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.
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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.
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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
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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.
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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
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Enterobacter cloaceae
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Enterobacter cloacae :: isolation & purification
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Rev Esp Anestesiol Reanim. 2009 Apr ;56 (4):239-44 19537264
[Severe perioperative thrombocytosis in a patient undergoing lung resection]
B Fernández Torres, E Ramos Martínez, R Rosendo Ríos, R Rodríguez Mejías, A Gutiérrez Guillén, M de las Mulas Béjar
Departamento de Anestesiología y Reanimación, Hospital Virgen Macarena, Sevilla. barfertor@tiscali.es
Severe thrombocytosis (platelet count > 1,000,000 microL(-1)) is a rare, usually reactive, process and few perioperative cases have been reported. We describe the management of a patient who developed severe reactive thrombocytosis in the preoperative period before undergoing segmentectomy to remove a malignant nodule. A platelet count of 2,086,000 microL(-1) was observed during the first few days after surgery; we therefore started antiplatelet therapy to prevent thrombotic complications. We analyze the factors that might have contributed to the development of severe thrombocytosis in this case and discuss the different treatment options that may affect perioperative outcomes in these patients.
Most cited papers:
Proc Natl Acad Sci U S A. 1995 Mar 14;92 (6):2081-5 7892228 Cit:129
FimH adhesin of type 1 pili is assembled into a fibrillar tip structure in the Enterobacteriaceae.
C H Jones, J S Pinkner, R Roth, J Heuser, A V Nicholes, S N Abraham, S J Hultgren
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.
Type 1 pili are heteropolymeric mannosebinding fibers produced by all members of the Enterobacteriaceae family. The bulk of the fiber is composed of FimA. Two macromolecular complexes responsible for mediating an interaction with mannose-containing receptors were purified from fimA- Escherichia coli by mannose affinity chromatography and ion-exchange chromatography. One complex contained only the mannose-binding adhesin, FimH, associated with FimG, a minor component of the type 1 pilus. In the other complex the FimG-FimH moiety was loosely associated with a chaperone-minor subunit complex (FimC-FimF), possibly representing an intermediate in tip fibrilla assembly. The FimC chaperone has also been shown to form a preassembly complex with FimH that has been purified and characterized previously. Purified FimC did not bind to the FimG-FimH complex but did recognize FimH dissociated from the FimG-FimH complex. Quick-freeze deep-etch electron microscopy revealed that the FimG-FimH complex had a thin fibrillar architecture. High-resolution electron microscopy of type 1 pili revealed that a 16-nm fibrillar tip structure with an architecture identical to that of the FimG-FimH complex was joined end-to-end to the pilus rod. In a fimH- deletion mutant, the tip fibrillae joined to pilus rods were approximately 3 nm in length. The full-length tip fibrilla was restored by complementation with the fimH gene in trans. The bipartite nature of the type 1 pilus was also demonstrated on pili purified from clinical isolates of members of the Enterobacteriaceae family arguing that it is a conserved feature of the type 1 pilus.
J Infect Dis. 1995 Sep ;172 (3):886-91 7658090 Cit:57
Antibiotic-induced endotoxin release in patients with gram-negative urosepsis: a double-blind study comparing imipenem and ceftazidime.
J M Prins, M A van Agtmael, E J Kuijper, S J van Deventer, P Speelman
Department of Internal Medicine, Academic Medical Center, Amsterdam, Netherlands.
The clinical significance of differences between antibiotics in endotoxin-liberating potential is unknown. Thirty patients with gram-negative urosepsis were randomized between imipenem and ceftazidime, which have, respectively, a low and a high endotoxin-liberating potential in vitro. In patients treated with ceftazidime, a slower defervescence was noticed. After 4 h of treatment, the blood endotoxin level decreased in all 3 endotoxemic patients receiving imipenem, whereas it increased in 2 of the 4 endotoxemic patients receiving ceftazidime, and in ceftazidime-treated patients, the endotoxin level in urine decreased less than in imipenem-treated subjects. Serum and urine cytokine levels increased 10%-40% after 4 h of ceftazidime treatment compared with no increase in the imipenem-treated patients (P >.05). Endotoxin release during antibiotic killing in vitro, assessed for all microorganisms, was 10-fold higher with ceftazidime (P <.001). These results indicate that differences between antibiotics in endotoxin release may affect the inflammatory response during treatment.
