Antimicrobial Susceptibility of Udder Pathogens Isolated from Dairy Herds in the West Littoral Region of Uruguay
RE Gianneechini,1 C Concha,2 and A Franklin3
1Department of Veterinary Microbiology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala, Sweden
2Department of Mastitis and Diagnostical Products, Uppsala, Sweden
3Department of Antibiotics, National Veterinary Institute, Uppsala, Sweden
Corresponding author.
Received March 9, 2001; Accepted November 12, 2001.
Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesAbstract
A total of 522 strains belonging to streptococci, enterococci and staphylococci isolated from sub-clinical and clinical cases of bovine mastitis from the west littoral region of Uruguay were analysed for their susceptibility to several antimicrobial agents. The susceptibility patterns were studied by agar disk diffusion methods (ADDM) and broth micro-dilution to determine the minimum inhibitory concentration (MIC). The concentration that inhibits 90% (MIC90) of the analysed strains reported in micrograms per millilitre, for Staphylococcus aureus were > 8, 8, ≤ 0.5, ≤ 4, ≤ 1, ≤ 0.5, > 64, ≤ 0.25, 0.5, ≤ 1 and ≤ 1 to penicillin, ampicillin, oxacillin, cephalotin, gentamicin, erythromycin, oxitetracycline, enrofloxacin, trimethoprim/sulfamethoxazole, neomycin, and clindamycin, respectively. Coagulase-negative staphylococci (CNS) had different values for penicillin (4) and ampicillin (2), while the other antimicrobial agents had the same MIC90 values as reported for S. aureus. The MIC90 values for streptococci were 0.12, 0.25, ≤ 4, 16, ≤ 0.25, 0.5, 0.25 for penicillin, ampicillin, cephalotin, gentamicin, erythromycin, oxytetracycline and trimethoprim-sulfamethoxazole, whereas MIC90 for enterococci were 4, 4, 4, ≤ 0.5, 2, > 8 for penicillin, ampicillin, gentamicin, erythromycin, oxytetracycline and trimethoprim-sulfamethoxazole, respectively. Of 336 strains of S. aureus, 160 (47.6%) were resistant to penicillin. For 41 CNS strains, 10 (27%) presented penicillin-resistance. All the streptococcal strains were susceptible to penicillin, while 3 (7%) of the 43 enteroccocal strains were resistant. Non significant statistical differences were found between the results obtained by ADDM and broth micro-dilution for classifying bacterial isolates as susceptible or resistant according to the National Committee of Clinical Laboratory Standards.Keywords: cow, mammary qland, bacteria, resistant, sensitive Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesIntroduction
Bovine mastitis is the major problem for milk producers throughout the world and responsible for substantial losses of revenue annually. Antibiotic therapy is an important tool in the scheme of mastitis control. The treatments are more effective when directed by veterinarians; for example correct drug selection can be enhanced using an appropriate antimicrobial susceptibility test. The misuse or intensive use of antibiotics can lead to the development of resistance among different bacterial strains and contamination of foodstuff, with animal and human health implications [20]. The antimicrobial resistance is the result of mutations or exchange of genetic material such as plasmids and transposons [26]. Such resistance determinants most probably are acquired by pathogenic bacteria from a pool of resistance genes in other microbial genera present in different environments [8]. Increased resistance of Staphylococcus aureus and coagulase-negative staphylococci (CNS) isolated from bovine mastitis cases to antimicrobial agents has been reported by [14,1] and [22].Milk production in Uruguay (South America) is important with a total of 410.000 dairy cows, yielding 1462 millions litres in 1999 [27]. In spite of the importance of this sector, only 3 surveys to evaluate the resistance of udder pathogens to antibiotics have been performed in Uruguay using agar disk diffusion (ADDM, [5]): 1) [9] testing S. aureus and Streptococcus agalactiae isolated from subclinical cases obtained from 43 dairy farms in the southern dairy region of Uruguay showed that 53% of S. aureus and 100% of Str. agalactiae were sensitive to penicillin. 2) [17] found 78% of S. aureus strains susceptible to penicillin in the dairy area around Tacuarembó city (north of Uruguay). 