):
This is an injectable narcotic drug that is one fifth as powerful as morphine. It is an excellent drug for controlling pain for short periods in dogs. I have used it to spare dogs that were critically injured by automobile and had no hope of recovery. It gave these pets relief from pain until the owners could except the situation. I have not used it in cats. It is not suitable for long term use because, with time, pets become resistant to its action. The dose of this drug in dogs is published as 2 mg/pound given intramuscularly or orally every 4 hours. A published dose in cats is 1-2 mg/pound.
I use up to four times this dose up to three times a day.
Oxymorphone (Numorphan):
This medication is a semi-synthetic opioid ten times as strong as morphine. It is given by injection and its effects last 4-6 hrs. I have personally never used it or seen it used but it is said to be a good drug for postoperative pain in dogs and cats.
Buspirone (Buspar):
Buspirone is a highly effective medication when used for treatment of anxiety and PMS syndrome in humans. It is very safe when given at correct dosages to dogs and cats. More recently, it has been evaluated for its pain-relieving effect. In experimental rats, It was effective in blocking the pain of burns, wounds and inflammation. These data suggest the potential use of buspirone in the management of various types of pain. I place it here because it acts on the same mu receptors through which the opium narcotics block pain. Until now, it has been used in dogs and cats to decrease phobias and generalized anxiety. Buspirone should not be used along with the MO class of antidepressants because dangerous increases in blood pressure can result.
Butorphanol (Stadol, Torbutrol, Torbugesic-SA, Ft. Dodge/Wyeth):
This mixed pain-receptor blocker is a less controlled form of narcotic (C-IV). This is one of the few opioid drugs that works consistently well in cats. This opioid agonist-antagonist is administered by injection or intranasal spray. It has been given to dogs and cats by subcutaneous injection three times a day. After twenty minutes, its pain-controlling properties take effect and last for eight hours. It can also be given by mouth. It causes less side effects than traditional opioids narcotics. Occasionally, butorphanol will cause eye dilation, confusion and pain at the site of injection - especially when given to cats. It is not considered to be useful in the management of chronic pain. Compounding pharmacies can prepare a transdermal gel of butorphanol that can be applied to the inner surface of the ear or a shaved spot on the neck of dogs and cats. The injectable liquid butorphanol can also be given by dropper to cats. Since it has no taste, cats will not fuss about a bitter taste. It is not absorbed well when given in this manner to dogs. I have read that butorphanol tends to make cats more affectionate. The only problems with this are that the cats may rub on you and purr, making it hard for you to sleep. Others have warned that cats may become more aggressive and likely to bite while on butorphanol. Some possible side effects are loss of appetite, vomiting, incoordination, and restlessness.
Buprenorphine (Temgesic, Vetergesic, Buprenex):
This partial mu agonists binds to them and prevents their activation in the sensation of pain.
Buprenorphine is approximately 30 times stronger than morphine. It is a bit expensive for some clients. However, it is a medication with very few serious side effects. It works best when pain is no more than moderate. It can also be prepared by compounding pharmacies as a transdermal patch and administered through the skin, similar to fentanyl. Although these patches take up to six hours, it maintains its pain-controlling effect for up to twelve hours. With subsequent patches, pain control becomes more uniform.
Mixed agonist/antagonists like butorphanol (CIV) are not considered useful in the management of chronic pain. First-pass effect destroys some of the drug, and the analgesia is considered to be relatively short-lived (1-2 hours). Because these drugs are kappa agonists and mu antagonists, the pain relief is often less than optimal for chronic discomfort. However, visceral nociception is considered to be more responsive to kappa agonism, leading some urologists to advocate butorphanol's use in chronic bladder pain (FLUTD).
Other Non-traditional Drugs:
Some sources claim that omega-3 fatty acids, obtained from fish, are helpful in controlling chronic pain. Many veterinarians dispense it because, as far as we know, it is completely safe when given in moderate amounts.
Nutritional centers often dispense bioflavinoids for chronic pain. I do not know on what data this is based. Other veterinarians administer acupuncture for chronic pain. I have not seen this to be effective.
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Ron Hines DVM PhD
Dear Reader,
You should never give your pet the medications I write about without expert, hands-on advice from your local veterinarian. Do not give doses higher than recommended. Although mixing medications is sometimes helpful, it should not be done without expert counsel. This article is for your general knowledge only.
Those of us who have and work with pets know that they experience pain similar to human beings. Although we cannot prove this scientifically, we are so closely attuned to our four-legged friends that we know when they are uncomfortable and troubled by pain. You, as the pet’s owner, are more likely to notice signs of pain than your veterinarian because you are more attuned and bonded to your own pet.
Pain can be a temporary problem or a persistent one. Acute or sudden pain can be the result of surgery or sudden damage to any of the major organs, muscles or bones of the body. However, Pain can be a temporary problem or a persistent one. Some causes of chronic pain in older pets are hip dysplasia, arthritis of the spine and joints instability.
All pain-relieving drugs are called analgesics. Controlling pain in your pet actually improves the outcome of many diseases or surgery. Whenever possible, it is better to give pain-controlling medications early rather than waiting until the pain becomes severe.
It is hard to objectively judge the severity of pain in human beings and even more difficult to do so in animals. Our thresholds to pain differ between one person and another and from one animal to another. Some sources claim a five-fold difference between individual pets. Pain perception depends partially on species, breed, age, gender, time of day and your pet’s individual temperament. Young animals tend to have a lower threshold to pain. Older and debilitated pets are more stoic and may not show as much response to pain - but they feel it just the same. Hunting and working breeds of dogs are more resistant to expressing pain than toy or miniature breeds. Signs of pain are subtler in cats than in dogs. When you take your pet in to an animal hospital, your pet’s is usually worrying about the visit and strange environment will often ignore the pain that you noticed at home. This can be very frustrating for owners who tell me “but he limped at home!”
Cats in pain are more stoic than dogs and they hide pain more effectively. Cats will often hunch up when the pain is in their tummies and be reluctant to move. Some cats hiss if a painful site is touched or become unresponsive to affection and petting. Other cats become aggressive and belligerent when in pain. Often their food consumption goes down. Some cats in acute pain meow pathetically. They often carry their ears down. Many pain control medications are bitter. It can be extremely hard to get cats to accept them. For this reason, medications are best when they are prepared in tasteless capsules by a compounding pharmacy.
In both cats and dogs, pain may lead to over-grooming the area that is painful. This can lead to hair loss and self-mutilation of the area. Some pets tremble and move with their stomachs tensed up. Others tremble. Some will show lameness of an affected leg while others become aggressive, pant or grimace. Any sudden behavior change can be a symptom of pain. Excessive salivation, licking of the lips, dilation of the eyes, rapid breathing and increased heart rate may all be attributable to pain. Some dogs in pain also eat less. Some become restless and do not sleep well. Some stop grooming and appear dejected. Pain can cause an increase in body temperature (fever), respiration, heart rate and blood pressure.
When the pain of hip dysplasia or spinal arthritis in dogs is severe, the dog may be unwilling to rear up on its back legs for a treat. I will often pinch the toe of a pet that appears to be in pain to judge the severity of the pain. If the cat or dog reacts to the toe pinch then I assume the severity of its other pain is less than that of the pinch.
Pain alone can actually change the results of blood chemistry analysis. Dogs and cats in pain may have elevated blood sugar. Their blood cortisol (steroid) and white cell levels often increase. Pain can also interfere with the immune system, increase the risk of infections and slowing the healing of wounds and surgical incisions.
You must understand that complete elimination of long-term pain may be impossible or even undesirable in your pet. But it can be minimized with a number of medications. There are five major classes of medicine that can be used to control pain in dogs and cats. Cats do not metabolize many drugs as well as other animals, so your options are fewer. All of the following medications must be used with extreme caution in cats. You and your veterinarian must weigh the advantages of pain medications in cats against the possible damage that might occur.
There are some general rules in using pain control medications in dogs and cats. The first is to try to give the medication early before the pain becomes too intense. The second is that it is usually safer and more effective to give lower doses of two or more pain control medications that have different modes of action rather than a higher dose of a single medication. Doses should always be kept to the bare minimum needed to give relief. Older patients should receive lower doses less frequently than younger more robust pets. It is also wise to check kidney and liver function before and during the use of pain control medications.
Pain control medications used in pets
The Non-steroidal Anti-inflammatory Agents (NSAIDs):
In 1989 scientists discovered the first of the NSAIDs. Pain sensation and inflammation rely on messenger chemicals called prostaglandins. There are two types of prostaglandins, the “good ones” called COX 1 and the “bad” ones called COX 2. Older NSAIDs prevent the formation both good and bad prostaglandins. “Good prostaglandins protect the lining of the stomach and small intestine and also assist clotting and kidney blood flow. “Bad” prostaglandins cause inflammation, swelling and pain. They have become the most frequently used pain medications used in pets. They are widely used in humans as well. There are basically two kinds of NSAIDs. I call the newer ones (COX 2 blockers)and the older ones (Cox 1 and Cox2 blockers). They both work by blocking an enzyme called cyclooxygenase (COX) which is necessary for both prostaglandin formation. The older NSAIDs cause more problems because they block both the pain-producing prostaglandins and the good, protective, prostaglandins. We suspect that NSAIDs also work directly on the brain to block pain sensation.
Now, the enzyme, Cyclooxygenase occurs in 2 forms: The ”good” COX 1 which controls the formation of “good” prostaglandins that protect the lining of the stomach and intestine, the blood clotting process and blood flow to the kidneys. The “bad” one, COX 2, encourages pain and inflammation. This is a simplified explanation but it should be sufficient for readers. Although all the newer NSAIDs work in the same way, some seem more effective in blocking COX 2 in a particular pet. So don’t give up if the first medication you use doesn’t appear to work.
All NSAIDs are well absorbed when given orally. They are eliminated from the body by the liver and kidneys.
When your pet is in acute (sudden) pain, your veterinarian will often use injectable forms of NSAIDs for fast relief.
The Special Problem Of Cats and NSAIDs
Cats are very special animals. They have a unique body biochemistry and liver function that make the NSAIDs more dangerous to them. Your cat’s liver does not have enough of a specific enzyme (bilirubin-glucuronide enzyme). Because of this, NSAIDs tend to linger in the cat’s blood stream. So NSAIDs must be given in very limited doses and they must be given less frequently.
It appears that two of the few NSAIDs that cats tolerate fairly well are meloxicam and ketoprofen. These medications come in flavored syrup as well as tablets. The volume of syrup needed is small cats so they usually accept it. Both compounds are registered for use in cats in Canada and Europe. There, they appear to be relatively safe especially when given for short periods of time. But neither medication is licensed by the FDA for use in cats in the United States.
Because there is a great deal of variation between individual cats as to the effects of these drugs, your veterinarian should monitor your cat closely for any side effects. This is especially true if either drug is given for extended periods of time.
Possible Side Effects in Pets
Because the liver plays an important roll in eliminating NSAIDs from the body, It is best not to use NSAIDs in the face of known liver disease, because animals with liver disease do not remove these drugs from their bodies normally. Also, on occasion, NSAIDs may cause sudden liver failure. According to the FDA’s records on the toxicity of approved products for dogs, Carprofen (Rimadyl) had had the most problems. However, this may be because it is the most widely prescribed.
From time to time, all of the NSAIDs will cause kidney damage in cats and dogs. This is because they can limit blood flow to the kidneys. This is not a problem if your pet has normal kidneys. But if your pet already has some kidney blood flow damage, more might result. This is especially true with the older NSAIDs. COX-2 enzyme increases blood circulation in the kidneys. When COX-2 is inhibited by the older NSAID versions blood flow through the kidneys can drop to dangerous levels.
Another common side effect is stomach and intestinal bleeding. Again, this is more of a problem with the older NSAIDs. When this occurs, your pet will experience vomiting and diarrhea. If this occurs the medication must be stopped or the dose decreased. This side effect occurs more in pets than in humans that receive NSAIDs. It is especially true of the older NSAIDs, which I listed below. An early warning sign of bleeding is a decreased number of circulating red blood cells. Your veterinarian can check for this periodically.
If I must use the older NSAIDs, I often suggest over-the-counter medications that limit stomach acidity (cimetidine, ranitidine, famotidine) Pantoprazole or omeprazole may also have this potential but I have not used them. These medications may minimize NSAID side effects. Some veterinarians give a synthetic prostaglandin, mistoprostol, with NSAIDs to coat and protect the pet’s stomach and intestines. Omeprazole may also be beneficial but I have found no data on its use in pets.
When administering NSAIDs, do not use two different ones at the same time. They should also not be given in combination with corticosteroids (prednisolone, prednisone, dexamethasone, etc).
Also, pets receiving diuretics such as Lasix (furosemide) are also more susceptible to NSAID side effect.
The Newer And Safer NSAIDs:
Most of this group were originally developed for use in humans. They are safer than the “old” NSAIDs. Because the newer ones are COX-2 selective, they are less likely to cause stomach or intestinal bleeding. This is the reason they were very popular in people until the Vioxx (rofecoxib) scare occurred. Luckily, dogs and cats do not share this risk because they do not commonly have heart attacks. Only Meloxicam injection is approved for short term use in cats.
These are dog or human products. None are approved in the United States for use in cats. But because veterinarians want to relieve suffering, we sometimes give them anyway. There are much fewer options in treating pain in cats. When we do use them in cats, it is called an “off label use”.
Before using these drugs, your veterinarian may suggest bloodwork to determine how well the liver and kidneys are functioning in your pet. If it is for long-term use, I would definitely suggest that. It is generally unwise to give this group of medications at the same time corticosteroid-type medications are given. The exception is in end-of-life situations.
Carprofen (Rimadyl, Pfizer):
Introduced in the USA in 1997 by Pfizer Animal Health Co for use in dogs, this NSAID is very similar to meloxicam. Carprofen is often given to dogs before surgery to decrease post-operative pain.
The other approved use is to combat the pain of arthritis. It is available for dogs in two oral forms, caplets and chewable tablets. In Europe, Canada and other countries, carprofen is also registered for short-term therapy in cats.
It will, on rare occasions (2 per 1000 dogs) , cause liver damage in dogs. Particularly Labrador Retrievers.
Ketoprofen (Orudis, Oruvai, Actron, Oruvail, Orudis-KT, Ketofen) :
This medication is sold for human use, over-the-counter at most pharmacies and super-centers. It is only approved in the US for use in people and horses. In Europe and Canada it is approved for use in dogs and cats. There it is available in tablet or injectable form.
All the factors and precautions I mention in the introduction to NSAIDs apply to ketoprofen.
As with the other NSAIDs, ketoprofen is processed in the liver to inactive byproducts that are eliminated by the kidneys. Side effects can includine intestinal upset with vomiting and diarrhea similar to other NSAIDs. Word-of-mouth and published articles recommend this medication to relieve short-term pain (up to 5 days) in dogs and cats. However, this is not an approved use in the United States. Once given by mouth, ketoprofen is rapidly absorbed. After 2-3 hours, blood levels of this medication are only half their original levels.
Side effects, including liver damage and kidney disease, have been reported in pets. Because ketoprofen can adversely affect blood clotting, I do not suggest it be given before or after surgery.
Etodolac (Etogesic, Wyeth Co):
Etodolac is approved for use in dogs in the United States. It is also quite similar to carprofen. Etodolac is given once a day to manage arthritis. It can be given with or without food.
The most commonly reported side effects to etodolac are diarrhea, vomiting, or mopyness and inactivity. When given at the recommended dose, side effects are rare. But if the dose is trebled, vomiting, intestinal bleeding, and weight loss often occur. Etodolac is eliminated by the liver and with the stool. All the cautions I mentioned in the introduction apply to etodolac.
Meloxicam (Metacam, Mobic, Borringer-Ingelheim, Merial Co):
This potent inhibitor of prostaglandin synthesis is used for the treatment of the acute and chronic pain associated with muscle disease and arthritis. It is also used in the management of surgical pain. Meloxicam is a favorite of mine in this group. This is probably because occasionally I myself have take the human brand of meloxicam called Mobic. It is marketed for use in dogs in the United States. It is available in oral tablet, suspension and injectable forms.
Meloxicam is approved for use in dogs. A larger, loading dose is given on the first day. On succeeding days, the dose should be lowered to the lowest possible amount that keeps the dog pain free or nearly so. Intestinal safety seems to be greater for meloxicam than for many other NSAIDs.
The injectable form of Metacam is approved for cats as a one-time, subcutaneous injection for post-surgical pain. If given more than one time or if other NSAIDs are given, kidney and liver toxicity may occur in cats. Both their kidney and liver function must be monitored frequently. If your veterinarian elects to use it in cats, it should not be given more than two or three days per week. Because of the nature of NSAIDs in cats, you must weight the potential risks against the benefits and make your own decision along with your veterinarian.
Deracoxib (Deramaxx, Novartis):
Deracoxib was first approved for controlling post-operative pain. In 2003, it won approval for the prevention of chronic arthritis pain. It is available in beef-flavored chewable tablets. When used to control the pain of surgery, it should be given by injection about two hours before the surgery. It can then be given for up to six days following the surgery. The dose of deracoxib is lowered when the drug is intended for long-term use in the treatment of arthritis.
Deracoxib is related to a class of antibiotic drugs called "sulfonamides" which means they contains a contain sulfur in their structure. However deracoxib is not an antibiotic. It should not be used in pets that have a history of problems taking sulfas. It should not be given after long periods of anesthesia. Do not use deracoxib in dogs that weigh less than 4 lbs. Do not administer it to pregnant dogs, nursing mothers or dogs under age 4 months of age. It is best to give this medication with food. It should never be given with corticosteroid medications. Do not use deracoxib in dogs that are dehydrated, or taking diuretics, or dogs that have preexisting kidney, liver, heart or circulatory problems. Symptoms of overdosage may include diarrhea, vomiting, and bloody stools.
Duracoxib is unique among the newer NSAIDs in the long length of time it controls pain. It persists in the blood stream longer than other NSAIDs. As with the other NSAIDs, the medication will occasionally cause life-threatening stomach punctures so dogs on this medication need to be monitored closely. It should not be used in dogs with an elevated BUN or Creatinine – signs of kidney disease. In such dogs it can exacerbate kidney failure and uremia. A recent study found that the risk of intestinal perforation was quite high when this product was given at doses exceeding the manufacturer's recomendations or when the dogs received corticosteroids while on the medication.
Studies, using deracoxib in cats have been run. A specially made liquid formula was accepted readily by the cats, and no adverse effects were observed. But several more scientific studies are needed before we know the effectiveness and safety of deracoxib in cats. Until then, you must consider the use of deracoxib in cats as an experiment with unknown risks and benefits.
Tepoxalin (Zubrin):
First marketed for dogs in 2003 by the Schering-Plough Animal Health Corporation,
This medication has properties similar to both carprofen and ketoprofen. This medication not only inhibits “bad” prostaglandin formation but also acts through different pathways to block pain.
Firocoxib (Prevacox):
Firocoxib is similar to dericoxib. It was recently approved in the United States and Europe for the control of pain and inflammation associated with arthritis in dogs. It is available in a chewable tablets.
Tolmetin (Tolectin, McNeil Co, Janssen-Ortho):
I have no experience with this medication. Tolmetin was approved for human use in 1997. It is used to treat rheumatoid arthritis. It is usually taken three times a day. I am not aquatinted with its use in pets. Studies in animals have shown that tolmetin to possess anti-inflammatory, analgesic, and anti-fever activity. In rats, tolmetin prevents the development of arthritis and also decreases inflammation. Tolmetin appears to reduce prostaglandin synthesis similar to other NSAIDs.
In humans it may cause headache, dizziness, nervousness, upset stomach, stomach pain or cramps, vomiting, diarrhea or constipation and gas.