J Clin Microbiol. 2002 Apr ;40 (4):1237-43 11923338 Cit:47
Epidemiology of extended-spectrum beta-lactamase-producing Enterobacter isolates in a Spanish hospital during a 12-year period.
Rafael Cantón, Antonio Oliver, Teresa M Coque, María del Carmen Varela, José Claudio Pérez-Díaz, Fernando Baquero
Servicio de Microbiología Hospital Ramón y Cajal, Madrid, Spain. rcanton@hrc.insalud.es
Fifteen Enterobacter clinical isolates (11 Enterobacter cloacae isolates, 3 Enterobacter aerogenes isolates, and 1 Enterobacter gergoviae isolate), representing 0.4% of all Enterobacter isolates recovered in our hospital from 1989 to 2000, were suspected of harboring an extended-spectrum beta-lactamase (ESBL). These isolates were recovered from 14 different patients. ESBLs were transferred by conjugation into an Escherichia coli recipient strain. Pulsed-field gel electrophoresis (PFGE) revealed a single clone of E. aerogenes and six different clones of E. cloacae. Four of these E. cloacae clonal types were represented by only one isolate each, but the other two were represented by three and four isolates, respectively. Isoelectric focusing, susceptibility phenotyping, PCR analysis, and sequencing demonstrated the presence of three different ESBLs. The most frequent was the recently characterized CTX-M-10 ESBL, which was found in the E. gergoviae isolate and in all but one of the E. cloacae isolates. The remaining E. cloacae isolate harbored a TEM-27 ESBL, and the three E. aerogenes isolates harbored a TEM-24 ESBL. PFGE revealed that our E. aerogenes strain was indistinguishable from the French TEM-24-producing E. aerogenes endemic clone. Although a low prevalence of ESBL-producing Enterobacter isolates was found in our institution over a 12-year period, a diversity of nonepidemic E. cloacae clones was detected, as was the persistence of the CTX-M-10 beta-lactamase. The presence of the TEM-24-producing E. aerogenes French clone in our institution also demonstrates the intercountry dissemination of ESBL-producing isolates.
J Clin Microbiol. 1994 Mar ;32 (3):596-602 8195364 Cit:34
Arbitrarily primed PCR, ribotyping, and plasmid pattern analysis applied to investigation of a nosocomial outbreak due to Enterobacter cloacae in a neonatal intensive care unit.
F Grattard, B Pozzetto, P Berthelot, I Rayet, A Ros, B Lauras, O G Gaudin
Laboratoire de Bactériologie-Virologie, Faculté de Médicine J. Lisfranc, Saint-Etienne, France.
In December 1992, Enterobacter cloacae was isolated from the oropharynx and respiratory tract of six ventilated neonates hospitalized in the intensive care unit (ICU) of our hospital. To establish the spread of the outbreak, 41 strains of E. cloacae were analyzed for genotypic markers by three methods: plasmid profile analysis, ribotyping with EcoRI or PvuII endonuclease, and arbitrarily primed (AP) PCR. The tested strains included 12 isolates from the 6 epidemic cases, 4 isolates from the respiratory tract of 4 children hospitalized in other wards during the same period, 13 isolates from 12 children hospitalized in pediatric units before or after the outbreak, and 12 epidemiologically unrelated isolates. Ribotyping and AP PCR demonstrated that each of the last 12 strains exhibited distinct genomic patterns, as did each of the strains isolated from neonates hospitalized before or after the epidemic peak. Conversely, two clones of strains were found among the isolates recovered in December, with concordant results being obtained by the three typing methods: the first clone included seven strains from five ventilated children in the ICU and two children from another ward; another clone was shared by one neonate in the ICU and an infant from another ward. These results indicate that ribotyping and AP PCR-the latter applied, to our knowledge, for the first time to the genotypic analysis of E. cloacae--represent very discriminatory tools for the investigation of nosocomial outbreaks caused by this species.
J Hosp Infect. 1994 Dec ;28 (4):273-86 7897189 Cit:31
Enterobacter cloacae in a neonatal intensive care unit: account of an outbreak and its relationship to use of third generation cephalosporins.
D Acolet, Z Ahmet, E Houang, R Hurley, M E Kaufmann
Hillingdon Hospital, London, UK.
After uneventful use of cefotaxime and ceftazidime as first line therapy for three years in our neonatal intensive care unit we isolated cephalosporin-resistant Enterobacter cloacae (CREC) strains which caused clusters of cases or colonization and/or serious neonatal infection. By using two or more typing methods, at least five different strains with similar patterns of antimicrobial sensitivities were identified. The results of a case-control study did not support the notion that the use of third generation cephalosporins was associated with colonization and infection by CREC. The outbreak was brought under control by interrupting the transmission of the epidemic strain D, by measures such as cohort nursing, diligent handwashing before and after procedures, and thorough environmental cleaning as well as by decontamination with glutaraldehyde after dismantling of the blood gas analyser believed to have acted as a persistent reservoir. Our experience highlights the danger of inadequate supervision and maintenance of equipment used for near-patient testing and the need to monitor such equipment not only in terms of its calibration and analytical performance but also microbiologically.
J Biol Chem. 1995 Mar 17;270 (11):5729-35 7890700 Cit:29
Molecular evolution of a class C beta-lactamase extending its substrate specificity.
M Nukaga, S Haruta, K Tanimoto, K Kogure, K Taniguchi, M Tamaki, T Sawai
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Enterobacter cloacae GC1, a clinical strain isolated in 1992 in Japan, was found to produce a chromosomal class C beta-lactamase with extended substrate specificity to oxyimino beta-lactam antibiotics, significantly differing from the known E. cloacae beta-lactamases such as the P99 beta-lactamase. The 1560 nucleotides including the GC1 beta-lactamase gene were sequenced, and the amino acid sequence of the mature enzyme comprising 364 amino acids was deduced. A comparison of the amino acid sequence with those of known E. cloacae beta-lactamases revealed the duplication of three amino acids at positions 208-213, i.e. Ala-Val-Arg-Ala-Val-Arg. This duplication was attributed to a tandem duplication of a 9-nucleotide sequence. The chimeric beta-lactamases produced by the chimeric genes from the GC1 and P99 beta-lactamase genes indicated that the extended substrate specificity is entirely attributed to the 3-amino acid insertion. Two mutant beta-lactamases were prepared from P99 beta-lactamase by site-directed mutagenesis, i.e. an Ala-Ala-Ala sequence was inserted before or after the native Ala-Val-Arg at positions 208-210. These mutant enzymes revealed that the Ala-Val-Arg located from positions 211 to 213 in the GC1 beta-lactamase are the newly inserted residues, and this phenomenon is independent of the characteristics of the amino acids inserted.
Clin Infect Dis. 1992 Jul ;15 (1):30-2 1352150 Cit:26
Molecular analysis provides evidence for the endogenous origin of bacteremia and meningitis due to Enterobacter cloacae in an infant.
N Lambert-Zechovsky, E Bingen, E Denamur, N Brahimi, P Brun, H Mathieu, J Elion
Laboratoire de Microbiologie, Hôpital Robert Debré, Paris, France.
We analyzed the restriction fragment length polymorphism (RFLP) of total DNA and of ribosomal DNA regions (ribotyping) to document the occurrence of endogenous, systemic bacteremia and meningitis due to Enterobacter cloacae in a newborn. Five strains of E. cloacae were isolated from this newborn. Three of these strains were recovered from stool at counts of 10(8), 10(9), and 10(9) organisms/g of feces, respectively; one strain was isolated from blood; and one strain was isolated from cerebrospinal fluid. In addition, five epidemiologically unrelated strains of E. cloacae were studied for comparison. Our study clearly shows the genetic relatedness of the strains isolated sequentially from cultures of stool, blood, and cerebrospinal fluid. RFLP analysis of total DNA and ribotyping seem particularly well suited to the study of the epidemiology of nosocomial E. cloacae strains.