Meclofenamic acid (Arquel):
Meclofenamic acid is FDA-approved for use in dogs This NSAID is available as an oral tablet. Meclofenamic acid has a therapeutic index that is lower than that of other NSAID, possibly due to the way it circulates in the liver. This means that the necessary dose for pain relief is quite close to the dose that can cause side effects. It is sold for the treatment of pain and inflammation - especially that associated with arthritis. It may take three or four days for pain relief to be seen in your dog. Side effects that can occur include vomiting, diarrhea, lack of appetite, bloody stool, black tarry stool, or ulcers in the stomach or small intestines. Less common side effects are depression, fever, behavior changes, fast breathing, edema inability to control urine, or irreversible anemia.
Do not use Meclofenamic acid in dogs that are hypersensitive (allergic) to this drug or any other NSAIDs. Do not use the medication at full dosage for more than 5-6 days. After that period, the dose should be decrease to the minimum dose and most infrequent administration that controls your pet’s pain. That is, give it only a few days per week if possible.
All the earlier warning I have given concerning NSAIDs apply to meclofenamic acid. Do not use this drug in dogs that have kidney or liver problems or heart disease. Tests must be run by your veterinarian to rule these conditions out. Give with a full meal. Do not give meclofenamic acid for a week prior to surgery or the week after surgery. Do not give to dehydrated pets or those taking diuretics for heart or lung problems. Do not use this product in dogs with clotting deficiencies such as Von Willebrand’s disease. Do not give the medication to pregnant or nursing mothers. Do not give to pets under eight months of age. This product is not approved for use in cats. Do not give with other NSAIDs or sulfa antibiotics, glipizide, or valproic acid or oral anticoagulants. In epileptic dogs, Meclofenamic acid may increase blood concentrations of phenytoin. If you exceed the recommended dosage you may see evidence of stomach ulcers and kidney damage.
Vedaprofen (Quadrisol-1 and Quadrisol-5, Intervet Co):
I have no personal experience with vedaprofen. However, reports out of the Netherlands, Portugal and Asia suggest to me that this medication may have significant potential uses in dogs and possibly cats. But because all NSAIDs have similar effects on the body, I do not expect it to be free of the side effects present with all NSAIs.
Intervet, on its European site, states that Quadrisol “has been tested in numerous field trials and has been proven to be safe, effective and well tolerated by dogs” and is said to be safe for use in nursing mothers. However, the possible side-effects are the same as the other newer NSAIDs. It is marketed for dogs for the relief of pain and control of inflammation associated with short-term injury or long-term for arthritic problems. It comes in gel form in a dosing syringe. It should be given with food. It is sold in two formulations: Quadrisol-1 is for use in dogs weighing under 5 pounds and Quadrisol-5 for dogs weighing over 5 pounds. In some countries Quadrisol-1 is also marketed for the management of fever and post-operative pain in cats. In studies in the Netherlands it was found to be a bit more effective than meloxicam.
Celecoxib (Celebrex, Pfizer Inc):
This drug is not approved for use in pets. In one study in beagles, there was dangerous variation in the length of time the drug stayed in the body as well as great variation in blood levels between dogs. I do not recommend its use until we understand it better.
Valdecoxib (Bextra):
This medication was introduced to the US market in 2001 as an arthritis remedy for humans. Valdecoxib is a potent and specific inhibitor of cyclooxygenase-2 (COX-2). However, side effects in humans led to it’s being banned in several countries. There is no data on its use in animals.
The Older, Less Safe NSAIDs:
These medications are much less specific than the newer NSAIDs. These medications reduce both the “good” and the bad prostaglandins. Bleeding is the most common side effect of these drugs. Despite this common side effects, the older NSAIDs are still used today in veterinary medicine because they are so much cheaper than the newer ones. Do not give these older medications after major surgery because they can lengthen the time that wounds bleed. Most dogs receiving these medications eventually develop gastro-intestinal problems and must stop taking the medications.
Aspirin (acetylsalicylic acid):
In 1899, the German company, Bayer, began marketing the new drug "Aspirin". Over the succeeding hundred years, aspirin became the most widely used anti-inflammatory drug in humans and dogs. It can be purchased in various forms including plain, buffered, and enteric-coated formulations as well as topical creams and rectal suppositories. It begins reducing pain in 1-2 hours after it is swallowed. Aspirin like drugs are called salicylates.
None of them, including aspirin, were ever approved by the FDA for use in pets. Aspirin is metabolized and eliminated by the kidneys after being processed in the liver. Before the newer NSAIDs were available, it was commonly used in dogs. It is more dangerous in cats, because they lack a liver enzyme, glucuronyl transferase. Because of this, cats have difficulty processing and eliminating aspirin. Aspirin lingers very long in the blood stream of cats (40hrs). Because of this, I would never give aspirin to cats - but some veterinarians do give the buffered form to cats. No human pill-form of aspirin should be given whole, to small pets. In dogs aspirin is eliminated within 7.5 hrs. Veterinarians used aspirin for the relief of pain associated with muscle or bone inflammation or arthritis. Aspirin should never be used in pets suffering from kidney disease or high blood pressure. I rarely give it because of the high rate of side effects from prolonged use. In cats, it has been used every two days to prevent and dissolve blood clots. Misoprostol helps in reducing stomach and intestinal ulceration associated with aspirin. Aspirin overdose in dogs or cats will result in salicylate poisoning. This is characterized by hemorrhage, severe blood acid-base abnormalities, coma, seizures, and death.
Ibuprofen (Advil, Motrin, Nuprin, Medipren):
Ibuprofen is an arylpropionic acid derivative that has been used in dogs as an anti-inflammatory agent. It is not approved for use in dogs or cats. Dogs are much more likely to develop gastro-intestinal side effects from ibuprofen administration than are humans. For this reason, I never give this medication to pets and don’t advise you doing so. At therapeutic doses, adverse effects observed in dogs include vomiting, diarrhea, gastro-intestinal bleeding, and kidney infection.
Phenylbutazone (Butazolodine, “bute”):
Phenylbutazone is another older NSAID agents that has been used in veterinary medicine for over thirty years to treat arthritis. It is primarily used in horses but was also administered to dogs. Veterinarians rarely administer it today. It is approved by the FDA for pain control in dogs but not cats. Its side effects in dogs can include ulceration and bleeding of the stomach and intestines as well as anemia. In dogs, phenylbutazone has been associated with bleeding disorders, liver damage, kidney damage, and rare cases of irreversible bone marrow suppression leading to death.
Piroxicam (Feldene, Pfizer):
Piroxicam is an older NSAID that is a member of the oxicam group of drugs. It is approved for use in humans only. Although it works well in humans I have found it to cause stomach distress in most dogs that I have tried this medication on. Piroxicam undergoes extensive recycling and processing in the liver of dogs This results in a prolonged presence in the plasma of dogs. Gastric and intestinal ulceration and bleeding and kidney damage have been observed in dogs receiving piroxicam.
Naproxen (Naprosyn. Aleve, Roche Co):
This NSAID is not approved for use in pets and I have never given it. One dose is said to lasts 45-92 hours in dogs. Dogs are extremely sensitive to its toxic effects. So, wjem it is used, it should only be given every second or third day. I am not familiar enough with this drug to suggest a safe dose. Because of the bad side effects that often occur, I do not recommend using naproxen in pets.
Flunixin meglumine (Banamine, Shering-Plough):
Flunixin meglumine is a potent injectable NSAID, which is particularly good for intestinal pain. In the United States, it is approved only for use in horses. Veterinarians have use it frequently in treating the pain associated with parvovirus intestinal disease in dogs and for treating post-surgical stomach pain. However, leading textbooks suggest it not be used at all in dogs. When given, it should not be given for more than two days. Recent research has shown that it can retard healing in dogs – especially when the intestines have been cut. The pain reducing effect of an injection of flunixin meglumine only lasts for a few hours. Long term administration of flunixin meglumine to dogs results in severe gastro-intestinal ulceration and kidney damage. It should not be given more than once or twice.
Ibuprofen (Advil, Motrin, Nuprin, Medipren):
This is a great anti-inflammatory drug in humans but it consistently causes ulcers in dogs after 2-6 weeks of use. In dogs, it will eventually cause ulcers of the stomach as well as vomiting. At a dose low enough to not have these side effects, the drug probably does not relieve pain. I do not recommend the use of ibuprofen in pets.
Indomethacin (Indocin, Indocin-SR):
When it is given to pets at doses high enough to relieve pain, Indomethacin is highly toxic to the gastro-intestinal tract of dogs. It often results in severe ulceration, bleeding, and dark, bloody stools. Do not use it in pets.
Pain Relievers Unrelated To NSAIDs :
Acetaminophen (Tylenol):
Acetaminophen is a para-aminophenol derivative with anti-fever and pain control activity activity, but very little anti-inflammatory effect. Acetaminophen does not produce stomach ulcers or retard blood clotting. Acetaminophen is more effective in inhibiting COX enzymes in the brain rather than in the body. I have never found it to be effective in reducing pain in dogs. In dogs, dose-dependent bad side effects include depression, vomiting, and destruction of blood hemoglobin. It should never be use in cats due to their lack of liver glucuronosyl transferase and the potential for hemolytic anemia and liver destruction. I have read that one extra-strength Tylenol will kill a cat.
Methocarbamol (Robaxin):
Methocarbamol is a muscle relaxant that exerts its effect by acting on the central nervous system (the nerves that control the muscles) rather than on the muscles themselves. It is a rather save drug. Although it does not directly lessen pain, it may relieve muscle tension associated with arthritis in pets. It has weak sedative properties and may make the urine appear darker.
Dissociative Anethetic Agents:
Ketamine (Ketaset, Vetalar):
This medication is commonly used as a general anesthetic in cats. However, studies have found that it reduces pain when it is applied to the skin as a gel or paste. A specialty compounding pharmacy can provide it. There have been some studies that found that ketamine, given by mouth, also alleviates pain. Of course, these studies work best in humans who can tell you if they are feeling less pain. It is also an NMDA receptor antagonist. NMDA is involved in the sensation of pain.
Alpha-2 agonists or Blockers:
Xylazine (Rompun):
Xylazine is an injectable sedative and anesthetic that is approved for use in dogs. It belongs to a class called alpha-2 agonists. When given by intramuscular injection at less than anesthetic doses, it is a very effective pain reliever. Anesthetic doses commonly cause dogs to vomit, but at low dosages, vomiting
and decreased breathing are rare.
I like to incorporate xylazine into my anesthetic protocols for dogs, because pain relief persists long after the surgery is completed. I have not personally used this xylazine in cats for pain relief but several sources suggest dosages that can be used in cats. I, personally, would not give xylazine to cats.
Medetomidine hydrochloride (Domitor, Novartis Co):
Medetomidine hydrochloride is approved as an anesthetic and pain reliever for use in dogs recovering from surgery. It also belongs to a class of drugs called alpha-2 adrenoreceptor agonists. At anesthetic doses this medication slows heart rate.
It is metabolized but the liver and excreted through the kidneys so it should not be given to dogs that have liver or kidney disease. It should also not be given to dogs at times that they are agitated and fearful at the animal hospital or dogs that might go into shock due to coexisting diseases.
NMDA Receptor Antagonists or Blockers:
NMDA receptor antagonists block pain by binding to the N-methyl-D-aspartate (NMDA) receptor. NMDA blocking medications include ketamine, dextromethorphan, memantine, and amantadine. The opioids methadone, dextropropoxyphene, and ketobemidone are also antagonists at the NMDA receptor.
Amantadine (Symmetrel):
Amantadine is the most commonly used oral drug in this class. It was originally developed as an antiviral compound, and has also been used to treat drug reactions that affect coordination (extrapyramidal reactions) and Parkinson's disease in humans. It appears to effectively block pain in dogs. There is positive information on its use in cats but I have never used it in cats. The dose of amantadine must be reduced in pets with poorly functioning kidneys. It has been given on a daily basis, but in most cases, it is given for one to two weeks and then discontinued until pain worsens. Side effects are uncommon, but may include agitation or diarrhea.
Dextromethorphan (Robitussin, Dexalone, Vicks Formula 44, etc):
This is a common cough suppressant used in humans and pets. But it is also an NMDA inhibitor. This is a rather safe medication when given at recommended dosage. It may have some potential in allieving pain in pets but I have never seen reference to this use.
Gabapentin (Neurontin):
Gabapentin has been used for many years to treat chronic pain in humans. It may offer some potential for use in dogs to lessen chronic pain. I have never personally used this medication in pets.
Tricyclic antidepressants (Elavil):
From studies in humans, we know that the tricyclic antidepressant, (Elavil), is sometimes effective in reducing chronic pain. I have administered it to dogs and cats without serious problems.
Joint Protective (Chondro-protective) agents (Cosequin, Arthroflex, etc.):
These nutritional supplements can be given safely to dogs and cats and many veterinarians dispense them. They supply the body with the building blocks of joint cartilage. In theory at least, they should be helpful in relieving some of the joint pain of arthritis. Similar products are sold over-the-counter at human pharmacies. They all seem to be non-toxic. They include polysulfated glycosoaminoglycans, glucosamine and chondroitin sulfate. However, I am a guinea pig in the Harvard Lifetime Study of Professionals. In a recent newsletter I received, glucosamine and chondroitin were found to be worthless in treating joint pain in humans. So I feel that the drugs effectiveness in dogs is now also in doubt.
Corticosteroids:
All of the corticosteroids mimic the effects of the body's own cortisone which is produced in the two adrenal gland. The are all the most effective blockers of inflammation and resulting pain. However, they all have major side effects when given over extended periods of time. These changes include, weight gain, fluid gain, increased thirst and urination, thinning of the skin, liver changes, decreased resistance to infectious disease, mood swings, and increased blood sugar. When they must be used, they should be given in the minimal amount that will control and inflammation and should not be given more than two or three times a week. A few corticosteroids used in humans for nasal allergies and one, beclomethasone, that is used on dogs topically can be applied every day. But they have very limited or no effects within the body.
Prednisone/Prednisolone (generic):
Prednisone and prednisolone are members of the glucocorticoid class of steroid hormones. They mimic the effects of natural cortisol, which is produced by the adrenal gland. They break down stored resources (fats, sugars and proteins) so that they may be used as fuels in times of stress. We do not use the glucocorticoids for their influences on glucose and protein metabolism; we use them because they are the second most effective anti-inflammatory, anti-pain medications other than Narcotic Opiate drugs. However, they have severe non-painful side effects and must only be given occasionally in measured doses or reserved for dog and cats in the twilight of their lives.
Dexamethasone (Azium, Voren):
Dexamethasone is another member of the cortisone class of hormones. This means they are steroids but, unlike the anabolic steroids that we hear about in sports which build muscle mass and strength, dexamethasone causes break-down of stored resources (fats, sugars and proteins). Dexamethazone is also a powerful anti-inflammation, anti-pain medication. However, the same side effects that limit prednisone/prednisolone apply to this medication.
It should only be given to control pain when all other medications have failed.
The Government Regulated Opiate Narcotics:
Opiates or Opioids are the most powerful pain-relieving compounds available for pets and humans. They all mimic natural brain chemicals that limit our perception of pain. However, they are highly addictive and should be reserved for pain that will not respond to other medications or when pets are in terminal condition. Also, with time, doses have to be increased to obtain comparable pain relief. Side effects include euphoria (joyous feeling), depression of breathing, physical dependence, and slowed heart rate constipation and itching. They may also cause contraction of the pupils of the eyes, sedation and unusual taste in foods. In humans, they are generally given to alleviate the pain of terminal cancer or painful nerve conditions. In pets, they are given to stop the pain of arthritis or cancer, when all other medications fail. In dogs in late hip dysplasia, corticosteroids are more likely to be given. Injectable and oral forms of opiates are rarely dispensed for pets in the United States. In the US, the Drug Enforcement Agency (DEA) frowns on any long-term use of opioids, fearing they might their diversion to illegal human use. The DEA calls all the opiates CII or class-2 narcotics. Veterinarians and physicians fear prosecution by the DEA and use them as little as possible. None of the controlled opioid class of drugs with the exception of fentanyl has FDA approval for use in pets. Because cats are deficient in glutathione liver enzyme, the half-life of some opioids in cats may be prolonged and doses must be smaller.
Fentanyl (Duragesic, Sublimaze, Ortho-McNeil Co):
Fentanyl patches are the most commonly used form of opiate narcotic used in pets. Fentanyl was first synthesized in the laboratory in the late 1950s to control pain in humans – particularly those suffering from cancer. It is about 90 times as potent as morphine. Unlike oral or injectable opioid narcotics, fentanyl patches slowly and continuously releases the drug giving smooth, continuous pain relief. Fentanyl passes through the pet’s skin and provides very steady pain relief. Veterinarians most often used fentanyl short-term, after surgery, in advanced cancer or subsequent to body injury. Fentanyl is sold as a transdermal adhesive patch which is placed on a hairless portion of the pet’s skin. In medium and larger dogs, complete patches can be given. In large cats, a whole patch can also be used. In smaller cats, only a portion of the plastic liner is folded back. Patches should never be cut. In dogs, one patch lasts for 72 hours but it takes 12-36 hours for enough of the drug to be absorbed. In cats, the effect of the patch can last up to 120 hours. Cats absorb enough of the drug to be effective within 5-8 hours. In dogs and cats, side effects include excitement or lethargy, poor appetite, and low body temperature. Fentanyl patches are sold in sizes that deliver 25, 50, 75 and 100 micrograms of the drug per hour. The 25 mcg/hr patches work well on 8-12 pound cats when only half the protective liner is removed. I find it best to cover the patch with an elastic bandage. Fur stubble interferes with delivery of the drug so that 50 mcg/hr human patches actually deliver about 37 mcg/hr in pets.
A common danger is the pet or a household member eating the patch or the patch being exposed to excessive heat, such as a heating pad, electric blanket, or a home heater vent. Should this occur, naloxone or Buprenorphine need to be given immediately. A suggested dose of naloxone is 0.2 mg/kg, repeated until breathing returns to normal (1). It should not be used in pets receiving Anipryl (selegiline hydrochloride) for Cushing’s disease or for Canine Cognitive Dysfunction Syndrome (doggy Altzheimers). Be very careful if you have small children. These patches can be fatal if swallowed.
Tramadol (Ultram):
This new synthetic opiate-like drug is a derivative of codeine. Because tramadol is not technically an opiate, it is not controlled by the Drug Enforcement Administration. It is one of the most promising drugs available to veterinarians for treating chronic pain in your pet. It has two modes of action: mu agonism and monoamine reuptake inhibition similar to the newer, human antidepressants. It appears to be comparable to meperidine or codeine in its pain-relieving affect. Administering tramadol with NSAIDs, or mu agonists increases the pain relief. . It tends not to cause vomiting as the opioids often do. The drug is inexpensive and available in generic form. Tramadol can be used for pain relief in both dogs and cats. Since most NSAIDs are dangerous in cats, tramadol provides a nice choice for cats with chronic pain issues). Because of it’s bitter taste, it should be prepared for cats in gelatin capsules. Side effects are uncommon but they include sleepiness and upset stomach and intestine. It must never be given with the newer, human, anti-depressants (SSRIs). In dogs, tramadol is usually given twice a day – but it can be given more frequently if required.
Morphine (Oramorph SR, Roxane):
Morphine sulfate (a CII regulated narcotic) is available in tablet, liquid and capsule preparations. Cats have been given the liquid formula - but most cats and some dog strongly dislike the bitter taste. I have never used this drug in my practice, although I saw it used when I was in veterinary school. Although it is a centuries-old drug, it is rarely used in animals in the United States. It is a very effective barrier to pain. Morphine was usually given to dogs by injection. It went into disfavor because clients occasionally took the pet’s medication themselves and because of strong government opposition to it’s use. This is because of the stigma of narcotics addiction in humans. Also, because the dog dose is so much higher than the human dose, owner fatalities occurred when the tablets were dispensed for dogs but taken by the pet owner.