J Hosp Infect. 1992 Jun ;21 (2):95-101 1353097 Cit:24
Rapid genotyping shows the absence of cross-contamination in Enterobacter cloacae nosocomial infections.
E Bingen, E Denamur, N Lambert-Zechovsky, N Brahimi, M el Lakany, J Elion
Laboratoire de Bactériologie, Hôpital Robert Debré, Paris, France.
Restriction fragment length polymorphism analysis (RFLP) of total DNA and rDNA regions was used for the epidemiological evaluation of 10 Enterobacter cloacae nosocomial isolates obtained from nine patients in our hospital. Five of these patients were hospitalized during overlapping periods, thus raising the question of cross-contamination. A single biochemical pattern and antibiotic susceptibility profile was observed for all isolates but one. In contrast, based on the results of total DNA and rDNA RFLP patterns, the genetic unrelatedness of the isolates was clearly shown, thus excluding a common source of contamination or patient-to-patient transfer.
J Antimicrob Chemother. 2002 Oct ;50 (4):503-11 12356794 Cit:21
Metallo-beta-lactamase-producing Enterobacteriaceae isolates in a university hospital in Taiwan: prevalence of IMP-8 in Enterobacter cloacae and first identification of VIM-2 in Citrobacter freundii.
Jing-Jou Yan, Wen-Chien Ko, Chin-Luan Chuang, Jiunn-Jong Wu
Department of Pathology, College of Medicine, National Cheng Kung University, No. 1 University Road, Tainan 70101, Taiwan.
A total of 9082 clinical isolates of Enterobacteriaceae other than Klebsiella spp. collected in 1999 and 2000 at a university hospital in Taiwan were investigated for the production of metallo- beta-lactamases (MBLs). Thirty-six (2.9%) of the 1261 Enterobacter cloacae isolates and one (0.3%) of the 340 Citrobacter freundii isolates were found to carry bla(IMP-8) and bla(VIM-2), respectively, by colony hybridization, PCR and sequence analysis. The IMP-8 producers were recovered from 20 patients and four of them had recently transferred from other hospitals, implying spread of IMP-8-producing E. cloacae among different healthcare settings. Of the 20 non-repetitive IMP-8 producers, 17 (85%) isolates also harboured bla(SHV-12), which was on the same transferable plasmids with bla(IMP-8). The bla(VIM-2)-positive isolate and all non-repetitive bla(IMP-8)-positive isolates appeared susceptible to imipenem (MICs < 8 mg/L) and meropenem (MICs < 4 mg/L), indicating the difficulty in detection of MBLs in Enterobacteriaceae by routine susceptibility testing. Ribotyping of the IMP-8-producing E. cloacae isolates indicated that the dissemination of bla(IMP-8) was due largely to the spread of an epidemic clone, but horizontal transfer among unrelated strains also occurred.
Antimicrob Agents Chemother. 1999 Aug ;43 (8):2051-5 10428935 Cit:21
Comparative in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin against Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes clinical isolates with alterations in GyrA and ParC proteins.
S Brisse, D Milatovic, A C Fluit, J Verhoef, N Martin, S Scheuring, K Köhrer, F J Schmitz
Eijkman-Winkler Institute, Utrecht University, 3584 CX, Utrecht, The Netherlands. sbrisse@lab.azu.nl
The in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin were tested against 72 ciprofloxacin-resistant and 28 ciprofloxacin-susceptible isolates of Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes. Irrespective of the alterations in GyrA and ParC proteins, clinafloxacin exhibited greater activity than all other fluoroquinolones tested against K. pneumoniae and E. aerogenes.