Meperidine (Demerol):
This is an injectable narcotic drug that is one fifth as powerful as morphine. It is an excellent drug for controlling pain for short periods in dogs. I have used it to spare dogs that were critically injured by automobile and had no hope of recovery. It gave these pets relief from pain until the owners could except the situation. I have not used it in cats. It is not suitable for long term use because, with time, pets become resistant to its action. The dose of this drug in dogs is published as 2 mg/pound given intramuscularly or orally every 4 hours. A published dose in cats is 1-2 mg/pound.
I use up to four times this dose up to three times a day.
Oxymorphone (Numorphan):
This medication is a semi-synthetic opioid ten times as strong as morphine. It is given by injection and its effects last 4-6 hrs. I have personally never used it or seen it used but it is said to be a good drug for postoperative pain in dogs and cats.
Buspirone (Buspar):
Buspirone is a highly effective medication when used for treatment of anxiety and PMS syndrome in humans. It is very safe when given at correct dosages to dogs and cats. More recently, it has been evaluated for its pain-relieving effect. In experimental rats, It was effective in blocking the pain of burns, wounds and inflammation. These data suggest the potential use of buspirone in the management of various types of pain. I place it here because it acts on the same mu receptors through which the opium narcotics block pain. Until now, it has been used in dogs and cats to decrease phobias and generalized anxiety. Buspirone should not be used along with the MO class of antidepressants because dangerous increases in blood pressure can result.
Butorphanol (Stadol, Torbutrol, Torbugesic-SA, Ft. Dodge/Wyeth):
This mixed pain-receptor blocker is a less controlled form of narcotic (C-IV). This is one of the few opioid drugs that works consistently well in cats. This opioid agonist-antagonist is administered by injection or intranasal spray. It has been given to dogs and cats by subcutaneous injection three times a day. After twenty minutes, its pain-controlling properties take effect and last for eight hours. It can also be given by mouth. It causes less side effects than traditional opioids narcotics. Occasionally, butorphanol will cause eye dilation, confusion and pain at the site of injection - especially when given to cats. It is not considered to be useful in the management of chronic pain. Compounding pharmacies can prepare a transdermal gel of butorphanol that can be applied to the inner surface of the ear or a shaved spot on the neck of dogs and cats. The injectable liquid butorphanol can also be given by dropper to cats. Since it has no taste, cats will not fuss about a bitter taste. It is not absorbed well when given in this manner to dogs. I have read that butorphanol tends to make cats more affectionate. The only problems with this are that the cats may rub on you and purr, making it hard for you to sleep. Others have warned that cats may become more aggressive and likely to bite while on butorphanol. Some possible side effects are loss of appetite, vomiting, incoordination, and restlessness.
Buprenorphine (Temgesic, Vetergesic, Buprenex):
This partial mu agonists binds to them and prevents their activation in the sensation of pain.
Buprenorphine is approximately 30 times stronger than morphine. It is a bit expensive for some clients. However, it is a medication with very few serious side effects. It works best when pain is no more than moderate. It can also be prepared by compounding pharmacies as a transdermal patch and administered through the skin, similar to fentanyl. Although these patches take up to six hours, it maintains its pain-controlling effect for up to twelve hours. With subsequent patches, pain control becomes more uniform.
Mixed agonist/antagonists like butorphanol (CIV) are not considered useful in the management of chronic pain. First-pass effect destroys some of the drug, and the analgesia is considered to be relatively short-lived (1-2 hours). Because these drugs are kappa agonists and mu antagonists, the pain relief is often less than optimal for chronic discomfort. However, visceral nociception is considered to be more responsive to kappa agonism, leading some urologists to advocate butorphanol's use in chronic bladder pain (FLUTD).
Other Non-traditional Drugs:
Some sources claim that omega-3 fatty acids, obtained from fish, are helpful in controlling chronic pain. Many veterinarians dispense it because, as far as we know, it is completely safe when given in moderate amounts.
Nutritional centers often dispense bioflavinoids for chronic pain. I do not know on what data this is based. Other veterinarians administer acupuncture for chronic pain. I have not seen this to be effective.
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Saturday, May 8, 2010
Saturday, May 1, 2010
pathogenic enterobacteria
Dr Alvin Fox
BACTERIOLOGY IMMUNOLOGY MYCOLOGY PARASITOLOGY VIROLOGY
SPANISH
BACTERIOLOGY - CHAPTER ELEVEN
ENTEROBACTERIACEAE, VIBRIO, CAMPYLOBACTER AND
HELICOBACTER
ALBANIAN
VIDEO LECTURE
SEARCH
CONTACT US
MICROBE RADIO
Sleuthing Salmonella
Phages Fight Food Poisoning
The Ocean and Cholera
Reading: Murray Third Edition: Chapters 29-31
KEY WORDS
Opportunistic Gastroenteritis
Diarrhea
Dysentery
Urinary tract infections
Lactose positive/negative
API strip
Enteropathogenic E. coli
Enterotoxigenic E. coli
Heat stable toxin
Heat labile toxin
Enteroinvasive E. coli
Enterohemorrhagic E. coli
Vero toxin (Shiga-like)
Hemolysin
Adhesive pili
Shigella
Bacillary dysentery
Shiga toxin
Salmonella typhi
Typhoid
Vi antigen
Salmonella enteritidis (salmonellosis)
Salmonella cholerae-suis
Vibrio cholerae
Cholera
Choleragen (cholera toxin)
Yersinia entercolitica
Campylobacter jejuni
Helicobacter pylori
ENTEROBACTERIACEAE
General
This group of organisms includes several that cause primary infections of the human gastrointestinal tract. Thus, they are referred to as enterics (regardless of whether they cause gut disorders). Bacteria that affect the gastrointestinal tract include certain strains of E. coli and Salmonella, all 4 species of Shigella, and Yersinia entercolitica. The rheumatic disease, Reiter's syndrome (associated with HLA-B27), can result from prior exposure to Salmonella, Shigella, or Yersinia. Other organisms that are not members of the Enterobacteriacae, including Campylobacter and Chlamydia, are also causative agents of Reiter's syndrome. Yersina pestis (the cause of "plague") will be considered separately with other zoonotic organisms.
Members of this family are major causes of opportunistic infection (including septicemia, pneumonia, meningitis and urinary tract infections). Examples of genera that cause opportunistic infections are: Citrobacter, Enterobacter, Escherichia, Hafnia, Morganella, Providencia and Serratia. Selection of antibiotic therapy is complex due to the diversity of organisms.
Some of the organisms additionally cause community-acquired disease in otherwise healthy people. Klebsiella pneumoniae is often involved in respiratory infections. The organism has a prominent capsule aiding pathogenicity . The commonest community acquired ("ascending") urinary tract infection is caused by E. coli. The vast majority of urinary tract infections are ascending, often from fecal contamination. Proteus is another common cause of urinary tract infection; the organism produces a urease that degrades urea producing an alkaline urine.
Isolation and identification of Enterobacteriaceae
These are Gram-negative facultative anerobic rods. They lack cytochrome oxidase and are referred to as oxidase negative. They are often isolated from fecal matter on agar containing lactose and a pH indicator. Colonies that ferment lactose will produce sufficient acid to cause a color shift in the indicator (Figure 1). E. coli is a fermenter of lactose, while Shigella, Salmonella and Yersinia are non-fermenters. "Non-pathogenic" strains of E. coli (and other lactose-positive enterics) are often present in normal feces. Since they are difficult to differentiate from "pathogenic" E. coli, lactose-negative colonies are often the only ones identified in feces. All Enterobacteriaceae isolated from other sites (which contain low numbers of bacteria [e.g. urine] or are normally sterile [e.g. blood]) are identified biochemically, for example using the API 20E system. Important serotypes can be differentiated by their O (lipopolysaccharide), H (flagellar) and K (capsular) antigens. However, serotyping is generally not performed in the routine clinical laboratory.
Figure 1A Reactions in TSI agar slants. For more information on this figure, please go here. © Neal R. Chamberlain, Kirksville College of Osteopathic Medicine, Kirksville, MO and The MicrobeLibrary
Figure 1B Nonlactose fermenter on Hektoen agar which contains bile salts and acid indicators (bromthymol blue and acid fuchsin). The gram-positive bacteria are inhibited so the agar is selective for gram-negative bacteria. The lactose fermenters form orange colonies while the nonfermenters appear green to blue-green. This is especially helpful in distinguishing potential pathogens from normal flora in stool specimens. However, it is difficult to tell the non-fermenters from each other. The organism on this plate could be Salmonella, Proteus, or Shigella. © Pat Johnson, Palm Beach Community College, Lake Worth, Florida and The MicrobeLibrary
Figure 1B Growth of a nonlactose fermenter on MacConkey agar which contains bile salts and crystal violet which inhibit the growth of gram-positive bacteria. The agar also contains lactose and a red dye that differentiates the lactose fermenters from the non-fermenters. Colonies of lactose fermenting bacteria are pink to red while the nonfermenters are colorless or transparent. This agar does not distinguish between the non-lactose fermenters; this growth could indicate several organisms - Proteus, Salmonella or Shigella, for example. In a stool specimen, it would be enough evidence to continue with further identification. © Pat Johnson, Palm Beach Community College, Lake Worth, Florida and The MicrobeLibrary
Figure 1C Growth of gram-negative bacteria that cannot ferment lactose on eosin methylene blue (EMB) agar which contains bile salts and dyes which inhibit growth of gram-positive bacteria. Growth on EMB agar is a useful diagnostic tool to distinguish between lactose fermenters and non-fermenters which will appear colorless. Salmonella and Shigella, both non-lactose fermenting pathogens, can be distinguished from the more common intestinal flora which ferment lactose. © Pat Johnson, Palm Beach Community College, Lake Worth, Florida and The MicrobeLibrary
Figure 2. Bacteria (rod), yeast (round), and fungal hyphae (filamentous) on a kitchen sponge
© Dennis Kunkel Microscopy, Inc. Used with permission
Figure 3 E. coli (0157:H7) hemorrhagic type. Gram-negative, enteric, facultatively anaerobic, rod prokaryote. Potentially fatal to humans, contracted when contaminated meat is cooked inadequately. © Dennis Kunkel Microscopy, Inc. Used with permission
Gastroenteritis, diarrhea and dysentery
(i) Escherichia coli (Figure 3)
At the species level, E. coli and Shigella are indistinguishable. For practical reasons (primarily to avoid confusion), they are not placed in the same genus. Not surprisingly there is a lot of overlap between diseases caused by the two organisms.
1) Enteropathogenic E. coli (EPEC). Certain serotypes are commonly found associated with infant diarrhea. The use of gene probes has confirmed these strains as different from other groups listed below. There is a characteristic morphological lesion with destruction of microvilli without invasion of the organism which suggests adhesion is important. Clinically, one observes fever, diarrhea, vomiting and nausea usually with non-bloody stools.
2) Enterotoxigenic E. coli (ETEC) produce diarrhea resembling cholera but much milder in degree. They also cause "travellers diarrhea". Two types of plasmid-encoded toxins are produced.
a) Heat labile toxins which are similar to choleragen (see cholera section below). Adenyl cyclase is activated with production of cyclic AMP and increased secretion of water and ions.
b) Heat stable toxins. Guanylate cyclase is activated which inhibits ionic uptake from the gut lumen. Watery diarrhea, fever and nausea result in both cases.
3) Enteroinvasive E. coli (EIEC ) produce a dysentery (indistinguishable clinically from shigellosis, see bacillary dysentery below).
4) Enterohemorrhagic E. coli (EHEC). These are usually serotype O157:H7 (figure 4).
Figure 4A Transmission electron micrograph of Escherichia coli O157:H7 CDC/Peggy S. Hayes psh1@cdc.gov
Figure 4B Chronology of E. coli O157:H7 infections, an emerging type of foodborne illness. CDC
These organisms can produce a hemorrhagic colitis (characterized by bloody and copious diarrhea with few leukocytes in afebrile patients). However, they are taking on increasing importance (figure 4) with the recognition of outbreaks caused by contaminated hamburger meat. The organisms can disseminate into the bloodstream producing systemic hemolytic-uremic syndrome (hemolytic anemia, thrombocytopenia and kidney failure). Production of Vero toxin (biochemically similar to shiga toxin - thus also known as "shiga-like") is highly associated with this group of organisms. The toxin is encoded by a lysogenic phage. Hemolysins (plasmid-encoded) are also important in pathogenesis.
As noted above, there are at least four etiologically distinct diseases. However, in the diagnostic laboratory, the groups are not generally differentiated and treatment is based on symptomatology. Usually, fluid replacement is the primary treatment. Antibiotics are generally not used except in severe disease or disease that has progressed to a systemic stage (e.g.hemolytic-uremia syndrome).
Two major classes of pili are produced by E. coli: mannose-sensitive and mannose-resistant pili. The former bind to mannose containing glyocoproteins and the latter to cerebrosides on the host epithelium, allowing attachment. This aids in colonization by E. coli.
Figure 5. Shigella dysenteriae - Gram-negative, enteric, facultatively anaerobic, rod prokaryote; causes bacterial dysentery. This species is most often found in water contaminated with human feces. © Dennis Kunkel Microscopy, Inc. Used with permission
(ii) Shigella
Shigella (4 species; S. flexneri, S. boydii, S. sonnei (figure 5), S. dysenteriae) all cause bacillary dysentery or shigellosis, (bloody feces associated with intestinal pain). The organism invades the epithelial lining layer but does not penetrate. Usually within 2-3 days, dysentery results from bacteria damaging the epithelial layers lining the intestine, often with release of mucus and blood (found in the feces) and attraction of leukocytes (also found in the feces as "pus"). However, watery diarrhea is frequently observed with no evidence of dysentery. Shiga toxin (chromosomally-encoded), which is neurotoxic, enterotoxic and cytotoxic, plays a role. Its enterotoxicity can make the disease clinically appear as a diarrhea. The toxin inhibits protein synthesis (acting on the 70S ribosome and lysing 28S rRNA). This is primarily a disease of young children occurring by fecal-oral contact. Adults can catch this disease from children, although it can be transmitted by infected adult food handlers who contaminate food. The source in each case is unwashed hands. Man is the only "reservoir".
Managing of dehydration is of primary concern. Indeed, mild diarrhea is often not recognized as shigellosis. Patients with severe dysentery are usually treated with antibiotics (e.g. ampicillin). In contrast to salmonellosis, patients respond to antibiotic therapy and disease duration is diminished.
Figure 6. Salmonella - rod prokaryote (dividing); note the flagella. Causes salmonellosis (food poisoning). (x 20,800) © Dennis Kunkel Microscopy, Inc. Used with permission
Figure 7a. Isolation Rate for Salmonella enteritidis by region, United States, 1974-1994 CDC
(iii) Salmonella (figure 6)
Based on genetic studies, there is a single species of Salmonella (Salmonella enterica). At the other extreme using appropriate antibodies, more than 2000 antigenic "types" have been recognized. There are, however, only a few types that are commonly associated with characteristic human diseases (most simply referred to as S. enteritidis, S. cholerae-suis and S. typhi).
Salmonellosis, the common salmonella infection, is caused by a variety of serotypes (most commonly S. enteritidis) and is transmitted from contaminated food (such as poultry and eggs) (figure 7a). It does not have a human reservoir and usually presents as a gastroenteritis (nausea, vomiting and non-bloody stools). The disease is usually self-limiting (2 - 5 days). Like Shigella, these organisms invade the epithelium and do not produce systemic infection. In uncomplicated cases of salmonellosis, which are the vast majority, antibiotic therapy is not useful. S. cholerae-suis (seen much less commonly) causes septicemia after invasion. In this case, antibiotic therapy is required.
The severest form of salmonella infections, "typhoid" (enteric fever), caused by Salmonella typhi, is rarely seen in the US, although it is one of the historical causes of widespread epidemics and still is in the third world. The organism is transmitted from a human reservoir or in the water supply (if sanitary conditions are poor) or in contaminated food. It initially invades the intestinal epithelium and, during this acute phase, gastrointestinal symptoms are noted. The organisms penetrates (usually within the first week) and passes into the bloodstream where it is disseminated in macrophages. Typical features of a systemic bacterial infection are noted. The septicemia usually is temporary with the organism finally lodging in the gall bladder. Organisms are shed into the intestine for some weeks. At this time, the gastroenteritis (including diarrhea) is noted again. The Vi (capsular) antigen plays a role in the pathogenesis of typhoid. A carrier state is common; thus one person (e.g. a food handler) can cause a lot of spread. Antibiotic therapy is essential. Vaccines are not widely effective and not generally used (see comments on cholera).
Figure 7b
Yersinia enterocolitica - Gram-negative, facultatively anaerobic, rod prokaryote (dividing). This bacterium releases a toxin that causes enteritis with pain resembling appendicitis. © Dennis Kunkel Microscopy, Inc. Used with permission
(iv) Yersinia
Yersinia entercolitica (figure 7b) is a major cause of gastroenteritis (the main clinical symptom) in Scandinavia and elsewhere and is seen in the US. The organisms are invasive (usually without systemic spread). Typically the infection is characterized by diarrhea, fever and abdominal pain. However, systemic symptoms, after bacteremia, are seen. This organism can be transmitted by fecal contamination of water or milk by domestic animals or from eating meat products. It is best isolated by "cold" enrichment: when refrigerated this organism survives while others do not. A similar, but less severe, disease is caused by Y. pseudotuberculosis. Antibiotic therapy is recommended.
VIBRIOS
These are Gram-negative rods. They are comma shaped, facultative anaerobes which are oxidase positive. The most important vibrio, Vibrio cholerae (figure 8 and 9), is the causative agent of cholera. It has simple nutritional requirements and is readily cultivated. V. cholerae is found in the feces of an infected individual and ends up in the water supply if sewage is untreated. The organism is thus transmitted by drinking contaminated water. The organism survives in fresh water and, like other vibrios, in salt water. Food, after water contamination, is another means of transmission. Thus, it is primarily a disease of the third world. In the US, it is observed in the occasional international traveler, although it is sometimes seen after ingestion of sea-food. Once in the gut, the organism adheres to the epithelium of the intestine without penetration. Adhesion to the microvilli is thus important in pathogenesis. Cholera toxin is then secreted.
Choleragen (cholera toxin) is chromosomally encoded and contains two types of subunit (A and B). The B subunit binds to gangliosides on epithelial cell surfaces allowing internalization of the A subunit. B subunits may provide a hydrophobic channel through which A penetrates. The A subunit catalyses ADP-ribosylation of a regulator complex which in turn activates adenylate cyclase present in the cell membrane of the epithelium of the gut. The overproduction of cyclic AMP in turn stimulates massive secretion of ions and water into the lumen. Dehydration and death (without treatment) result. Thus, fluid replacement is the major component of treatment. Antibiotic therapy (including tetracycline) is additionally used. Vaccination is only partially effective and not generally recommended. It is most commonly used by international travelers.
Vibrio parahemolyticus is usually transmitted by ingestion of raw sea-food and thus is not commonly seen in the US. The organism grows best in high concentrations of salt. A non-bloody diarrhea is observed but it is not as severe as cholera
ANIMATION
Pathology of Cholera
© Alan House and Mike Hyman, Department of Microbiology, North Carolina State University, Raleigh, N.C. and The MicrobeLibrary
Figure 8a
Vibrio parahaemolyticus - halophilic, facultative anerobic, rod bacterium that causes a food-borne illness known as seafood poisoning. Usually transmitted through eating raw or undercooked seafood such as oysters. Less commonly, this organism can cause an infection in the skin when an open wound is exposed to warm seawater. © Dennis Kunkel Microscopy, Inc. Used with permission
Figure 8b
Vibrio parahaemolyticus - halophilic, facultative anerobic, rod bacterium that causes a food-borne illness known as seafood poisoning. Usually transmitted through eating raw or undercooked seafood such as oysters. Less commonly, this organism can cause an infection in the skin when an open wound is exposed to warm seawater. © Dennis Kunkel Microscopy, Inc. Used with permission
Figure 8c Vibrio cholerae. Leifson flagella stain (digitally colorized). CDC/Dr. William A. Clark
Figure 9. Vibrio cholerae - Gram-negative, facultatively anaerobic, curved (vibrio-shaped), rod prokaryote; causes Asiatic cholera. © Dennis Kunkel Microscopy, Inc. Used with permission
Figure 10a. Campylobacter fetus. Leifson flagella stain (digitally colorized). CDC/Dr. William A. Clark
Figure 10b
Campylobacter jejuni is an enteric, curved-rod prokaryote (bacterium). It is the bacterium that causes campylobacteriosis, one of the most common bacterial causes of diarrheal illness in the United States. It is a relatively fragile bacterium that is easily killed by cold or hot temperatures. Birds are carriers due to their body temperature being just right to host the bacteria. Improper handling of raw poultry or undercooked fowl is usually the source of infection in humans. © Dennis Kunkel Microscopy, Inc. Used with permission
CAMPYLOBACTER AND HELICOBACTER
These two groups of Gram-negative organisms are both curved or spiral shaped and are genetically related.
The most common of the Campylobacter (figure 10) causing human disease are C. jejuni. The organism infects the intestinal tract of several animal species (including cattle and sheep) and is a major cause of cause of abortions. The organism is transmitted to man in milk and meat products. Watery diarrhea predominates but dysentery is common. The organism is invasive but generally less so than Shigella. Malaise, fever and abdominal pain are other disease features. Bacteremia is observed in a small minority of cases. The organism is microaerophilic and grows best at 42oC. It is frequently isolated under these conditions using selective media . It can be treated with antibiotics but is usually a self-limiting disease.
Helicobacter pylori (figure 11) has been accepted in the last few years as the major cause of stomach ulcers. The organism chronically lives in and on the stomach mucosa of man. Culture is the preferred method of diagnosis but may miss a number of cases. The organism characteristically produces a urease which generates ammonia and carbon dioxide. This aids in detecting and identifying the isolated organism. Urease is produced in such large amounts that it can be directly detected in mucosa sampled after endoscopy. Alternatively, 13C or 14C labeled CO2 is detected in the breath after feeding labeled urea. Production of ammonia is a factor in pathogenesis (in locally neutralizing stomach acid). Antibiotic therapy eliminates the organism, peptic ulcers heal and relapses are generally avoided.
Conclusion
Sanitary measures protect the water supply, avoiding contamination with sewage. This is the primary reason that epidemics with life-threatening pathogens (e.g cholera and typhoid) are rarely seen in western countries but are commonly seen in the third world. Other less severe diseases (e.g. salmonellosis, EHEC) are still common from eating contaminated animal products, which has been less well controlled. Shigella, which has a human host, would be even more difficult to eradicate. Vaccination is rarely used and, indeed, is an expensive way to go compared to sewage treatment. In severe diarrhea, fluid replacement is essential. Antibiotic therapy is used in severe local infection and always in systemic disease.
Figure 10c
Campylobacter jejuni - Gram-negative, enteric, curved (vibrio-shaped), rod prokaryote. Found in the gastrointestinal tract of humans and animals, it can travel to the oral cavity and genitourinary tract. Causes gastroenteritis, especially in infants. © Dennis Kunkel Microscopy, Inc. Used with permission
Figure 11a
Helicobacter pylori electron micrographs; fastidious microaerophile; typical helical shape shown in EM; causative agent of chronic gastritis, peptic ulcers and gastric cancer. Image can be used to describe the helical morphology of the organism. Average size: 1micron by 2-5 microns. Organism is
in log phase of growth. © Cindy R. DeLoney, Loyola University of Chicago, Chicago, Illinois and The MicrobeLibrary
Figure 11b
Helicobacter pylori - Gram-negative, spiral to pleomorphic, spiral rod prokaryote. It can move by means of tiny flagella at the end of the cell. There are many strains of H. pylori which are distinguished by the human disease with which they cause. H. pylori infection is the main cause of chronic superficial gastritis and it is associated with both gastric and duodenal ulcers. It lives in the interface between the surface of gastric epithelial cells (the lining of the stomach). It often clusters at the junctions of epithelial cells. © Dennis Kunkel Microscopy, Inc. Used with permission
Figure 11c
Helicobacter pylori - Gram-negative, spiral to pleomorphic, spiral rod prokaryote. © Dennis Kunkel Microscopy, Inc. Used with permission
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BACTERIOLOGY IMMUNOLOGY MYCOLOGY PARASITOLOGY VIROLOGY
SPANISH
BACTERIOLOGY - CHAPTER ELEVEN
ENTEROBACTERIACEAE, VIBRIO, CAMPYLOBACTER AND
HELICOBACTER
ALBANIAN
VIDEO LECTURE
SEARCH
CONTACT US
MICROBE RADIO
Sleuthing Salmonella
Phages Fight Food Poisoning
The Ocean and Cholera
Reading: Murray Third Edition: Chapters 29-31
KEY WORDS
Opportunistic Gastroenteritis
Diarrhea
Dysentery
Urinary tract infections
Lactose positive/negative
API strip
Enteropathogenic E. coli
Enterotoxigenic E. coli
Heat stable toxin
Heat labile toxin
Enteroinvasive E. coli
Enterohemorrhagic E. coli
Vero toxin (Shiga-like)
Hemolysin
Adhesive pili
Shigella
Bacillary dysentery
Shiga toxin
Salmonella typhi
Typhoid
Vi antigen
Salmonella enteritidis (salmonellosis)
Salmonella cholerae-suis
Vibrio cholerae
Cholera
Choleragen (cholera toxin)
Yersinia entercolitica
Campylobacter jejuni
Helicobacter pylori
ENTEROBACTERIACEAE
General
This group of organisms includes several that cause primary infections of the human gastrointestinal tract. Thus, they are referred to as enterics (regardless of whether they cause gut disorders). Bacteria that affect the gastrointestinal tract include certain strains of E. coli and Salmonella, all 4 species of Shigella, and Yersinia entercolitica. The rheumatic disease, Reiter's syndrome (associated with HLA-B27), can result from prior exposure to Salmonella, Shigella, or Yersinia. Other organisms that are not members of the Enterobacteriacae, including Campylobacter and Chlamydia, are also causative agents of Reiter's syndrome. Yersina pestis (the cause of "plague") will be considered separately with other zoonotic organisms.
Members of this family are major causes of opportunistic infection (including septicemia, pneumonia, meningitis and urinary tract infections). Examples of genera that cause opportunistic infections are: Citrobacter, Enterobacter, Escherichia, Hafnia, Morganella, Providencia and Serratia. Selection of antibiotic therapy is complex due to the diversity of organisms.
Some of the organisms additionally cause community-acquired disease in otherwise healthy people. Klebsiella pneumoniae is often involved in respiratory infections. The organism has a prominent capsule aiding pathogenicity . The commonest community acquired ("ascending") urinary tract infection is caused by E. coli. The vast majority of urinary tract infections are ascending, often from fecal contamination. Proteus is another common cause of urinary tract infection; the organism produces a urease that degrades urea producing an alkaline urine.
Isolation and identification of Enterobacteriaceae
These are Gram-negative facultative anerobic rods. They lack cytochrome oxidase and are referred to as oxidase negative. They are often isolated from fecal matter on agar containing lactose and a pH indicator. Colonies that ferment lactose will produce sufficient acid to cause a color shift in the indicator (Figure 1). E. coli is a fermenter of lactose, while Shigella, Salmonella and Yersinia are non-fermenters. "Non-pathogenic" strains of E. coli (and other lactose-positive enterics) are often present in normal feces. Since they are difficult to differentiate from "pathogenic" E. coli, lactose-negative colonies are often the only ones identified in feces. All Enterobacteriaceae isolated from other sites (which contain low numbers of bacteria [e.g. urine] or are normally sterile [e.g. blood]) are identified biochemically, for example using the API 20E system. Important serotypes can be differentiated by their O (lipopolysaccharide), H (flagellar) and K (capsular) antigens. However, serotyping is generally not performed in the routine clinical laboratory.
Figure 1A Reactions in TSI agar slants. For more information on this figure, please go here. © Neal R. Chamberlain, Kirksville College of Osteopathic Medicine, Kirksville, MO and The MicrobeLibrary
Figure 1B Nonlactose fermenter on Hektoen agar which contains bile salts and acid indicators (bromthymol blue and acid fuchsin). The gram-positive bacteria are inhibited so the agar is selective for gram-negative bacteria. The lactose fermenters form orange colonies while the nonfermenters appear green to blue-green. This is especially helpful in distinguishing potential pathogens from normal flora in stool specimens. However, it is difficult to tell the non-fermenters from each other. The organism on this plate could be Salmonella, Proteus, or Shigella. © Pat Johnson, Palm Beach Community College, Lake Worth, Florida and The MicrobeLibrary
Figure 1B Growth of a nonlactose fermenter on MacConkey agar which contains bile salts and crystal violet which inhibit the growth of gram-positive bacteria. The agar also contains lactose and a red dye that differentiates the lactose fermenters from the non-fermenters. Colonies of lactose fermenting bacteria are pink to red while the nonfermenters are colorless or transparent. This agar does not distinguish between the non-lactose fermenters; this growth could indicate several organisms - Proteus, Salmonella or Shigella, for example. In a stool specimen, it would be enough evidence to continue with further identification. © Pat Johnson, Palm Beach Community College, Lake Worth, Florida and The MicrobeLibrary
Figure 1C Growth of gram-negative bacteria that cannot ferment lactose on eosin methylene blue (EMB) agar which contains bile salts and dyes which inhibit growth of gram-positive bacteria. Growth on EMB agar is a useful diagnostic tool to distinguish between lactose fermenters and non-fermenters which will appear colorless. Salmonella and Shigella, both non-lactose fermenting pathogens, can be distinguished from the more common intestinal flora which ferment lactose. © Pat Johnson, Palm Beach Community College, Lake Worth, Florida and The MicrobeLibrary
Figure 2. Bacteria (rod), yeast (round), and fungal hyphae (filamentous) on a kitchen sponge
© Dennis Kunkel Microscopy, Inc. Used with permission
Figure 3 E. coli (0157:H7) hemorrhagic type. Gram-negative, enteric, facultatively anaerobic, rod prokaryote. Potentially fatal to humans, contracted when contaminated meat is cooked inadequately. © Dennis Kunkel Microscopy, Inc. Used with permission
Gastroenteritis, diarrhea and dysentery
(i) Escherichia coli (Figure 3)
At the species level, E. coli and Shigella are indistinguishable. For practical reasons (primarily to avoid confusion), they are not placed in the same genus. Not surprisingly there is a lot of overlap between diseases caused by the two organisms.
1) Enteropathogenic E. coli (EPEC). Certain serotypes are commonly found associated with infant diarrhea. The use of gene probes has confirmed these strains as different from other groups listed below. There is a characteristic morphological lesion with destruction of microvilli without invasion of the organism which suggests adhesion is important. Clinically, one observes fever, diarrhea, vomiting and nausea usually with non-bloody stools.
2) Enterotoxigenic E. coli (ETEC) produce diarrhea resembling cholera but much milder in degree. They also cause "travellers diarrhea". Two types of plasmid-encoded toxins are produced.
a) Heat labile toxins which are similar to choleragen (see cholera section below). Adenyl cyclase is activated with production of cyclic AMP and increased secretion of water and ions.
b) Heat stable toxins. Guanylate cyclase is activated which inhibits ionic uptake from the gut lumen. Watery diarrhea, fever and nausea result in both cases.
3) Enteroinvasive E. coli (EIEC ) produce a dysentery (indistinguishable clinically from shigellosis, see bacillary dysentery below).
4) Enterohemorrhagic E. coli (EHEC). These are usually serotype O157:H7 (figure 4).
Figure 4A Transmission electron micrograph of Escherichia coli O157:H7 CDC/Peggy S. Hayes psh1@cdc.gov
Figure 4B Chronology of E. coli O157:H7 infections, an emerging type of foodborne illness. CDC
These organisms can produce a hemorrhagic colitis (characterized by bloody and copious diarrhea with few leukocytes in afebrile patients). However, they are taking on increasing importance (figure 4) with the recognition of outbreaks caused by contaminated hamburger meat. The organisms can disseminate into the bloodstream producing systemic hemolytic-uremic syndrome (hemolytic anemia, thrombocytopenia and kidney failure). Production of Vero toxin (biochemically similar to shiga toxin - thus also known as "shiga-like") is highly associated with this group of organisms. The toxin is encoded by a lysogenic phage. Hemolysins (plasmid-encoded) are also important in pathogenesis.
As noted above, there are at least four etiologically distinct diseases. However, in the diagnostic laboratory, the groups are not generally differentiated and treatment is based on symptomatology. Usually, fluid replacement is the primary treatment. Antibiotics are generally not used except in severe disease or disease that has progressed to a systemic stage (e.g.hemolytic-uremia syndrome).
Two major classes of pili are produced by E. coli: mannose-sensitive and mannose-resistant pili. The former bind to mannose containing glyocoproteins and the latter to cerebrosides on the host epithelium, allowing attachment. This aids in colonization by E. coli.
Figure 5. Shigella dysenteriae - Gram-negative, enteric, facultatively anaerobic, rod prokaryote; causes bacterial dysentery. This species is most often found in water contaminated with human feces. © Dennis Kunkel Microscopy, Inc. Used with permission
(ii) Shigella
Shigella (4 species; S. flexneri, S. boydii, S. sonnei (figure 5), S. dysenteriae) all cause bacillary dysentery or shigellosis, (bloody feces associated with intestinal pain). The organism invades the epithelial lining layer but does not penetrate. Usually within 2-3 days, dysentery results from bacteria damaging the epithelial layers lining the intestine, often with release of mucus and blood (found in the feces) and attraction of leukocytes (also found in the feces as "pus"). However, watery diarrhea is frequently observed with no evidence of dysentery. Shiga toxin (chromosomally-encoded), which is neurotoxic, enterotoxic and cytotoxic, plays a role. Its enterotoxicity can make the disease clinically appear as a diarrhea. The toxin inhibits protein synthesis (acting on the 70S ribosome and lysing 28S rRNA). This is primarily a disease of young children occurring by fecal-oral contact. Adults can catch this disease from children, although it can be transmitted by infected adult food handlers who contaminate food. The source in each case is unwashed hands. Man is the only "reservoir".
Managing of dehydration is of primary concern. Indeed, mild diarrhea is often not recognized as shigellosis. Patients with severe dysentery are usually treated with antibiotics (e.g. ampicillin). In contrast to salmonellosis, patients respond to antibiotic therapy and disease duration is diminished.
Figure 6. Salmonella - rod prokaryote (dividing); note the flagella. Causes salmonellosis (food poisoning). (x 20,800) © Dennis Kunkel Microscopy, Inc. Used with permission
Figure 7a. Isolation Rate for Salmonella enteritidis by region, United States, 1974-1994 CDC
(iii) Salmonella (figure 6)
Based on genetic studies, there is a single species of Salmonella (Salmonella enterica). At the other extreme using appropriate antibodies, more than 2000 antigenic "types" have been recognized. There are, however, only a few types that are commonly associated with characteristic human diseases (most simply referred to as S. enteritidis, S. cholerae-suis and S. typhi).
Salmonellosis, the common salmonella infection, is caused by a variety of serotypes (most commonly S. enteritidis) and is transmitted from contaminated food (such as poultry and eggs) (figure 7a). It does not have a human reservoir and usually presents as a gastroenteritis (nausea, vomiting and non-bloody stools). The disease is usually self-limiting (2 - 5 days). Like Shigella, these organisms invade the epithelium and do not produce systemic infection. In uncomplicated cases of salmonellosis, which are the vast majority, antibiotic therapy is not useful. S. cholerae-suis (seen much less commonly) causes septicemia after invasion. In this case, antibiotic therapy is required.
The severest form of salmonella infections, "typhoid" (enteric fever), caused by Salmonella typhi, is rarely seen in the US, although it is one of the historical causes of widespread epidemics and still is in the third world. The organism is transmitted from a human reservoir or in the water supply (if sanitary conditions are poor) or in contaminated food. It initially invades the intestinal epithelium and, during this acute phase, gastrointestinal symptoms are noted. The organisms penetrates (usually within the first week) and passes into the bloodstream where it is disseminated in macrophages. Typical features of a systemic bacterial infection are noted. The septicemia usually is temporary with the organism finally lodging in the gall bladder. Organisms are shed into the intestine for some weeks. At this time, the gastroenteritis (including diarrhea) is noted again. The Vi (capsular) antigen plays a role in the pathogenesis of typhoid. A carrier state is common; thus one person (e.g. a food handler) can cause a lot of spread. Antibiotic therapy is essential. Vaccines are not widely effective and not generally used (see comments on cholera).
Figure 7b
Yersinia enterocolitica - Gram-negative, facultatively anaerobic, rod prokaryote (dividing). This bacterium releases a toxin that causes enteritis with pain resembling appendicitis. © Dennis Kunkel Microscopy, Inc. Used with permission
(iv) Yersinia
Yersinia entercolitica (figure 7b) is a major cause of gastroenteritis (the main clinical symptom) in Scandinavia and elsewhere and is seen in the US. The organisms are invasive (usually without systemic spread). Typically the infection is characterized by diarrhea, fever and abdominal pain. However, systemic symptoms, after bacteremia, are seen. This organism can be transmitted by fecal contamination of water or milk by domestic animals or from eating meat products. It is best isolated by "cold" enrichment: when refrigerated this organism survives while others do not. A similar, but less severe, disease is caused by Y. pseudotuberculosis. Antibiotic therapy is recommended.
VIBRIOS
These are Gram-negative rods. They are comma shaped, facultative anaerobes which are oxidase positive. The most important vibrio, Vibrio cholerae (figure 8 and 9), is the causative agent of cholera. It has simple nutritional requirements and is readily cultivated. V. cholerae is found in the feces of an infected individual and ends up in the water supply if sewage is untreated. The organism is thus transmitted by drinking contaminated water. The organism survives in fresh water and, like other vibrios, in salt water. Food, after water contamination, is another means of transmission. Thus, it is primarily a disease of the third world. In the US, it is observed in the occasional international traveler, although it is sometimes seen after ingestion of sea-food. Once in the gut, the organism adheres to the epithelium of the intestine without penetration. Adhesion to the microvilli is thus important in pathogenesis. Cholera toxin is then secreted.
Choleragen (cholera toxin) is chromosomally encoded and contains two types of subunit (A and B). The B subunit binds to gangliosides on epithelial cell surfaces allowing internalization of the A subunit. B subunits may provide a hydrophobic channel through which A penetrates. The A subunit catalyses ADP-ribosylation of a regulator complex which in turn activates adenylate cyclase present in the cell membrane of the epithelium of the gut. The overproduction of cyclic AMP in turn stimulates massive secretion of ions and water into the lumen. Dehydration and death (without treatment) result. Thus, fluid replacement is the major component of treatment. Antibiotic therapy (including tetracycline) is additionally used. Vaccination is only partially effective and not generally recommended. It is most commonly used by international travelers.