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Papers 148Most cited
Enterobacter cloacae :: isolation & purification
Latest Paper:
Rev Esp Anestesiol Reanim. 2009 Apr ;56 (4):239-44 19537264
[Severe perioperative thrombocytosis in a patient undergoing lung resection]
B Fernández Torres, E Ramos Martínez, R Rosendo Ríos, R Rodríguez Mejías, A Gutiérrez Guillén, M de las Mulas Béjar
Departamento de Anestesiología y Reanimación, Hospital Virgen Macarena, Sevilla. barfertor@tiscali.es
Severe thrombocytosis (platelet count > 1,000,000 microL(-1)) is a rare, usually reactive, process and few perioperative cases have been reported. We describe the management of a patient who developed severe reactive thrombocytosis in the preoperative period before undergoing segmentectomy to remove a malignant nodule. A platelet count of 2,086,000 microL(-1) was observed during the first few days after surgery; we therefore started antiplatelet therapy to prevent thrombotic complications. We analyze the factors that might have contributed to the development of severe thrombocytosis in this case and discuss the different treatment options that may affect perioperative outcomes in these patients.
Most cited papers:
Proc Natl Acad Sci U S A. 1995 Mar 14;92 (6):2081-5 7892228 Cit:129
FimH adhesin of type 1 pili is assembled into a fibrillar tip structure in the Enterobacteriaceae.
C H Jones, J S Pinkner, R Roth, J Heuser, A V Nicholes, S N Abraham, S J Hultgren
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110.
Type 1 pili are heteropolymeric mannosebinding fibers produced by all members of the Enterobacteriaceae family. The bulk of the fiber is composed of FimA. Two macromolecular complexes responsible for mediating an interaction with mannose-containing receptors were purified from fimA- Escherichia coli by mannose affinity chromatography and ion-exchange chromatography. One complex contained only the mannose-binding adhesin, FimH, associated with FimG, a minor component of the type 1 pilus. In the other complex the FimG-FimH moiety was loosely associated with a chaperone-minor subunit complex (FimC-FimF), possibly representing an intermediate in tip fibrilla assembly. The FimC chaperone has also been shown to form a preassembly complex with FimH that has been purified and characterized previously. Purified FimC did not bind to the FimG-FimH complex but did recognize FimH dissociated from the FimG-FimH complex. Quick-freeze deep-etch electron microscopy revealed that the FimG-FimH complex had a thin fibrillar architecture. High-resolution electron microscopy of type 1 pili revealed that a 16-nm fibrillar tip structure with an architecture identical to that of the FimG-FimH complex was joined end-to-end to the pilus rod. In a fimH- deletion mutant, the tip fibrillae joined to pilus rods were approximately 3 nm in length. The full-length tip fibrilla was restored by complementation with the fimH gene in trans. The bipartite nature of the type 1 pilus was also demonstrated on pili purified from clinical isolates of members of the Enterobacteriaceae family arguing that it is a conserved feature of the type 1 pilus.
J Infect Dis. 1995 Sep ;172 (3):886-91 7658090 Cit:57
Antibiotic-induced endotoxin release in patients with gram-negative urosepsis: a double-blind study comparing imipenem and ceftazidime.
J M Prins, M A van Agtmael, E J Kuijper, S J van Deventer, P Speelman
Department of Internal Medicine, Academic Medical Center, Amsterdam, Netherlands.
The clinical significance of differences between antibiotics in endotoxin-liberating potential is unknown. Thirty patients with gram-negative urosepsis were randomized between imipenem and ceftazidime, which have, respectively, a low and a high endotoxin-liberating potential in vitro. In patients treated with ceftazidime, a slower defervescence was noticed. After 4 h of treatment, the blood endotoxin level decreased in all 3 endotoxemic patients receiving imipenem, whereas it increased in 2 of the 4 endotoxemic patients receiving ceftazidime, and in ceftazidime-treated patients, the endotoxin level in urine decreased less than in imipenem-treated subjects. Serum and urine cytokine levels increased 10%-40% after 4 h of ceftazidime treatment compared with no increase in the imipenem-treated patients (P >.05). Endotoxin release during antibiotic killing in vitro, assessed for all microorganisms, was 10-fold higher with ceftazidime (P <.001). These results indicate that differences between antibiotics in endotoxin release may affect the inflammatory response during treatment.
J Clin Microbiol. 2002 Apr ;40 (4):1237-43 11923338 Cit:47
Epidemiology of extended-spectrum beta-lactamase-producing Enterobacter isolates in a Spanish hospital during a 12-year period.
Rafael Cantón, Antonio Oliver, Teresa M Coque, María del Carmen Varela, José Claudio Pérez-Díaz, Fernando Baquero
Servicio de Microbiología Hospital Ramón y Cajal, Madrid, Spain. rcanton@hrc.insalud.es
Fifteen Enterobacter clinical isolates (11 Enterobacter cloacae isolates, 3 Enterobacter aerogenes isolates, and 1 Enterobacter gergoviae isolate), representing 0.4% of all Enterobacter isolates recovered in our hospital from 1989 to 2000, were suspected of harboring an extended-spectrum beta-lactamase (ESBL). These isolates were recovered from 14 different patients. ESBLs were transferred by conjugation into an Escherichia coli recipient strain. Pulsed-field gel electrophoresis (PFGE) revealed a single clone of E. aerogenes and six different clones of E. cloacae. Four of these E. cloacae clonal types were represented by only one isolate each, but the other two were represented by three and four isolates, respectively. Isoelectric focusing, susceptibility phenotyping, PCR analysis, and sequencing demonstrated the presence of three different ESBLs. The most frequent was the recently characterized CTX-M-10 ESBL, which was found in the E. gergoviae isolate and in all but one of the E. cloacae isolates. The remaining E. cloacae isolate harbored a TEM-27 ESBL, and the three E. aerogenes isolates harbored a TEM-24 ESBL. PFGE revealed that our E. aerogenes strain was indistinguishable from the French TEM-24-producing E. aerogenes endemic clone. Although a low prevalence of ESBL-producing Enterobacter isolates was found in our institution over a 12-year period, a diversity of nonepidemic E. cloacae clones was detected, as was the persistence of the CTX-M-10 beta-lactamase. The presence of the TEM-24-producing E. aerogenes French clone in our institution also demonstrates the intercountry dissemination of ESBL-producing isolates.
J Clin Microbiol. 1994 Mar ;32 (3):596-602 8195364 Cit:34
Arbitrarily primed PCR, ribotyping, and plasmid pattern analysis applied to investigation of a nosocomial outbreak due to Enterobacter cloacae in a neonatal intensive care unit.
F Grattard, B Pozzetto, P Berthelot, I Rayet, A Ros, B Lauras, O G Gaudin
Laboratoire de Bactériologie-Virologie, Faculté de Médicine J. Lisfranc, Saint-Etienne, France.
In December 1992, Enterobacter cloacae was isolated from the oropharynx and respiratory tract of six ventilated neonates hospitalized in the intensive care unit (ICU) of our hospital. To establish the spread of the outbreak, 41 strains of E. cloacae were analyzed for genotypic markers by three methods: plasmid profile analysis, ribotyping with EcoRI or PvuII endonuclease, and arbitrarily primed (AP) PCR. The tested strains included 12 isolates from the 6 epidemic cases, 4 isolates from the respiratory tract of 4 children hospitalized in other wards during the same period, 13 isolates from 12 children hospitalized in pediatric units before or after the outbreak, and 12 epidemiologically unrelated isolates. Ribotyping and AP PCR demonstrated that each of the last 12 strains exhibited distinct genomic patterns, as did each of the strains isolated from neonates hospitalized before or after the epidemic peak. Conversely, two clones of strains were found among the isolates recovered in December, with concordant results being obtained by the three typing methods: the first clone included seven strains from five ventilated children in the ICU and two children from another ward; another clone was shared by one neonate in the ICU and an infant from another ward. These results indicate that ribotyping and AP PCR-the latter applied, to our knowledge, for the first time to the genotypic analysis of E. cloacae--represent very discriminatory tools for the investigation of nosocomial outbreaks caused by this species.
J Hosp Infect. 1994 Dec ;28 (4):273-86 7897189 Cit:31
Enterobacter cloacae in a neonatal intensive care unit: account of an outbreak and its relationship to use of third generation cephalosporins.