Vibrio parahemolyticus is usually transmitted by ingestion of raw sea-food and thus is not commonly seen in the US. The organism grows best in high concentrations of salt. A non-bloody diarrhea is observed but it is not as severe as cholera
ANIMATION
Pathology of Cholera
© Alan House and Mike Hyman, Department of Microbiology, North Carolina State University, Raleigh, N.C. and The MicrobeLibrary
Figure 8a
Vibrio parahaemolyticus - halophilic, facultative anerobic, rod bacterium that causes a food-borne illness known as seafood poisoning. Usually transmitted through eating raw or undercooked seafood such as oysters. Less commonly, this organism can cause an infection in the skin when an open wound is exposed to warm seawater. © Dennis Kunkel Microscopy, Inc. Used with permission
Figure 8b
Vibrio parahaemolyticus - halophilic, facultative anerobic, rod bacterium that causes a food-borne illness known as seafood poisoning. Usually transmitted through eating raw or undercooked seafood such as oysters. Less commonly, this organism can cause an infection in the skin when an open wound is exposed to warm seawater. © Dennis Kunkel Microscopy, Inc. Used with permission
Figure 8c Vibrio cholerae. Leifson flagella stain (digitally colorized). CDC/Dr. William A. Clark
Figure 9. Vibrio cholerae - Gram-negative, facultatively anaerobic, curved (vibrio-shaped), rod prokaryote; causes Asiatic cholera. © Dennis Kunkel Microscopy, Inc. Used with permission
Figure 10a. Campylobacter fetus. Leifson flagella stain (digitally colorized). CDC/Dr. William A. Clark
Figure 10b
Campylobacter jejuni is an enteric, curved-rod prokaryote (bacterium). It is the bacterium that causes campylobacteriosis, one of the most common bacterial causes of diarrheal illness in the United States. It is a relatively fragile bacterium that is easily killed by cold or hot temperatures. Birds are carriers due to their body temperature being just right to host the bacteria. Improper handling of raw poultry or undercooked fowl is usually the source of infection in humans. © Dennis Kunkel Microscopy, Inc. Used with permission
CAMPYLOBACTER AND HELICOBACTER
These two groups of Gram-negative organisms are both curved or spiral shaped and are genetically related.
The most common of the Campylobacter (figure 10) causing human disease are C. jejuni. The organism infects the intestinal tract of several animal species (including cattle and sheep) and is a major cause of cause of abortions. The organism is transmitted to man in milk and meat products. Watery diarrhea predominates but dysentery is common. The organism is invasive but generally less so than Shigella. Malaise, fever and abdominal pain are other disease features. Bacteremia is observed in a small minority of cases. The organism is microaerophilic and grows best at 42oC. It is frequently isolated under these conditions using selective media . It can be treated with antibiotics but is usually a self-limiting disease.
Helicobacter pylori (figure 11) has been accepted in the last few years as the major cause of stomach ulcers. The organism chronically lives in and on the stomach mucosa of man. Culture is the preferred method of diagnosis but may miss a number of cases. The organism characteristically produces a urease which generates ammonia and carbon dioxide. This aids in detecting and identifying the isolated organism. Urease is produced in such large amounts that it can be directly detected in mucosa sampled after endoscopy. Alternatively, 13C or 14C labeled CO2 is detected in the breath after feeding labeled urea. Production of ammonia is a factor in pathogenesis (in locally neutralizing stomach acid). Antibiotic therapy eliminates the organism, peptic ulcers heal and relapses are generally avoided.
Conclusion
Sanitary measures protect the water supply, avoiding contamination with sewage. This is the primary reason that epidemics with life-threatening pathogens (e.g cholera and typhoid) are rarely seen in western countries but are commonly seen in the third world. Other less severe diseases (e.g. salmonellosis, EHEC) are still common from eating contaminated animal products, which has been less well controlled. Shigella, which has a human host, would be even more difficult to eradicate. Vaccination is rarely used and, indeed, is an expensive way to go compared to sewage treatment. In severe diarrhea, fluid replacement is essential. Antibiotic therapy is used in severe local infection and always in systemic disease.
Figure 10c
Campylobacter jejuni - Gram-negative, enteric, curved (vibrio-shaped), rod prokaryote. Found in the gastrointestinal tract of humans and animals, it can travel to the oral cavity and genitourinary tract. Causes gastroenteritis, especially in infants. © Dennis Kunkel Microscopy, Inc. Used with permission
Figure 11a
Helicobacter pylori electron micrographs; fastidious microaerophile; typical helical shape shown in EM; causative agent of chronic gastritis, peptic ulcers and gastric cancer. Image can be used to describe the helical morphology of the organism. Average size: 1micron by 2-5 microns. Organism is
in log phase of growth. © Cindy R. DeLoney, Loyola University of Chicago, Chicago, Illinois and The MicrobeLibrary
Figure 11b
Helicobacter pylori - Gram-negative, spiral to pleomorphic, spiral rod prokaryote. It can move by means of tiny flagella at the end of the cell. There are many strains of H. pylori which are distinguished by the human disease with which they cause. H. pylori infection is the main cause of chronic superficial gastritis and it is associated with both gastric and duodenal ulcers. It lives in the interface between the surface of gastric epithelial cells (the lining of the stomach). It often clusters at the junctions of epithelial cells. © Dennis Kunkel Microscopy, Inc. Used with permission
Figure 11c
Helicobacter pylori - Gram-negative, spiral to pleomorphic, spiral rod prokaryote. © Dennis Kunkel Microscopy, Inc. Used with permission
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Tuesday, April 13, 2010
water testing for coliforms
Bacteriology 102:
A General Overview of Coliforms
YOU ARE HERE:
John L's Bacteriology Pages >
Bact.102 Website–Fall 2006 >
Coliforms Page Last
Modified:
6/5/09
COLIFORMS AND THE CONCEPT OF INDICATOR ORGANISMS
Due to the wide variety of intestinal pathogens (bacteria, viruses, protozoa) that could be found in feces, sewage and (ultimately) water, it would not be cost-effective to test specifically for the presence of each different pathogen. This is where the concept of the indicator organism applies. Generally, an "innocent" organism which often finds itself associated with (i.e., in the same natural habitat as) problem-causing organisms can be, when isolated from a site, an indication that the problem organisms may also be present at that site. Such an associated organism is therefore an indicator of the possible problem. Specifically, if pollution of a water resource by sewage or other fecal pollution (and the associated intestinal pathogens) is suspected, a desirable indicator organism for such pollution would follow these criteria:
It is always present in the feces from both normal and infected individuals.
It is not found anywhere in the environment except where there is contamination by fecal pollution.
It survives longer in the environment than pathogens, but not so long as to indicate "historical" contamination.
It is easy to detect in the laboratory in that its characteristics can be exploited to make it easily enriched for and isolated (according to principles we learned in Exp. 11).
It should not cause a "problem" itself and pose a health risk for laboratory workers.
In the United States, significant indicator organisms for certain kinds of contamination of water and food are the coliforms. The usual definition for coliforms that one may encounter is that they are gram-negative rods that ferment lactose rapidly with the production of gas (insoluble gas that is detectable in a Durham tube) in Lactose Lauryl Tryptose Broth and Brilliant Green Bile Broth at 35°C. Those which additionally do so in EC Broth at 44.5°C belong to the subset of coliforms called "fecal coliforms." Coliforms happen to be easy to detect with the appropriate selective-differential media. These media tend to inhibit gram-positive bacteria, and the presence of coliforms is suggested by gas from lactose fermentation in the Durham tube of the enrichment media and by acidic colonies on EMB Agar which is the first step in the actual isolation process. Naturally any isolate must at least show gram-negativity and the ability to ferment lactose to acid and gas – two hallmarks of identification as a coliform. Most often a coliform isolate is ultimately identified as a species of Escherichia, Enterobacter, Klebsiella or Citrobacter, and one that has come through the EC Broth enrichment at 44.5°C is usually identified as Escherichia coli.
The presence of E. coli is considered a definitive indication of fecal pollution (with the possibility of the associated intestinal pathogens), as the natural habitat of these organisms is the intestinal tract of humans and higher animals. Coliforms identified otherwise may be found throughout the environment such as in soil or on plants. Finding these "non-fecal coliforms" may not indicate fecal contamination, but their presence in drinking water may indicate contamination by organisms from soil where there could be significant chemical or biological contamination. So, coliforms find their utility in being indicator organisms – and not just for fecal contamination.
Coliforms do not constitute a discrete taxonomic group. Many strains of the aforementioned genera may not ferment lactose at all and would therefore not be called coliforms. For example, some pathogenic E. coli strains do not ferment lactose, and it is sometimes very difficult to differentiate them from Shigella. Also, most strains of Citrobacter (such as the one we use in Experiments 14 and 17) very weakly attack lactose and may be initially confused with Salmonella when isolations are made from clinical or natural samples. So, considering the absolute definition of this non-taxonomic term, there can be no such thing as a non-lactose-fermenting coliform.
TESTING FOR COLIFORMS
From the foregoing it can be seen that in order to enrich for coliforms selectively, one can formulate media which (1) inhibit gram-positive bacteria as much as possible and (2) allow for the enrichment and detection of those organisms which ferment lactose to acid and gas, the latter being detectable in a Durham tube.
In the traditional coliform testing procedure, a water sample is inoculated into a tube of Lactose Lauryl Tryptose Broth (LLTB) to set up the selective enrichment for coliforms which is termed the Presumptive Test. If growth and gas are seen in the tube after incubation for up to 2 days at 35°C, one can presume that at least one coliform was originally inoculated into the medium, multiplying into a large population of cells while fermenting lactose with the production of acid and gas. Hydrogen is the insoluble gas that collects in the Durham tube; carbon dioxide tends to be soluble although some can be found in the gas bubble. (No pH indicator is included in the medium as detection of acid would be unnecessary.) The culture in the tube is certainly not a pure culture. Many non-coliform organisms such as Pseudomonas can also grow in the medium. In order to achieve maximum recovery of coliforms – possibly "damaged" by deleterious agents in the environment and thereby sensitive to selective agents in media – LLTB was made a medium of low-selectivity. It may not inhibit totally the gram-positive organisms in the sample, as strains of Bacillus and Clostridium which ferment lactose to acid and gas may grow.
Each positive tube (i.e., showing growth and gas) is then inoculated (by loop) into a tube each of two media for the Confirmatory Test. These media are strongly selective for gram-negative organisms and may even inhibit some enterics. Growth and gas constitute a positive result as for LLTB.
For the selective enrichment (and detection) of the "true" (gram-negative) coliforms, Brilliant Green Lactose Bile (BGLB) Broth is used and incubated for up to 2 days at 35°C. Thus, if any cells in the inoculum were coliforms, they should multiply, fermenting lactose and producing gas as in LLTB. Any of the above-mentioned lactose-positive strains of Bacillus and Clostridium which had contributed to a positive result in the Presumptive Test are inhibited in this medium. One should not expect only coliforms in BGLB, however, as Pseudomonas and many other gram-negative organisms can grow in the medium.
For the selective enrichment (and detection) of the fecal coliforms, a subset of the coliforms (not a separate group!), EC Broth is inoculated and incubated for up to 2 days at 44.5°C. Growth and gas together indicate a probability of E. coli and associated fecal contamination being present.
For quantitative coliform analysis, the Most Probable Number (MPN) method is applied with the dilution of the sample and inoculation of the LLTB Broth from the dilutions for the Presumptive Test, with each positive tube then being "confirmed" in the next step of the procedure.
A summary table of the aforementioned selective enrichment media follows. Note that these media increase in selectivity for the desired organisms from left to right. Of course, we would not expect pure cultures of anything in any enrichment medium (selective or otherwise), so the subsequent isolation is important as in any enrichment-isolation procedure as we have learned in Experiment 11. For a tube which shows growth and gas, suggesting the presence of coliforms, all it might have taken for a population of coliforms to develop in the tube could have been just one coliform cell in the inoculum. One way or another in the enrichment and isolation process, the non-coliforms will be "sorted out."
Chemoheterotrophic
Organisms in Water Growth Response of Organisms in Coliform Enrichment Media
LLTB BGLB EC Broth at 44.5°C
I. The true coliforms: "fecal" and others growth & gas growth & gas growth & gas for fecal coliforms (no growth for non-fecal coliforms)
II. Gram-negative bacteria other than coliforms growth
growth
little or no growth
III. Lactose-fermenting (to acid & gas) strains of Bacillus and Clostridium ("false coliforms") growth & gas no growth no growth
IV. Gram-positive bacteria other than those in the row above possible growth
no growth no growth
Now it is desired to obtain pure cultures of any coliforms present and identify them to some degree. Positive tubes from the Confirmatory Tests are streaked onto Eosin Methylene Blue (EMB) Agar to begin the Completed Test (actually a series of tests). Dark colonies on EMB Agar indicate probable coliforms (see explanation of the medium in Period 4). Representative colonies may be inoculated into Nutrient Agar slants and Lactose Fermentation Broth to check for basic coliform characteristics (gram-negative rods, fermentation of lactose to acid and gas). Traditionally the IMViC tests are done for each isolate; the four major letters of this acronym stand for the indole, methyl red, Voges-Proskauer and citrate tests. A certain combination of results usually leads to a probable identification, although additional tests must be run if definitive identification is required.
GO
TO: Selected Groups of Bacteria
Bacteriology 102 Website
Index of the General Bacteriology Pages
Page last modified on 6/5/09 at 8:00 PM, CDT.
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison
A General Overview of Coliforms
YOU ARE HERE:
John L's Bacteriology Pages >
Bact.102 Website–Fall 2006 >
Coliforms Page Last
Modified:
6/5/09
COLIFORMS AND THE CONCEPT OF INDICATOR ORGANISMS
Due to the wide variety of intestinal pathogens (bacteria, viruses, protozoa) that could be found in feces, sewage and (ultimately) water, it would not be cost-effective to test specifically for the presence of each different pathogen. This is where the concept of the indicator organism applies. Generally, an "innocent" organism which often finds itself associated with (i.e., in the same natural habitat as) problem-causing organisms can be, when isolated from a site, an indication that the problem organisms may also be present at that site. Such an associated organism is therefore an indicator of the possible problem. Specifically, if pollution of a water resource by sewage or other fecal pollution (and the associated intestinal pathogens) is suspected, a desirable indicator organism for such pollution would follow these criteria:
It is always present in the feces from both normal and infected individuals.
It is not found anywhere in the environment except where there is contamination by fecal pollution.
It survives longer in the environment than pathogens, but not so long as to indicate "historical" contamination.
It is easy to detect in the laboratory in that its characteristics can be exploited to make it easily enriched for and isolated (according to principles we learned in Exp. 11).
It should not cause a "problem" itself and pose a health risk for laboratory workers.
In the United States, significant indicator organisms for certain kinds of contamination of water and food are the coliforms. The usual definition for coliforms that one may encounter is that they are gram-negative rods that ferment lactose rapidly with the production of gas (insoluble gas that is detectable in a Durham tube) in Lactose Lauryl Tryptose Broth and Brilliant Green Bile Broth at 35°C. Those which additionally do so in EC Broth at 44.5°C belong to the subset of coliforms called "fecal coliforms." Coliforms happen to be easy to detect with the appropriate selective-differential media. These media tend to inhibit gram-positive bacteria, and the presence of coliforms is suggested by gas from lactose fermentation in the Durham tube of the enrichment media and by acidic colonies on EMB Agar which is the first step in the actual isolation process. Naturally any isolate must at least show gram-negativity and the ability to ferment lactose to acid and gas – two hallmarks of identification as a coliform. Most often a coliform isolate is ultimately identified as a species of Escherichia, Enterobacter, Klebsiella or Citrobacter, and one that has come through the EC Broth enrichment at 44.5°C is usually identified as Escherichia coli.
The presence of E. coli is considered a definitive indication of fecal pollution (with the possibility of the associated intestinal pathogens), as the natural habitat of these organisms is the intestinal tract of humans and higher animals. Coliforms identified otherwise may be found throughout the environment such as in soil or on plants. Finding these "non-fecal coliforms" may not indicate fecal contamination, but their presence in drinking water may indicate contamination by organisms from soil where there could be significant chemical or biological contamination. So, coliforms find their utility in being indicator organisms – and not just for fecal contamination.
Coliforms do not constitute a discrete taxonomic group. Many strains of the aforementioned genera may not ferment lactose at all and would therefore not be called coliforms. For example, some pathogenic E. coli strains do not ferment lactose, and it is sometimes very difficult to differentiate them from Shigella. Also, most strains of Citrobacter (such as the one we use in Experiments 14 and 17) very weakly attack lactose and may be initially confused with Salmonella when isolations are made from clinical or natural samples. So, considering the absolute definition of this non-taxonomic term, there can be no such thing as a non-lactose-fermenting coliform.
TESTING FOR COLIFORMS
From the foregoing it can be seen that in order to enrich for coliforms selectively, one can formulate media which (1) inhibit gram-positive bacteria as much as possible and (2) allow for the enrichment and detection of those organisms which ferment lactose to acid and gas, the latter being detectable in a Durham tube.
In the traditional coliform testing procedure, a water sample is inoculated into a tube of Lactose Lauryl Tryptose Broth (LLTB) to set up the selective enrichment for coliforms which is termed the Presumptive Test. If growth and gas are seen in the tube after incubation for up to 2 days at 35°C, one can presume that at least one coliform was originally inoculated into the medium, multiplying into a large population of cells while fermenting lactose with the production of acid and gas. Hydrogen is the insoluble gas that collects in the Durham tube; carbon dioxide tends to be soluble although some can be found in the gas bubble. (No pH indicator is included in the medium as detection of acid would be unnecessary.) The culture in the tube is certainly not a pure culture. Many non-coliform organisms such as Pseudomonas can also grow in the medium. In order to achieve maximum recovery of coliforms – possibly "damaged" by deleterious agents in the environment and thereby sensitive to selective agents in media – LLTB was made a medium of low-selectivity. It may not inhibit totally the gram-positive organisms in the sample, as strains of Bacillus and Clostridium which ferment lactose to acid and gas may grow.
Each positive tube (i.e., showing growth and gas) is then inoculated (by loop) into a tube each of two media for the Confirmatory Test. These media are strongly selective for gram-negative organisms and may even inhibit some enterics. Growth and gas constitute a positive result as for LLTB.
For the selective enrichment (and detection) of the "true" (gram-negative) coliforms, Brilliant Green Lactose Bile (BGLB) Broth is used and incubated for up to 2 days at 35°C. Thus, if any cells in the inoculum were coliforms, they should multiply, fermenting lactose and producing gas as in LLTB. Any of the above-mentioned lactose-positive strains of Bacillus and Clostridium which had contributed to a positive result in the Presumptive Test are inhibited in this medium. One should not expect only coliforms in BGLB, however, as Pseudomonas and many other gram-negative organisms can grow in the medium.
For the selective enrichment (and detection) of the fecal coliforms, a subset of the coliforms (not a separate group!), EC Broth is inoculated and incubated for up to 2 days at 44.5°C. Growth and gas together indicate a probability of E. coli and associated fecal contamination being present.
For quantitative coliform analysis, the Most Probable Number (MPN) method is applied with the dilution of the sample and inoculation of the LLTB Broth from the dilutions for the Presumptive Test, with each positive tube then being "confirmed" in the next step of the procedure.
A summary table of the aforementioned selective enrichment media follows. Note that these media increase in selectivity for the desired organisms from left to right. Of course, we would not expect pure cultures of anything in any enrichment medium (selective or otherwise), so the subsequent isolation is important as in any enrichment-isolation procedure as we have learned in Experiment 11. For a tube which shows growth and gas, suggesting the presence of coliforms, all it might have taken for a population of coliforms to develop in the tube could have been just one coliform cell in the inoculum. One way or another in the enrichment and isolation process, the non-coliforms will be "sorted out."
Chemoheterotrophic
Organisms in Water Growth Response of Organisms in Coliform Enrichment Media
LLTB BGLB EC Broth at 44.5°C
I. The true coliforms: "fecal" and others growth & gas growth & gas growth & gas for fecal coliforms (no growth for non-fecal coliforms)
II. Gram-negative bacteria other than coliforms growth
growth
little or no growth
III. Lactose-fermenting (to acid & gas) strains of Bacillus and Clostridium ("false coliforms") growth & gas no growth no growth
IV. Gram-positive bacteria other than those in the row above possible growth
no growth no growth
Now it is desired to obtain pure cultures of any coliforms present and identify them to some degree. Positive tubes from the Confirmatory Tests are streaked onto Eosin Methylene Blue (EMB) Agar to begin the Completed Test (actually a series of tests). Dark colonies on EMB Agar indicate probable coliforms (see explanation of the medium in Period 4). Representative colonies may be inoculated into Nutrient Agar slants and Lactose Fermentation Broth to check for basic coliform characteristics (gram-negative rods, fermentation of lactose to acid and gas). Traditionally the IMViC tests are done for each isolate; the four major letters of this acronym stand for the indole, methyl red, Voges-Proskauer and citrate tests. A certain combination of results usually leads to a probable identification, although additional tests must be run if definitive identification is required.
GO
TO: Selected Groups of Bacteria
Bacteriology 102 Website
Index of the General Bacteriology Pages
Page last modified on 6/5/09 at 8:00 PM, CDT.
John Lindquist, Department of Bacteriology,
University of Wisconsin – Madison
Friday, April 2, 2010
It's official: Men are obsessed with sex, hide their emotions, and cheat - Yahoo! India News
ANI
It's official: Men are obsessed with sex, hide their emotions, and cheat
Thu, Apr 1 12:25 PM
London, Apr 1 (ANI): Expert Dr Louann Brizendine has dived inside a man's mind and confirmed what most women long suspected: men are obsessed with sex, hide their emotions, and cheat.
According to Brizendine, testosterone causes the "man trance", where blokes have to stare at boobs, reports The Daily Star.
She says: "The best advice I have for women is make peace with the male brain. Let men be men."
Some of the other findings in the expert's new book Male Brain: A Breakthrough Understanding Of How Men And Boys Think are:
1 Men really are sex-crazed
The brain's part inked to sexual pursuit is two-and-a-half times larger in males than females.
2 They're programmed to perv
The testosterone drives what Louann calls the "man trance" - a glazed-eye stare at breasts. She says: "I wish I could say that men can stop themselves from entering this trance. But the truth is, they can't."
3 Men want more partners
According to the book, men want an average of 14 sexual partners in their lifetime. Women want one or two.
Louann says: "It's postcoital narcolepsy. During orgasm, males release a huge amount of oxytocin in their brains, and it is very sedating. It's not that he doesn't love you."
4 Men lie more about sex
Biologically speaking, men are more comfortable lying to the opposite sex.
5 Foreplay round the clock
In case of women, foreplay is everything that happens in the 24 hours before intercourse. For men it's what happens three minutes before entry
Louann says: "The male brain's initial emotional reaction can be stronger than the female. But within 2.5 seconds his face changes to hide the emotion, or even reverse it."
The expert doesn't reckon her book justifies bad behaviour. She says: "This is not giving men an excuse to rape and pillage. But men do have a right to give voice to their biological predisposition and have it come in to the dialogue." (ANI)
It's official: Men are obsessed with sex, hide their emotions, and cheat
Thu, Apr 1 12:25 PM
London, Apr 1 (ANI): Expert Dr Louann Brizendine has dived inside a man's mind and confirmed what most women long suspected: men are obsessed with sex, hide their emotions, and cheat.
According to Brizendine, testosterone causes the "man trance", where blokes have to stare at boobs, reports The Daily Star.
She says: "The best advice I have for women is make peace with the male brain. Let men be men."
Some of the other findings in the expert's new book Male Brain: A Breakthrough Understanding Of How Men And Boys Think are:
1 Men really are sex-crazed
The brain's part inked to sexual pursuit is two-and-a-half times larger in males than females.
2 They're programmed to perv
The testosterone drives what Louann calls the "man trance" - a glazed-eye stare at breasts. She says: "I wish I could say that men can stop themselves from entering this trance. But the truth is, they can't."
3 Men want more partners
According to the book, men want an average of 14 sexual partners in their lifetime. Women want one or two.
Louann says: "It's postcoital narcolepsy. During orgasm, males release a huge amount of oxytocin in their brains, and it is very sedating. It's not that he doesn't love you."
4 Men lie more about sex
Biologically speaking, men are more comfortable lying to the opposite sex.
5 Foreplay round the clock
In case of women, foreplay is everything that happens in the 24 hours before intercourse. For men it's what happens three minutes before entry
Louann says: "The male brain's initial emotional reaction can be stronger than the female. But within 2.5 seconds his face changes to hide the emotion, or even reverse it."
The expert doesn't reckon her book justifies bad behaviour. She says: "This is not giving men an excuse to rape and pillage. But men do have a right to give voice to their biological predisposition and have it come in to the dialogue." (ANI)
Saturday, March 13, 2010
AST Milk
Antimicrobial Susceptibility of Udder Pathogens Isolated from Dairy Herds in the West Littoral Region of Uruguay
RE Gianneechini,1 C Concha,2 and A Franklin3
1Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala, Sweden
2Department of Mastitis and Diagnostical Products, Uppsala, Sweden
3Department of Antibiotics, National Veterinary Institute, Uppsala, Sweden
Corresponding author.
Received March 9, 2001; Accepted November 12, 2001.
Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesAbstract
A total of 522 strains belonging to streptococci, enterococci and staphylococci isolated from sub-clinical and clinical cases of bovine mastitis from the west littoral region of Uruguay were analysed for their susceptibility to several antimicrobial agents. The susceptibility patterns were studied by agar disk diffusion methods (ADDM) and broth micro-dilution to determine the minimum inhibitory concentration (MIC). The concentration that inhibits 90% (MIC90) of the analysed strains reported in micrograms per millilitre, for Staphylococcus aureus were > 8, 8, ≤ 0.5, ≤ 4, ≤ 1, ≤ 0.5, > 64, ≤ 0.25, 0.5, ≤ 1 and ≤ 1 to penicillin, ampicillin, oxacillin, cephalotin, gentamicin, erythromycin, oxitetracycline, enrofloxacin, trimethoprim/sulfamethoxazole, neomycin, and clindamycin, respectively. Coagulase-negative staphylococci (CNS) had different values for penicillin (4) and ampicillin (2), while the other antimicrobial agents had the same MIC90 values as reported for S. aureus. The MIC90 values for streptococci were 0.12, 0.25, ≤ 4, 16, ≤ 0.25, 0.5, 0.25 for penicillin, ampicillin, cephalotin, gentamicin, erythromycin, oxytetracycline and trimethoprim-sulfamethoxazole, whereas MIC90 for enterococci were 4, 4, 4, ≤ 0.5, 2, > 8 for penicillin, ampicillin, gentamicin, erythromycin, oxytetracycline and trimethoprim-sulfamethoxazole, respectively. Of 336 strains of S. aureus, 160 (47.6%) were resistant to penicillin. For 41 CNS strains, 10 (27%) presented penicillin-resistance. All the streptococcal strains were susceptible to penicillin, while 3 (7%) of the 43 enteroccocal strains were resistant. Non significant statistical differences were found between the results obtained by ADDM and broth micro-dilution for classifying bacterial isolates as susceptible or resistant according to the National Committee of Clinical Laboratory Standards.Keywords: cow, mammary qland, bacteria, resistant, sensitive Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesIntroduction
Bovine mastitis is the major problem for milk producers throughout the world and responsible for substantial losses of revenue annually. Antibiotic therapy is an important tool in the scheme of mastitis control. The treatments are more effective when directed by veterinarians; for example correct drug selection can be enhanced using an appropriate antimicrobial susceptibility test. The misuse or intensive use of antibiotics can lead to the development of resistance among different bacterial strains and contamination of foodstuff, with animal and human health implications [20]. The antimicrobial resistance is the result of mutations or exchange of genetic material such as plasmids and transposons [26]. Such resistance determinants most probably are acquired by pathogenic bacteria from a pool of resistance genes in other microbial genera present in different environments [8]. Increased resistance of Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolated from bovine mastitis cases to antimicrobial agents has been reported by [14,1] and [22].Milk production in Uruguay (South America) is important with a total of 410.000 dairy cows, yielding 1462 millions litres in 1999 [27]. In spite of the importance of this sector, only 3 surveys to evaluate the resistance of udder pathogens to antibiotics have been performed in Uruguay using agar disk diffusion (ADDM, [5]): 1) [9] testing S. aureus and Streptococcus agalactiae isolated from subclinical cases obtained from 43 dairy farms in the southern dairy region of Uruguay showed that 53% of S. aureus and 100% of Str. agalactiae were sensitive to penicillin. 2) [17] found 78% of S. aureus strains susceptible to penicillin in the dairy area around Tacuarembó city (north of Uruguay). 3) [6] studied the resistance patterns of S. aureus and CNS isolated in the laboratory routine during 4 years from milk samples collected in the southwestern region of Uruguay for penicillin, cloxacillin, nafcillin, rifampin and tetracycline obtaining: 58%, 16%, 5%, 6%, 29% of resistance for S.aureus and 75%, 42%, 17%, 12%, 26% for CNS, respectively.The sale of antibiotics is free in Uruguay, while the mastitis treatment is usually performed by the herd dairyman, and the antimicrobial agents most commonly used are tetracyclines, beta-lactams, macrolides, and aminoglycosides.The methods for susceptibility testing used to choose the appropriate drug are ADDM qualitative test and quantitative determinations by means of micro-dilution to determine the minimum inhibitory concentration (MIC) [3,2]. These methods can be interpreted following the National Committee for Clinical Laboratory Standards criteria [24] or guidelines proposed by other national antibiogram committees [2].The purposes of this work were: To determine the phenotypic expression of in vitro susceptibility of antimicrobials for pathogens (staphylococci, streptococci, and enterococci) isolated from dairy herds in Uruguay, and to compare the results obtained by the ADDM vs. broth micro-dilution method according to the NCCLS criteria. Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesMaterials and methods
Sample
A total of 522 strains including streptococci, enterococci and staphylococci were used in the study. The strains were isolated from sub-clinical and clinical cases of bovine mastitis from a survey carried out in the west littoral region of Uruguay [15], where quarter foremilk samples from 1077 milking cows and 40 milk samples from clinical cases detected in one month were collected in 29 randomly selected dairy farms. All strains were identified according to the procedures of the laboratory at the Department of Mastitis and Diagnostical Products, National Veterinary Institute (SVA), Uppsala, Sweden [25]. The isolates were maintained frozen at -20°C in Trypticase soy broth (Difco Laboratories, Michigan, USA) containing 10% glycerol until testing.
Susceptibility testing
Prior to the susceptibility testing all isolates were sub-cultured on Blood-esculin agar and incubated for 24 h at 37°C. Two different tests were carried out to determine the drug susceptibility for all strains:
1 – The ADDM was conducted and interpreted according to the recommendations and criteria of the NCCLS for bacteria isolated from animals [24]. The following disks (Becton Dickinson Microbiology System, Cockeysville, Maryland, USA) were used: penicillin, 10 μg; ampicillin, 10 μg; oxacillin, 1 μg; amoxicillin – clavulanic acid, 20 μg + 10 μg; cephalotin, 30 μg; gentamicin, 10 μg; erythromycin, 15 μg; enrofloxacin, 5 μg; tetracycline, 30 μg; neomycin, 30 μg; trimethoprim-sulfamethoxazole, 1.25 μg + 23.75 μg.
The staphylococci were tested against all the drugs above, while the streptococci against only 6 of these antimicrobial agents (penicillin, ampicillin, cephalotin, gentamicin, erythromycin and tetracycline), and enterococci against penicillin, ampicillin, gentamicin, erythromycin and tetracycline. The medium used was Mueller-Hinton Agar (Difco Laboratories, Detroit, USA) for sthaphylococci and Mueller-Hinton agar supplemented with 5% sheep blood for streptococci. S. aureus ATCC 25923, E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were included as quality control strains. The plates were read after 18 h incubation at 37°C under aerobic conditions. The isolates were categorised as susceptible, intermediate and resistant by measuring the inhibition zone.
2 – The MIC was determined using a commercially available micro-dilution system (VetMIC™ +/- panels, SVA, Uppsala, Sweden). The tests were performed by manufacturer's instruction and interpreted according to international standards [24] using Mueller-Hinton broth (Oxoid Limited, Basingstoke Hants, England) and S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and E. coli ATCC 25922, as quality control strains.
When the streptococcal strains were tested, 100 μl were inoculated in each well with erythromycin to obtain the following dilution: 0.25; 0.5; 1 and 2 μg/ml of the antimicrobial agent. These modifications were carried out to adapt to the breakpoints suggested by [24] for erythromycin. All panels were read on the same conditions as in the ADDM. The lowest dilution with no visible growth was considered as MIC for each strain. The concentration at which 50% and 90% of the isolates were inhibited, as well as the minimum and maximum range were determined.
The breakpoints suggested by the [24] for kanamycin were used for neomycin in both tests.
Oxacillin resistance testing
In order to confirm the presence of oxacillin resistance among staphylococci, VetMIC™ GP_mo panels (SVA, Uppsala, Sweden) were used as recommended by [24]. The procedures were conducted following the manufacturer's recommendations: the inoculum was prepared with colony material directly from the plate incubated 24 h before. A 1 μl loop with colony material was suspended in 4 ml of distilled water plus 0.02% Twin 80. From this suspension 100 μl were transferred to 10 ml Mueller Hinton Broth + 2% NaCl [4], which achieved about 103 to 104 cfu/50 μl. Each oxacillin and control well of the panel was inoculated with 50 μl of this final bacterial suspension. The panel was incubated at 30°C during 24 h under aerobic conditions. The strains S. aureus ATCC 29886 and S. aureus ATCC 29887 were included as negative and positive control strains, respectively.
β-Lactamase Testing (Cloverleaf Method)
The assay to determine the production of β-lactamase by staphylococci was described previously by [12]. Briefly, the non-β-lactamase-producing S. aureus Oxford strain 209 is used as indicator. This strain is inoculated on PDM II agar plates (AB Biodisk, Solna, Sweden) to yield an almost confluent growth on the agar surface. In the centre of the agar plate a disk containing 10 μg of penicillin G (PDM Antibiotics Sensitive II, AB Biodisk) is placed in order to induce β-lactamase production in the studied strain. The staphylococci to be tested were streaked in a line from the edge of the plate towards the centre of the penicillin disk. When the investigated strain was positive β-lactamase producer, the indicator strain grew alongside this strain towards the penicillin disk, into the inhibited one. The S. aureus strains ATCC 29213 and ATCC 25923 were included as positive and negative control respectively.
Statistics analyses
The Z-test [21] was performed to compare the proportions of resistant strains to each antimicrobial agents obtained by means of both test.
Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesResults
All values obtained with control strains in both tests were within the expected ranges for all antimicrobial agents analysed. The ranges of MIC of each of the antimicrobial agents tested, MIC50 and MIC90 of the tested strains, and the percentage of resistance obtained by both micro-dilutions and ADDM are presented here for S. aureus (Table 1), CNS (Table 2), Str. agalactiae (Table 3), Streptococcus dysgalactiae (Table 4), Streptococcus uberis (Table 5) and Enterococcus sp (Table 6). Table 1
In vitro susceptibility of 336 strains of Staphylococcus aureus obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
Table 2
In vitro susceptibility of 41 strains of Coagulase Negative Staphylococcus obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
Table 3
In vitro susceptibility of 60 strains of Streptococcus agalactiae obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
Table 4
In vitro susceptibility of 9 strains of Streptococcus dysgalactiae obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
Table 5
In vitro susceptibility of 33 strains of Streptococcus uberis obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
Table 6
In vitro susceptibility of 43 strains of Enterococcus sp. obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
The differences found between both tests corresponding to each antimicrobial agent were not significant (p > 0.05). Of 336 strains of S. aureus, 215 (64%) were resistant to one or more antimicrobial agents in both tests. There was no resistance to oxacillin, cephalotin, gentamicin, enrofloxacin, clindamycin, and the combination of amoxicillin-clavulanic acid, whereas 160 (47.6%), 157 (46.7%), 45 (13.4%), 10 (3%), 2 (0.6%) and 1 strain (0.3%) were resistant to penicillin, ampicillin, tetracycline, erythromycin, neomycin and trimethoprim-sulphametoxazole, respectively. One hundred and fifty-six S. aureus isolates (46.4%) were β-lactamase producers. While of 41 CNS strains, 10 (27%) presented resistance to penicillin and 9 strains (22.5%) were β-lactamase producers. Seven suspected oxacillin resistant strains of S. aureus on the ADDM were susceptible in the confirmatory test.All isolates of Str. agalactiae, Str. dysgalactiae and Str. uberis were susceptible to penicillin and ampicillin, while 3 (7%) of 43 strains of Enterococcus sp. were resistant to penicillin. Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesDiscussion
The β-lactams (penicillins and cephalosporins) have become the first line of antimicrobial agents used for treatment of bovine mastitis in Uruguay. Within this class, penicillin, amoxicillin, cloxacillin and ampicillin are the mostly used agents. In the Nordic countries penicillin is used as the first-line antibiotic treatment of bovine mastitis, because of a low resistance rate and narrow spectrum. This is an important tool to limit the development of antibiotic resistance as much as possible [1]. In our study 47.6% of S. aureus were classified as penicillin resistant, MIC ≥ 0.25 μg/ml (Table 2), 96% of which produced β-lactamase. This was the same comparing the proportion of resistance (47%) as obtained by [9] in the southern dairy area of Uruguay. The comparison between these results obtained in Uruguay over the years demonstrated that the situation in general has not changed during the last 25 years in relation to penicillin resistance. Whereas, the prevalence of resistance to penicillin was similar in Argentina (40%) [13] and Finland (50.7%) [22]. However, it was higher than in Norway, 4.2% from clinical cases and 18% from sub-clinical cases [16], and Sweden, 6% [11].In relation to CNS, 27% of 41 isolates were penicillin resistant (Table 1). Results from Finland 37% [22] and Norway 26% [16] agree with our findings. The MIC90 of penicillin was 4 μg/ml for our survey, and another study determined 0.5 μg/ml in New Zealand [31].The detection of β-lactamase production in staphylococci is a useful and rapid method to detect penicillin resistance. At the National Veterinary Institute, Uppsala, β-lactamase results are used as rapid screen to indicate penicillin resistance [25]. In this study 96% and 90% of penicillin resistant strains of S.aureus and CNS were positive as indicated by the cloverleaf method. Test for β-lactamase producing should always be done to obtain the true picture of resistance to penicillin in staphylococci.The streptococci and enterococci showed high susceptibility (streptococci 100%) to penicillin in our study (Tables 3, 4, 5 and 6). This agree with the results from monitoring studies done in the Scandinavian countries, where the streptococci populations isolated from mastitis were highly susceptible to penicillin [28]. Only 7% of the Enterococcus sp. strains were classified as resistant against penicillin (> 8 μg/ml). The MIC90 of penicillin for Str. agalactiae, Str. dysgalactiae, and Str. uberis was 0.12 μg/ml in each case and for enterococci 4 μg/ml.Oxacillin was included here as recommended by the [24] to detect methicillin-resistant strains of S. aureus and CNS. In our study oxacillin resistance was not found in staphylococci. However, CNS strains with higher MIC than > 0.5 *g/ml of oxacillin should be tested for possible carriage of the mecA gene, in order to verify the occurrence of this gene [18].Cephalotin was included to determine the resistance against the first-generation cephalosporin class for all bacterial species except Enterococcus sp. All tested microorganisms were 100% sensitive to cephalotin and MIC90 was ≤ 4 μg/ml.Despite their structural differences macrolide and lincosamides antimicrobials have similar biological properties, including their mechanism of action against the 50S subunit of the bacterial ribosome. These common properties easily allow the development of cross-resistance [29].Erythromycin and clindamycin were included here to evaluate the resistance against these groups. Clindamycin was used in our survey to test resistance againts lincosamides in staphylococci and no strains were resistant. For our strains the MIC90 value was < 1 μg/ml, a result remarkably different as compared with 8 μg/ml obtained by [10] for S. aureus strains isolated in the United States.For erythromycin our findings (Tables 1 and 2) showed scarce resistance in S. aureus (3%) and in CNS (0%), similar to the result (2.4%) reported by [9]. The results were lower than reported by [13] for S. aureus in Argentina (11.6%). In Finland, [22] found 2.6% and 11.5% resistance among S. aureus and CNS, respectively, while in Sweden, [11] reported 1% resistance in S. aureus. The MIC90 of erythromycin in our study was ≤ 0.5 μg/ml to staphylococci.Streptococci showed high erythromycin susceptibility, only 3.4% of Str. agalactiae and 4.6% of Enterococcus sp. were resistant in our study. Substantial differences were found in relation to results obtained in Finland (17%) for enterococci [22], but no differences with respect to the erythromycin susceptibility result in streptococci (2,8%) obtained by [9]. Our MIC90 value of erythromycin for streptococci was ≤ 0.25 μg/ml except for Str. uberis (0.5 μg/ml), while for enterococci was ≤ 0.5 μg/ml.Aminoglycosides are used with precaution in dairy animals in order to avoid the risk of prolonged residues in milk. However, products for direct infusion into mammary gland containing neomycin are used because of the limited systemic effect caused by this way of administration [30]. The MIC90 (2 μg/ml) of neomycin (Table 1) in our survey for S. aureus was slightly different compared to the results obtained with S. aureus from different countries [10].The S. aureus and CNS bacteria were not gentamicin-resistant and the MIC90 values were ≤ 1 μg/ml for both. This was similar to the results obtained for S. aureus in Argentina [13]. As expected we found high MICs of gentamicin in Str. agalactiae and Str. uberis (Tables 3 and 5), while Str. uberis and Enterococcus sp. had lower MICs (Tables 4 and 6). Aminoglycosides are not the antimicrobials agents of choice for streptococcal mastitis because streptococci have inherited resistance to this class [28].Our results regarding tetracycline-resistance for S. aureus (13.4%) and CNS (13.9%) were similar to those in Finland [22], but higher than the results obtained in Norway for S. aureus (0.2 %) and CNS (3%) [16]. The results were twofold higher than the 6% reported by [9] in Uruguay. A possible explanation for this phenomenon could be that for many years tetracyclines have been the most widely antimicrobial class used by the farmers to treat any infection.In general the streptococci and enterococci were susceptible to oxytetracycline, with the exception of Str. dysgalactiae (Table 4). [28] stated that Str. dysgalactiae strains are less susceptible to tetracyclyne than Str. uberis strains, as also reported by [7]Staphylococci and streptococci were susceptible to trimethoprim-sulfamethoxazole, whereas enterococci were resistant (Table 6).Enrofloxacin is approved for systemic administration to treat bovine mastitis in some Scandinavian countries. We found a high susceptibility in staphylococci (Tables 1 and 2) and a similar situation was found by [22].Both antimicrobial susceptibility tests, ADDM and broth micro-dilution, used in this survey were performed according to the approved standard for bacteria isolated from animals and the interpretative criteria for veterinary use according to [24]. The ADDM is most commonly used in the veterinary laboratories in Uruguay and many other countries. There were no significant differences between the methods when classifying bacterial isolates as susceptible or resistant according to NLCCS (Tables 1, 2, 3, 4, 5 and 6). The results from ADDM could be influenced by several factors, such as: compositions of agar medium, pH, inoculum density, agar depth, timing of drug applications, incubation time, etc [2]. However, [23] have obtained high correlation coefficient (0.875 to 0.975) between both methods in agreement with our results. [19] considered ADDM as a useful tool when the level of compliance with NCCLS guidelines was evaluated periodically. Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesConclusion
This study did not show changes with respect to the penicillin and erythromycin resistance level of udder pathogens (staphylococci and streptococci) during the last 25 years in Uruguay, while a clear increase in tetracycline resistance was found for S. aureus.The Agar Disk Diffusion Method was a good tool, inexpensive, and readily available for regional veterinary laboratories. However, considering the necessity to maintain the surveillance over antimicrobial resistance in a country, it is important to periodically evaluate the compliance with guidelines such as National Committee for Clinical Laboratory Standards guidelines. It is also important to monitor regularly the minimum inhibitory concentrations for the isolated strains from different regions of the country. A responsible antibiotic policy would be highly relevant in a future programme for mastitis control and udder health in Uruguay.Acknowledgements
The authors thank Margareta Horn af Rantzein for her generous support of this work. The authors also acknowledge the staff of the mastitis laboratory, Department of Mastitis and Diagnostical Products, National Veterinary Institute, Uppsala, Sweden, where the work was carried out. R. E. Gianneechini was awarded a scholarship by the Swedish Foundation for International Co-operation in Research and Higher Education (STINT) and a grant from Instituto Nacional de Investigaciones Agropecuarias (INIA), Uruguay.