D Acolet, Z Ahmet, E Houang, R Hurley, M E Kaufmann
Hillingdon Hospital, London, UK.
After uneventful use of cefotaxime and ceftazidime as first line therapy for three years in our neonatal intensive care unit we isolated cephalosporin-resistant Enterobacter cloacae (CREC) strains which caused clusters of cases or colonization and/or serious neonatal infection. By using two or more typing methods, at least five different strains with similar patterns of antimicrobial sensitivities were identified. The results of a case-control study did not support the notion that the use of third generation cephalosporins was associated with colonization and infection by CREC. The outbreak was brought under control by interrupting the transmission of the epidemic strain D, by measures such as cohort nursing, diligent handwashing before and after procedures, and thorough environmental cleaning as well as by decontamination with glutaraldehyde after dismantling of the blood gas analyser believed to have acted as a persistent reservoir. Our experience highlights the danger of inadequate supervision and maintenance of equipment used for near-patient testing and the need to monitor such equipment not only in terms of its calibration and analytical performance but also microbiologically.
J Biol Chem. 1995 Mar 17;270 (11):5729-35 7890700 Cit:29
Molecular evolution of a class C beta-lactamase extending its substrate specificity.
M Nukaga, S Haruta, K Tanimoto, K Kogure, K Taniguchi, M Tamaki, T Sawai
Division of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Enterobacter cloacae GC1, a clinical strain isolated in 1992 in Japan, was found to produce a chromosomal class C beta-lactamase with extended substrate specificity to oxyimino beta-lactam antibiotics, significantly differing from the known E. cloacae beta-lactamases such as the P99 beta-lactamase. The 1560 nucleotides including the GC1 beta-lactamase gene were sequenced, and the amino acid sequence of the mature enzyme comprising 364 amino acids was deduced. A comparison of the amino acid sequence with those of known E. cloacae beta-lactamases revealed the duplication of three amino acids at positions 208-213, i.e. Ala-Val-Arg-Ala-Val-Arg. This duplication was attributed to a tandem duplication of a 9-nucleotide sequence. The chimeric beta-lactamases produced by the chimeric genes from the GC1 and P99 beta-lactamase genes indicated that the extended substrate specificity is entirely attributed to the 3-amino acid insertion. Two mutant beta-lactamases were prepared from P99 beta-lactamase by site-directed mutagenesis, i.e. an Ala-Ala-Ala sequence was inserted before or after the native Ala-Val-Arg at positions 208-210. These mutant enzymes revealed that the Ala-Val-Arg located from positions 211 to 213 in the GC1 beta-lactamase are the newly inserted residues, and this phenomenon is independent of the characteristics of the amino acids inserted.
Clin Infect Dis. 1992 Jul ;15 (1):30-2 1352150 Cit:26
Molecular analysis provides evidence for the endogenous origin of bacteremia and meningitis due to Enterobacter cloacae in an infant.
N Lambert-Zechovsky, E Bingen, E Denamur, N Brahimi, P Brun, H Mathieu, J Elion
Laboratoire de Microbiologie, Hôpital Robert Debré, Paris, France.
We analyzed the restriction fragment length polymorphism (RFLP) of total DNA and of ribosomal DNA regions (ribotyping) to document the occurrence of endogenous, systemic bacteremia and meningitis due to Enterobacter cloacae in a newborn. Five strains of E. cloacae were isolated from this newborn. Three of these strains were recovered from stool at counts of 10(8), 10(9), and 10(9) organisms/g of feces, respectively; one strain was isolated from blood; and one strain was isolated from cerebrospinal fluid. In addition, five epidemiologically unrelated strains of E. cloacae were studied for comparison. Our study clearly shows the genetic relatedness of the strains isolated sequentially from cultures of stool, blood, and cerebrospinal fluid. RFLP analysis of total DNA and ribotyping seem particularly well suited to the study of the epidemiology of nosocomial E. cloacae strains.
J Hosp Infect. 1992 Jun ;21 (2):95-101 1353097 Cit:24
Rapid genotyping shows the absence of cross-contamination in Enterobacter cloacae nosocomial infections.