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AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesReferences
1.Aarestrup FM, Jensen NE. Development of penicillin resistance among Staphylococcus aureus isolated from bovine mastitis in Denmark and other countries. Microbial Drug Resistance. 1998;4:247–256. [PubMed]
2.Acar JF, Goldstain FW. Antibiotics in Laboratory Medicine. 4. V. Lorian, Williams and Wilkins, Baltimore, MD, USA; 1996. Disk susceptibility test; pp. 1–51.
3.Amsterdam D. Antibiotics in Laboratory Medicine. 4. V. Lorian, Williams and Wilkins, Baltimore, MD, USA; 1996. Susceptibility testing of antimicrobials in liquid media; pp. 52–111.
4.Baker CN, Huang MB, Tenover FC. Optimizing testing of methicillin-resistant Staphylococcus species. Diagn Microbiol Infect Dis. 1994;19:167–170. doi: 10.1016/0732-8893(94)90061-2. [PubMed]
5.Bauer AW, Kirby WMM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J of Clinical Path. 1966;45:493–496. [PubMed]
6.Bouman M, Irigoyen D, Bertón A. Analisis de los resultados de 427 muestras remitidas para aislamiento de bacterias de mastitis y antibiograma. (Study of results from 427 milk samples remitted for bacteriologic cultures and susceptibility testing against antimicrobial agents). Jornadas de Salud de Ubre, Nva Helvecia, Uruguay. 1999. pp. 59–68. (In Spanish).
7.Brown MB, Roberts MC. Tetracycline resistance determinants in streptococcal species isolated from the bovine mammary gland. Vet Microbiol. 1991;29:173–180. doi: 10.1016/0378-1135(91)90124-X. [PubMed]
8.Davies J. Inactivation of antibiotics and the dissemination of resistance genes. Science. 1994;264:375–382. doi: 10.1126/science.8153624. [PubMed]
9.Del Baglivi L, Bonilla M, Laborde M. Investigación sobre mastitis subclinica en rodeos lecheros del Uruguay. (Subclinical mastitis research in dairy herds of Uruguay). Medicina-Veterinaria Montevideo, Uruguay. 1976;12:61,69–77. (In Spanish).
10.De Oliveira AP, Watts JL, Salmon SA, Aarestrup FM. Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Europe and United States. J Dairy Sci. 2000;83:855–862. [PubMed]
11.Franklin A. Antibiotic policy and ocurrence of resistance in Sweden. Proceedings of 25th International Dairy Congress, Aarhus, Denmark. 1998. pp. 229–234.
12.Franklin A, Wierup M. Evaluation of the sensititre method adapted for antimicrobial drug susceptibility testing in veterinary medicine. Vet Microbiol. 1982;7:447–454. doi: 10.1016/0378-1135(82)90061-X. [PubMed]
13.Gentilini E, Denamiel G, Llorente P, Godaly S, Rebuelto M, De Gregorio O. Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Argentina. J Dairy Sci. 2000;83:1224–1227. [PubMed]
14.Gentilini E, Denamiel G, Tirante L, Chavez C, Godaly MS. Bovine Mastitis: β-lactamase production. Staphylococcus aureus antibiotic resistance evolution. Proceedings of the 3rd International Mastitis Seminar, Tel Aviv, Israel. 1995;session 2:84–85.
15.Gianneechini R. Thesis. International Master of Science Programme, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala, Sweden; 2001. Occurrence and aetiology of clinical and subclinical mastitis, and antimicrobial susceptibility of udder pathogens in dairy herds in a region of Uruguay; pp. 31–43. ISSN 1403-2201, Report no. 25.
16.Hofshager M, Kruse H, Lassen J, Stavnes T-L, Essun K, Holstad G, Mørk T, Schau J, Grave K. Resistance in bacteria from infections in animals. In: Hilde Kruse, editor. Usage of antimicrobial agents in animals and occurrence of antimicrobal resistance in bacteria from animals, feed, and food in Norway 1999, NORM-VET, Oslo, Norway. 1999. pp. 16–18. ISSN-1502-4695.
17.Herrera B. Etiología de las mastitis subclínicas y estudio de la cuenca lechera de Tacuarembó. (Ethiology of subclinical mastitis and study of the dairy area of Tacuarembó). III Congreso Nacional de Veterinaria, Montevideo, Uruguay. 1982. pp. 495–505. In Spanish.
18.Hussain Z, Stoakes L, Massey V, Diagre D, Fitzgerald V, El Sayed S, Lannigan R. Correlation of oxacillin MIC with mecA gene carriage in Coagulase-negative staphylococci. J Clin Microbiol. 2000;38:752–754. [PubMed]
19.Kiehlbauch JA, Hannett GE, Salfinger M, Archinal W, Monserrat C, Carlyn C. Use of the National Committee for Clinical Laboratory Standards guidelines for disk diffusion susceptibility testing in New York State Laboratories. J Clin Microbiol. 2000;38:3341–3348. [PubMed]
20.Lingaas E. The use of antimicrobials in animal production – a threat to humans? NKVet Symposium, Helsinki, Finland. 1998. pp. 26–27.
21.Milton S. Statistical methods in the biological and health sciences. 2. chapter 8. New York, McGraw-Hill; 1992.
22.Myllys V, Asplund K, Brofeldt E, Hirvelä-Koski V, Honkanen-Buzalske T, Junttila J, Kulkas L, Myllykangas O, Niskanen M, Saloniemi H, Sandholm M, Saranpää T. Bovine mastitis in Finland in 1988 and 1995 – Changes in prevalence and antimicrobial resistance. Acta vet Scand. 1998;39:119–126. [PubMed]
23.Myllys V, Louhi M, Ali-Vehmas T. Comparison of penicillin-G susceptibility testing methods of Staphylococci isolated from bovine mastitis. J Vet Med B. 1992;39:723–731.
24.National Committee of Clinical Laboratories Standards. Performance Standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard. NCCLS. 1999;19:11,60. Document M31-A.
25.National Veterinary Institute. Department of Mastitis, Accreditation Certificate, Uppsala, Sweden. pp. 1–29.
26.Neu HC. The crisis in antibiotic resistance. Science. 1992;257:1064–1073. doi: 10.1126/science.257.5073.1064. [PubMed]
27.OPYPA. Oficina de Planeamiento y Producción Agropecuaria. Ministerio de Ganadería Agricultura y Pesca. Montevideo-Uruguay. 2000.
28.Pyörälä S, Myllys V. Resistance of bacteria to antimicrobials. The bovine udder and mastitis, University of Helsinki, Faculty of Veterinary Medicine, Helsinki, Finland. 1995. pp. 194–200.
29.Prescott JF. Lincosamides, Macrolides, and Pleuromutilins. In: Prescott JF, Baggot JD, Walker RD, editor. Antimicrobial therapy in veterinary medicine. 3. Iowa State University Press, Ames, Iowa, USA; 2000. pp. 229–262.
30.Prescott JF. Aminoglycosides and Aminocyclitols. In: Prescott JF, Bagott JD, Walker RD, editor. Antimicrobial therapy in veterinary medicine. 3. Iowa State University Press, Ames, Iowa.USA; 2000. pp. 191–228.
31.Salmon SA, Watts JL, Aarestrup JW, Yancey RJ., Jr Minimum Inhibitory Concentrations for selected antimicrobial agents against organisms isolated from the mammary glands of dairy heifers in New Zealand and Denmark. J Dairy Sci. 1998;81:570–578. [PubMed]
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Articles from Acta Veterinaria Scandinavica are provided here courtesy of
BioMed Central
RE Gianneechini,1 C Concha,2 and A Franklin3
1Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala, Sweden
2Department of Mastitis and Diagnostical Products, Uppsala, Sweden
3Department of Antibiotics, National Veterinary Institute, Uppsala, Sweden
Corresponding author.
Received March 9, 2001; Accepted November 12, 2001.
Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesAbstract
A total of 522 strains belonging to streptococci, enterococci and staphylococci isolated from sub-clinical and clinical cases of bovine mastitis from the west littoral region of Uruguay were analysed for their susceptibility to several antimicrobial agents. The susceptibility patterns were studied by agar disk diffusion methods (ADDM) and broth micro-dilution to determine the minimum inhibitory concentration (MIC). The concentration that inhibits 90% (MIC90) of the analysed strains reported in micrograms per millilitre, for Staphylococcus aureus were > 8, 8, ≤ 0.5, ≤ 4, ≤ 1, ≤ 0.5, > 64, ≤ 0.25, 0.5, ≤ 1 and ≤ 1 to penicillin, ampicillin, oxacillin, cephalotin, gentamicin, erythromycin, oxitetracycline, enrofloxacin, trimethoprim/sulfamethoxazole, neomycin, and clindamycin, respectively. Coagulase-negative staphylococci (CNS) had different values for penicillin (4) and ampicillin (2), while the other antimicrobial agents had the same MIC90 values as reported for S. aureus. The MIC90 values for streptococci were 0.12, 0.25, ≤ 4, 16, ≤ 0.25, 0.5, 0.25 for penicillin, ampicillin, cephalotin, gentamicin, erythromycin, oxytetracycline and trimethoprim-sulfamethoxazole, whereas MIC90 for enterococci were 4, 4, 4, ≤ 0.5, 2, > 8 for penicillin, ampicillin, gentamicin, erythromycin, oxytetracycline and trimethoprim-sulfamethoxazole, respectively. Of 336 strains of S. aureus, 160 (47.6%) were resistant to penicillin. For 41 CNS strains, 10 (27%) presented penicillin-resistance. All the streptococcal strains were susceptible to penicillin, while 3 (7%) of the 43 enteroccocal strains were resistant. Non significant statistical differences were found between the results obtained by ADDM and broth micro-dilution for classifying bacterial isolates as susceptible or resistant according to the National Committee of Clinical Laboratory Standards.Keywords: cow, mammary qland, bacteria, resistant, sensitive Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesIntroduction
Bovine mastitis is the major problem for milk producers throughout the world and responsible for substantial losses of revenue annually. Antibiotic therapy is an important tool in the scheme of mastitis control. The treatments are more effective when directed by veterinarians; for example correct drug selection can be enhanced using an appropriate antimicrobial susceptibility test. The misuse or intensive use of antibiotics can lead to the development of resistance among different bacterial strains and contamination of foodstuff, with animal and human health implications [20]. The antimicrobial resistance is the result of mutations or exchange of genetic material such as plasmids and transposons [26]. Such resistance determinants most probably are acquired by pathogenic bacteria from a pool of resistance genes in other microbial genera present in different environments [8]. Increased resistance of Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolated from bovine mastitis cases to antimicrobial agents has been reported by [14,1] and [22].Milk production in Uruguay (South America) is important with a total of 410.000 dairy cows, yielding 1462 millions litres in 1999 [27]. In spite of the importance of this sector, only 3 surveys to evaluate the resistance of udder pathogens to antibiotics have been performed in Uruguay using agar disk diffusion (ADDM, [5]): 1) [9] testing S. aureus and Streptococcus agalactiae isolated from subclinical cases obtained from 43 dairy farms in the southern dairy region of Uruguay showed that 53% of S. aureus and 100% of Str. agalactiae were sensitive to penicillin. 2) [17] found 78% of S. aureus strains susceptible to penicillin in the dairy area around Tacuarembó city (north of Uruguay). 3) [6] studied the resistance patterns of S. aureus and CNS isolated in the laboratory routine during 4 years from milk samples collected in the southwestern region of Uruguay for penicillin, cloxacillin, nafcillin, rifampin and tetracycline obtaining: 58%, 16%, 5%, 6%, 29% of resistance for S.aureus and 75%, 42%, 17%, 12%, 26% for CNS, respectively.The sale of antibiotics is free in Uruguay, while the mastitis treatment is usually performed by the herd dairyman, and the antimicrobial agents most commonly used are tetracyclines, beta-lactams, macrolides, and aminoglycosides.The methods for susceptibility testing used to choose the appropriate drug are ADDM qualitative test and quantitative determinations by means of micro-dilution to determine the minimum inhibitory concentration (MIC) [3,2]. These methods can be interpreted following the National Committee for Clinical Laboratory Standards criteria [24] or guidelines proposed by other national antibiogram committees [2].The purposes of this work were: To determine the phenotypic expression of in vitro susceptibility of antimicrobials for pathogens (staphylococci, streptococci, and enterococci) isolated from dairy herds in Uruguay, and to compare the results obtained by the ADDM vs. broth micro-dilution method according to the NCCLS criteria. Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesMaterials and methods
Sample
A total of 522 strains including streptococci, enterococci and staphylococci were used in the study. The strains were isolated from sub-clinical and clinical cases of bovine mastitis from a survey carried out in the west littoral region of Uruguay [15], where quarter foremilk samples from 1077 milking cows and 40 milk samples from clinical cases detected in one month were collected in 29 randomly selected dairy farms. All strains were identified according to the procedures of the laboratory at the Department of Mastitis and Diagnostical Products, National Veterinary Institute (SVA), Uppsala, Sweden [25]. The isolates were maintained frozen at -20°C in Trypticase soy broth (Difco Laboratories, Michigan, USA) containing 10% glycerol until testing.
Susceptibility testing
Prior to the susceptibility testing all isolates were sub-cultured on Blood-esculin agar and incubated for 24 h at 37°C. Two different tests were carried out to determine the drug susceptibility for all strains:
1 – The ADDM was conducted and interpreted according to the recommendations and criteria of the NCCLS for bacteria isolated from animals [24]. The following disks (Becton Dickinson Microbiology System, Cockeysville, Maryland, USA) were used: penicillin, 10 μg; ampicillin, 10 μg; oxacillin, 1 μg; amoxicillin – clavulanic acid, 20 μg + 10 μg; cephalotin, 30 μg; gentamicin, 10 μg; erythromycin, 15 μg; enrofloxacin, 5 μg; tetracycline, 30 μg; neomycin, 30 μg; trimethoprim-sulfamethoxazole, 1.25 μg + 23.75 μg.
The staphylococci were tested against all the drugs above, while the streptococci against only 6 of these antimicrobial agents (penicillin, ampicillin, cephalotin, gentamicin, erythromycin and tetracycline), and enterococci against penicillin, ampicillin, gentamicin, erythromycin and tetracycline. The medium used was Mueller-Hinton Agar (Difco Laboratories, Detroit, USA) for sthaphylococci and Mueller-Hinton agar supplemented with 5% sheep blood for streptococci. S. aureus ATCC 25923, E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were included as quality control strains. The plates were read after 18 h incubation at 37°C under aerobic conditions. The isolates were categorised as susceptible, intermediate and resistant by measuring the inhibition zone.
2 – The MIC was determined using a commercially available micro-dilution system (VetMIC™ +/- panels, SVA, Uppsala, Sweden). The tests were performed by manufacturer's instruction and interpreted according to international standards [24] using Mueller-Hinton broth (Oxoid Limited, Basingstoke Hants, England) and S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and E. coli ATCC 25922, as quality control strains.
When the streptococcal strains were tested, 100 μl were inoculated in each well with erythromycin to obtain the following dilution: 0.25; 0.5; 1 and 2 μg/ml of the antimicrobial agent. These modifications were carried out to adapt to the breakpoints suggested by [24] for erythromycin. All panels were read on the same conditions as in the ADDM. The lowest dilution with no visible growth was considered as MIC for each strain. The concentration at which 50% and 90% of the isolates were inhibited, as well as the minimum and maximum range were determined.
The breakpoints suggested by the [24] for kanamycin were used for neomycin in both tests.
Oxacillin resistance testing
In order to confirm the presence of oxacillin resistance among staphylococci, VetMIC™ GP_mo panels (SVA, Uppsala, Sweden) were used as recommended by [24]. The procedures were conducted following the manufacturer's recommendations: the inoculum was prepared with colony material directly from the plate incubated 24 h before. A 1 μl loop with colony material was suspended in 4 ml of distilled water plus 0.02% Twin 80. From this suspension 100 μl were transferred to 10 ml Mueller Hinton Broth + 2% NaCl [4], which achieved about 103 to 104 cfu/50 μl. Each oxacillin and control well of the panel was inoculated with 50 μl of this final bacterial suspension. The panel was incubated at 30°C during 24 h under aerobic conditions. The strains S. aureus ATCC 29886 and S. aureus ATCC 29887 were included as negative and positive control strains, respectively.
β-Lactamase Testing (Cloverleaf Method)
The assay to determine the production of β-lactamase by staphylococci was described previously by [12]. Briefly, the non-β-lactamase-producing S. aureus Oxford strain 209 is used as indicator. This strain is inoculated on PDM II agar plates (AB Biodisk, Solna, Sweden) to yield an almost confluent growth on the agar surface. In the centre of the agar plate a disk containing 10 μg of penicillin G (PDM Antibiotics Sensitive II, AB Biodisk) is placed in order to induce β-lactamase production in the studied strain. The staphylococci to be tested were streaked in a line from the edge of the plate towards the centre of the penicillin disk. When the investigated strain was positive β-lactamase producer, the indicator strain grew alongside this strain towards the penicillin disk, into the inhibited one. The S. aureus strains ATCC 29213 and ATCC 25923 were included as positive and negative control respectively.
Statistics analyses
The Z-test [21] was performed to compare the proportions of resistant strains to each antimicrobial agents obtained by means of both test.
Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesResults
All values obtained with control strains in both tests were within the expected ranges for all antimicrobial agents analysed. The ranges of MIC of each of the antimicrobial agents tested, MIC50 and MIC90 of the tested strains, and the percentage of resistance obtained by both micro-dilutions and ADDM are presented here for S. aureus (Table 1), CNS (Table 2), Str. agalactiae (Table 3), Streptococcus dysgalactiae (Table 4), Streptococcus uberis (Table 5) and Enterococcus sp (Table 6). Table 1
In vitro susceptibility of 336 strains of Staphylococcus aureus obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
Table 2
In vitro susceptibility of 41 strains of Coagulase Negative Staphylococcus obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
Table 3
In vitro susceptibility of 60 strains of Streptococcus agalactiae obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
Table 4
In vitro susceptibility of 9 strains of Streptococcus dysgalactiae obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
Table 5
In vitro susceptibility of 33 strains of Streptococcus uberis obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
Table 6
In vitro susceptibility of 43 strains of Enterococcus sp. obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
The differences found between both tests corresponding to each antimicrobial agent were not significant (p > 0.05). Of 336 strains of S. aureus, 215 (64%) were resistant to one or more antimicrobial agents in both tests. There was no resistance to oxacillin, cephalotin, gentamicin, enrofloxacin, clindamycin, and the combination of amoxicillin-clavulanic acid, whereas 160 (47.6%), 157 (46.7%), 45 (13.4%), 10 (3%), 2 (0.6%) and 1 strain (0.3%) were resistant to penicillin, ampicillin, tetracycline, erythromycin, neomycin and trimethoprim-sulphametoxazole, respectively. One hundred and fifty-six S. aureus isolates (46.4%) were β-lactamase producers. While of 41 CNS strains, 10 (27%) presented resistance to penicillin and 9 strains (22.5%) were β-lactamase producers. Seven suspected oxacillin resistant strains of S. aureus on the ADDM were susceptible in the confirmatory test.All isolates of Str. agalactiae, Str. dysgalactiae and Str. uberis were susceptible to penicillin and ampicillin, while 3 (7%) of 43 strains of Enterococcus sp. were resistant to penicillin. Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesDiscussion
The β-lactams (penicillins and cephalosporins) have become the first line of antimicrobial agents used for treatment of bovine mastitis in Uruguay. Within this class, penicillin, amoxicillin, cloxacillin and ampicillin are the mostly used agents. In the Nordic countries penicillin is used as the first-line antibiotic treatment of bovine mastitis, because of a low resistance rate and narrow spectrum. This is an important tool to limit the development of antibiotic resistance as much as possible [1]. In our study 47.6% of S. aureus were classified as penicillin resistant, MIC ≥ 0.25 μg/ml (Table 2), 96% of which produced β-lactamase. This was the same comparing the proportion of resistance (47%) as obtained by [9] in the southern dairy area of Uruguay. The comparison between these results obtained in Uruguay over the years demonstrated that the situation in general has not changed during the last 25 years in relation to penicillin resistance. Whereas, the prevalence of resistance to penicillin was similar in Argentina (40%) [13] and Finland (50.7%) [22]. However, it was higher than in Norway, 4.2% from clinical cases and 18% from sub-clinical cases [16], and Sweden, 6% [11].In relation to CNS, 27% of 41 isolates were penicillin resistant (Table 1). Results from Finland 37% [22] and Norway 26% [16] agree with our findings. The MIC90 of penicillin was 4 μg/ml for our survey, and another study determined 0.5 μg/ml in New Zealand [31].The detection of β-lactamase production in staphylococci is a useful and rapid method to detect penicillin resistance. At the National Veterinary Institute, Uppsala, β-lactamase results are used as rapid screen to indicate penicillin resistance [25]. In this study 96% and 90% of penicillin resistant strains of S.aureus and CNS were positive as indicated by the cloverleaf method. Test for β-lactamase producing should always be done to obtain the true picture of resistance to penicillin in staphylococci.The streptococci and enterococci showed high susceptibility (streptococci 100%) to penicillin in our study (Tables 3, 4, 5 and 6). This agree with the results from monitoring studies done in the Scandinavian countries, where the streptococci populations isolated from mastitis were highly susceptible to penicillin [28]. Only 7% of the Enterococcus sp. strains were classified as resistant against penicillin (> 8 μg/ml). The MIC90 of penicillin for Str. agalactiae, Str. dysgalactiae, and Str. uberis was 0.12 μg/ml in each case and for enterococci 4 μg/ml.Oxacillin was included here as recommended by the [24] to detect methicillin-resistant strains of S. aureus and CNS. In our study oxacillin resistance was not found in staphylococci. However, CNS strains with higher MIC than > 0.5 *g/ml of oxacillin should be tested for possible carriage of the mecA gene, in order to verify the occurrence of this gene [18].Cephalotin was included to determine the resistance against the first-generation cephalosporin class for all bacterial species except Enterococcus sp. All tested microorganisms were 100% sensitive to cephalotin and MIC90 was ≤ 4 μg/ml.Despite their structural differences macrolide and lincosamides antimicrobials have similar biological properties, including their mechanism of action against the 50S subunit of the bacterial ribosome. These common properties easily allow the development of cross-resistance [29].Erythromycin and clindamycin were included here to evaluate the resistance against these groups. Clindamycin was used in our survey to test resistance againts lincosamides in staphylococci and no strains were resistant. For our strains the MIC90 value was < 1 μg/ml, a result remarkably different as compared with 8 μg/ml obtained by [10] for S. aureus strains isolated in the United States.For erythromycin our findings (Tables 1 and 2) showed scarce resistance in S. aureus (3%) and in CNS (0%), similar to the result (2.4%) reported by [9]. The results were lower than reported by [13] for S. aureus in Argentina (11.6%). In Finland, [22] found 2.6% and 11.5% resistance among S. aureus and CNS, respectively, while in Sweden, [11] reported 1% resistance in S. aureus. The MIC90 of erythromycin in our study was ≤ 0.5 μg/ml to staphylococci.Streptococci showed high erythromycin susceptibility, only 3.4% of Str. agalactiae and 4.6% of Enterococcus sp. were resistant in our study. Substantial differences were found in relation to results obtained in Finland (17%) for enterococci [22], but no differences with respect to the erythromycin susceptibility result in streptococci (2,8%) obtained by [9]. Our MIC90 value of erythromycin for streptococci was ≤ 0.25 μg/ml except for Str. uberis (0.5 μg/ml), while for enterococci was ≤ 0.5 μg/ml.Aminoglycosides are used with precaution in dairy animals in order to avoid the risk of prolonged residues in milk. However, products for direct infusion into mammary gland containing neomycin are used because of the limited systemic effect caused by this way of administration [30]. The MIC90 (2 μg/ml) of neomycin (Table 1) in our survey for S. aureus was slightly different compared to the results obtained with S. aureus from different countries [10].The S. aureus and CNS bacteria were not gentamicin-resistant and the MIC90 values were ≤ 1 μg/ml for both. This was similar to the results obtained for S. aureus in Argentina [13]. As expected we found high MICs of gentamicin in Str. agalactiae and Str. uberis (Tables 3 and 5), while Str. uberis and Enterococcus sp. had lower MICs (Tables 4 and 6). Aminoglycosides are not the antimicrobials agents of choice for streptococcal mastitis because streptococci have inherited resistance to this class [28].Our results regarding tetracycline-resistance for S. aureus (13.4%) and CNS (13.9%) were similar to those in Finland [22], but higher than the results obtained in Norway for S. aureus (0.2 %) and CNS (3%) [16]. The results were twofold higher than the 6% reported by [9] in Uruguay. A possible explanation for this phenomenon could be that for many years tetracyclines have been the most widely antimicrobial class used by the farmers to treat any infection.In general the streptococci and enterococci were susceptible to oxytetracycline, with the exception of Str. dysgalactiae (Table 4). [28] stated that Str. dysgalactiae strains are less susceptible to tetracyclyne than Str. uberis strains, as also reported by [7]Staphylococci and streptococci were susceptible to trimethoprim-sulfamethoxazole, whereas enterococci were resistant (Table 6).Enrofloxacin is approved for systemic administration to treat bovine mastitis in some Scandinavian countries. We found a high susceptibility in staphylococci (Tables 1 and 2) and a similar situation was found by [22].Both antimicrobial susceptibility tests, ADDM and broth micro-dilution, used in this survey were performed according to the approved standard for bacteria isolated from animals and the interpretative criteria for veterinary use according to [24]. The ADDM is most commonly used in the veterinary laboratories in Uruguay and many other countries. There were no significant differences between the methods when classifying bacterial isolates as susceptible or resistant according to NLCCS (Tables 1, 2, 3, 4, 5 and 6). The results from ADDM could be influenced by several factors, such as: compositions of agar medium, pH, inoculum density, agar depth, timing of drug applications, incubation time, etc [2]. However, [23] have obtained high correlation coefficient (0.875 to 0.975) between both methods in agreement with our results. [19] considered ADDM as a useful tool when the level of compliance with NCCLS guidelines was evaluated periodically. Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesConclusion
This study did not show changes with respect to the penicillin and erythromycin resistance level of udder pathogens (staphylococci and streptococci) during the last 25 years in Uruguay, while a clear increase in tetracycline resistance was found for S. aureus.The Agar Disk Diffusion Method was a good tool, inexpensive, and readily available for regional veterinary laboratories. However, considering the necessity to maintain the surveillance over antimicrobial resistance in a country, it is important to periodically evaluate the compliance with guidelines such as National Committee for Clinical Laboratory Standards guidelines. It is also important to monitor regularly the minimum inhibitory concentrations for the isolated strains from different regions of the country. A responsible antibiotic policy would be highly relevant in a future programme for mastitis control and udder health in Uruguay.Acknowledgements
The authors thank Margareta Horn af Rantzein for her generous support of this work. The authors also acknowledge the staff of the mastitis laboratory, Department of Mastitis and Diagnostical Products, National Veterinary Institute, Uppsala, Sweden, where the work was carried out. R. E. Gianneechini was awarded a scholarship by the Swedish Foundation for International Co-operation in Research and Higher Education (STINT) and a grant from Instituto Nacional de Investigaciones Agropecuarias (INIA), Uruguay.
Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesReferences
1.Aarestrup FM, Jensen NE. Development of penicillin resistance among Staphylococcus aureus isolated from bovine mastitis in Denmark and other countries. Microbial Drug Resistance. 1998;4:247–256. [PubMed]
2.Acar JF, Goldstain FW. Antibiotics in Laboratory Medicine. 4. V. Lorian, Williams and Wilkins, Baltimore, MD, USA; 1996. Disk susceptibility test; pp. 1–51.
3.Amsterdam D. Antibiotics in Laboratory Medicine. 4. V. Lorian, Williams and Wilkins, Baltimore, MD, USA; 1996. Susceptibility testing of antimicrobials in liquid media; pp. 52–111.
4.Baker CN, Huang MB, Tenover FC. Optimizing testing of methicillin-resistant Staphylococcus species. Diagn Microbiol Infect Dis. 1994;19:167–170. doi: 10.1016/0732-8893(94)90061-2. [PubMed]
5.Bauer AW, Kirby WMM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J of Clinical Path. 1966;45:493–496. [PubMed]
6.Bouman M, Irigoyen D, Bertón A. Analisis de los resultados de 427 muestras remitidas para aislamiento de bacterias de mastitis y antibiograma. (Study of results from 427 milk samples remitted for bacteriologic cultures and susceptibility testing against antimicrobial agents). Jornadas de Salud de Ubre, Nva Helvecia, Uruguay. 1999. pp. 59–68. (In Spanish).
7.Brown MB, Roberts MC. Tetracycline resistance determinants in streptococcal species isolated from the bovine mammary gland. Vet Microbiol. 1991;29:173–180. doi: 10.1016/0378-1135(91)90124-X. [PubMed]
8.Davies J. Inactivation of antibiotics and the dissemination of resistance genes. Science. 1994;264:375–382. doi: 10.1126/science.8153624. [PubMed]
9.Del Baglivi L, Bonilla M, Laborde M. Investigación sobre mastitis subclinica en rodeos lecheros del Uruguay. (Subclinical mastitis research in dairy herds of Uruguay). Medicina-Veterinaria Montevideo, Uruguay. 1976;12:61,69–77. (In Spanish).
10.De Oliveira AP, Watts JL, Salmon SA, Aarestrup FM. Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Europe and United States. J Dairy Sci. 2000;83:855–862. [PubMed]
11.Franklin A. Antibiotic policy and ocurrence of resistance in Sweden. Proceedings of 25th International Dairy Congress, Aarhus, Denmark. 1998. pp. 229–234.
12.Franklin A, Wierup M. Evaluation of the sensititre method adapted for antimicrobial drug susceptibility testing in veterinary medicine. Vet Microbiol. 1982;7:447–454. doi: 10.1016/0378-1135(82)90061-X. [PubMed]
13.Gentilini E, Denamiel G, Llorente P, Godaly S, Rebuelto M, De Gregorio O. Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Argentina. J Dairy Sci. 2000;83:1224–1227. [PubMed]
14.Gentilini E, Denamiel G, Tirante L, Chavez C, Godaly MS. Bovine Mastitis: β-lactamase production. Staphylococcus aureus antibiotic resistance evolution. Proceedings of the 3rd International Mastitis Seminar, Tel Aviv, Israel. 1995;session 2:84–85.
15.Gianneechini R. Thesis. International Master of Science Programme, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala, Sweden; 2001. Occurrence and aetiology of clinical and subclinical mastitis, and antimicrobial susceptibility of udder pathogens in dairy herds in a region of Uruguay; pp. 31–43. ISSN 1403-2201, Report no. 25.
16.Hofshager M, Kruse H, Lassen J, Stavnes T-L, Essun K, Holstad G, Mørk T, Schau J, Grave K. Resistance in bacteria from infections in animals. In: Hilde Kruse, editor. Usage of antimicrobial agents in animals and occurrence of antimicrobal resistance in bacteria from animals, feed, and food in Norway 1999, NORM-VET, Oslo, Norway. 1999. pp. 16–18. ISSN-1502-4695.
17.Herrera B. Etiología de las mastitis subclínicas y estudio de la cuenca lechera de Tacuarembó. (Ethiology of subclinical mastitis and study of the dairy area of Tacuarembó). III Congreso Nacional de Veterinaria, Montevideo, Uruguay. 1982. pp. 495–505. In Spanish.
18.Hussain Z, Stoakes L, Massey V, Diagre D, Fitzgerald V, El Sayed S, Lannigan R. Correlation of oxacillin MIC with mecA gene carriage in Coagulase-negative staphylococci. J Clin Microbiol. 2000;38:752–754. [PubMed]
19.Kiehlbauch JA, Hannett GE, Salfinger M, Archinal W, Monserrat C, Carlyn C. Use of the National Committee for Clinical Laboratory Standards guidelines for disk diffusion susceptibility testing in New York State Laboratories. J Clin Microbiol. 2000;38:3341–3348. [PubMed]
20.Lingaas E. The use of antimicrobials in animal production – a threat to humans? NKVet Symposium, Helsinki, Finland. 1998. pp. 26–27.
21.Milton S. Statistical methods in the biological and health sciences. 2. chapter 8. New York, McGraw-Hill; 1992.
22.Myllys V, Asplund K, Brofeldt E, Hirvelä-Koski V, Honkanen-Buzalske T, Junttila J, Kulkas L, Myllykangas O, Niskanen M, Saloniemi H, Sandholm M, Saranpää T. Bovine mastitis in Finland in 1988 and 1995 – Changes in prevalence and antimicrobial resistance. Acta vet Scand. 1998;39:119–126. [PubMed]
23.Myllys V, Louhi M, Ali-Vehmas T. Comparison of penicillin-G susceptibility testing methods of Staphylococci isolated from bovine mastitis. J Vet Med B. 1992;39:723–731.
24.National Committee of Clinical Laboratories Standards. Performance Standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard. NCCLS. 1999;19:11,60. Document M31-A.
25.National Veterinary Institute. Department of Mastitis, Accreditation Certificate, Uppsala, Sweden. pp. 1–29.
26.Neu HC. The crisis in antibiotic resistance. Science. 1992;257:1064–1073. doi: 10.1126/science.257.5073.1064. [PubMed]
27.OPYPA. Oficina de Planeamiento y Producción Agropecuaria. Ministerio de Ganadería Agricultura y Pesca. Montevideo-Uruguay. 2000.
28.Pyörälä S, Myllys V. Resistance of bacteria to antimicrobials. The bovine udder and mastitis, University of Helsinki, Faculty of Veterinary Medicine, Helsinki, Finland. 1995. pp. 194–200.
29.Prescott JF. Lincosamides, Macrolides, and Pleuromutilins. In: Prescott JF, Baggot JD, Walker RD, editor. Antimicrobial therapy in veterinary medicine. 3. Iowa State University Press, Ames, Iowa, USA; 2000. pp. 229–262.
30.Prescott JF. Aminoglycosides and Aminocyclitols. In: Prescott JF, Bagott JD, Walker RD, editor. Antimicrobial therapy in veterinary medicine. 3. Iowa State University Press, Ames, Iowa.USA; 2000. pp. 191–228.
31.Salmon SA, Watts JL, Aarestrup JW, Yancey RJ., Jr Minimum Inhibitory Concentrations for selected antimicrobial agents against organisms isolated from the mammary glands of dairy heifers in New Zealand and Denmark. J Dairy Sci. 1998;81:570–578. [PubMed]
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