E Bingen, E Denamur, N Lambert-Zechovsky, N Brahimi, M el Lakany, J Elion
Laboratoire de Bactériologie, Hôpital Robert Debré, Paris, France.
Restriction fragment length polymorphism analysis (RFLP) of total DNA and rDNA regions was used for the epidemiological evaluation of 10 Enterobacter cloacae nosocomial isolates obtained from nine patients in our hospital. Five of these patients were hospitalized during overlapping periods, thus raising the question of cross-contamination. A single biochemical pattern and antibiotic susceptibility profile was observed for all isolates but one. In contrast, based on the results of total DNA and rDNA RFLP patterns, the genetic unrelatedness of the isolates was clearly shown, thus excluding a common source of contamination or patient-to-patient transfer.
J Antimicrob Chemother. 2002 Oct ;50 (4):503-11 12356794 Cit:21
Metallo-beta-lactamase-producing Enterobacteriaceae isolates in a university hospital in Taiwan: prevalence of IMP-8 in Enterobacter cloacae and first identification of VIM-2 in Citrobacter freundii.
Jing-Jou Yan, Wen-Chien Ko, Chin-Luan Chuang, Jiunn-Jong Wu
Department of Pathology, College of Medicine, National Cheng Kung University, No. 1 University Road, Tainan 70101, Taiwan.
A total of 9082 clinical isolates of Enterobacteriaceae other than Klebsiella spp. collected in 1999 and 2000 at a university hospital in Taiwan were investigated for the production of metallo- beta-lactamases (MBLs). Thirty-six (2.9%) of the 1261 Enterobacter cloacae isolates and one (0.3%) of the 340 Citrobacter freundii isolates were found to carry bla(IMP-8) and bla(VIM-2), respectively, by colony hybridization, PCR and sequence analysis. The IMP-8 producers were recovered from 20 patients and four of them had recently transferred from other hospitals, implying spread of IMP-8-producing E. cloacae among different healthcare settings. Of the 20 non-repetitive IMP-8 producers, 17 (85%) isolates also harboured bla(SHV-12), which was on the same transferable plasmids with bla(IMP-8). The bla(VIM-2)-positive isolate and all non-repetitive bla(IMP-8)-positive isolates appeared susceptible to imipenem (MICs < 8 mg/L) and meropenem (MICs < 4 mg/L), indicating the difficulty in detection of MBLs in Enterobacteriaceae by routine susceptibility testing. Ribotyping of the IMP-8-producing E. cloacae isolates indicated that the dissemination of bla(IMP-8) was due largely to the spread of an epidemic clone, but horizontal transfer among unrelated strains also occurred.
Antimicrob Agents Chemother. 1999 Aug ;43 (8):2051-5 10428935 Cit:21
Comparative in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin against Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes clinical isolates with alterations in GyrA and ParC proteins.
S Brisse, D Milatovic, A C Fluit, J Verhoef, N Martin, S Scheuring, K Köhrer, F J Schmitz
Eijkman-Winkler Institute, Utrecht University, 3584 CX, Utrecht, The Netherlands. sbrisse@lab.azu.nl
The in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin were tested against 72 ciprofloxacin-resistant and 28 ciprofloxacin-susceptible isolates of Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes. Irrespective of the alterations in GyrA and ParC proteins, clinafloxacin exhibited greater activity than all other fluoroquinolones tested against K. pneumoniae and E. aerogenes.
Science news
Vital Signs:
Childhood: 1 in 4 Parents Link Autism to Vaccines
feeds.nytimes.com
NASA's Fermi
probes 'dragons' of the gamma-ray sky
feeds.sciencedaily.com
Q&A: Making
Movies in Windows 7
feeds.nytimes.com
Three-way swap
news.bbc.co.uk
iPhone
Addictive, Survey Reveals
feeds.livescience.com
Genome-wide
study of alcohol dependence points to chromosome 11
feeds.sciencedaily.com
Drinkers 'may
gain less weight'
news.bbc.co.uk
More news
© BioInfoBank Institute
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