3) [6] studied the resistance patterns of S. aureus and CNS isolated in the laboratory routine during 4 years from milk samples collected in the southwestern region of Uruguay for penicillin, cloxacillin, nafcillin, rifampin and tetracycline obtaining: 58%, 16%, 5%, 6%, 29% of resistance for S.aureus and 75%, 42%, 17%, 12%, 26% for CNS, respectively.The sale of antibiotics is free in Uruguay, while the mastitis treatment is usually performed by the herd dairyman, and the antimicrobial agents most commonly used are tetracyclines, beta-lactams, macrolides, and aminoglycosides.The methods for susceptibility testing used to choose the appropriate drug are ADDM qualitative test and quantitative determinations by means of micro-dilution to determine the minimum inhibitory concentration (MIC) [3,2]. These methods can be interpreted following the National Committee for Clinical Laboratory Standards criteria [24] or guidelines proposed by other national antibiogram committees [2].The purposes of this work were: To determine the phenotypic expression of in vitro susceptibility of antimicrobials for pathogens (staphylococci, streptococci, and enterococci) isolated from dairy herds in Uruguay, and to compare the results obtained by the ADDM vs. broth micro-dilution method according to the NCCLS criteria. Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesMaterials and methods
Sample
A total of 522 strains including streptococci, enterococci and staphylococci were used in the study. The strains were isolated from sub-clinical and clinical cases of bovine mastitis from a survey carried out in the west littoral region of Uruguay [15], where quarter foremilk samples from 1077 milking cows and 40 milk samples from clinical cases detected in one month were collected in 29 randomly selected dairy farms. All strains were identified according to the procedures of the laboratory at the Department of Mastitis and Diagnostical Products, National Veterinary Institute (SVA), Uppsala, Sweden [25]. The isolates were maintained frozen at -20°C in Trypticase soy broth (Difco Laboratories, Michigan, USA) containing 10% glycerol until testing.
Susceptibility testing
Prior to the susceptibility testing all isolates were sub-cultured on Blood-esculin agar and incubated for 24 h at 37°C. Two different tests were carried out to determine the drug susceptibility for all strains:
1 – The ADDM was conducted and interpreted according to the recommendations and criteria of the NCCLS for bacteria isolated from animals [24]. The following disks (Becton Dickinson Microbiology System, Cockeysville, Maryland, USA) were used: penicillin, 10 μg; ampicillin, 10 μg; oxacillin, 1 μg; amoxicillin – clavulanic acid, 20 μg + 10 μg; cephalotin, 30 μg; gentamicin, 10 μg; erythromycin, 15 μg; enrofloxacin, 5 μg; tetracycline, 30 μg; neomycin, 30 μg; trimethoprim-sulfamethoxazole, 1.25 μg + 23.75 μg.
The staphylococci were tested against all the drugs above, while the streptococci against only 6 of these antimicrobial agents (penicillin, ampicillin, cephalotin, gentamicin, erythromycin and tetracycline), and enterococci against penicillin, ampicillin, gentamicin, erythromycin and tetracycline. The medium used was Mueller-Hinton Agar (Difco Laboratories, Detroit, USA) for sthaphylococci and Mueller-Hinton agar supplemented with 5% sheep blood for streptococci. S. aureus ATCC 25923, E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were included as quality control strains. The plates were read after 18 h incubation at 37°C under aerobic conditions. The isolates were categorised as susceptible, intermediate and resistant by measuring the inhibition zone.
2 – The MIC was determined using a commercially available micro-dilution system (VetMIC™ +/- panels, SVA, Uppsala, Sweden). The tests were performed by manufacturer's instruction and interpreted according to international standards [24] using Mueller-Hinton broth (Oxoid Limited, Basingstoke Hants, England) and S. aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and E. coli ATCC 25922, as quality control strains.
When the streptococcal strains were tested, 100 μl were inoculated in each well with erythromycin to obtain the following dilution: 0.25; 0.5; 1 and 2 μg/ml of the antimicrobial agent. These modifications were carried out to adapt to the breakpoints suggested by [24] for erythromycin. All panels were read on the same conditions as in the ADDM. The lowest dilution with no visible growth was considered as MIC for each strain. The concentration at which 50% and 90% of the isolates were inhibited, as well as the minimum and maximum range were determined.
The breakpoints suggested by the [24] for kanamycin were used for neomycin in both tests.
Oxacillin resistance testing
In order to confirm the presence of oxacillin resistance among staphylococci, VetMIC™ GP_mo panels (SVA, Uppsala, Sweden) were used as recommended by [24]. The procedures were conducted following the manufacturer's recommendations: the inoculum was prepared with colony material directly from the plate incubated 24 h before. A 1 μl loop with colony material was suspended in 4 ml of distilled water plus 0.02% Twin 80. From this suspension 100 μl were transferred to 10 ml Mueller Hinton Broth + 2% NaCl [4], which achieved about 103 to 104 cfu/50 μl. Each oxacillin and control well of the panel was inoculated with 50 μl of this final bacterial suspension. The panel was incubated at 30°C during 24 h under aerobic conditions. The strains S. aureus ATCC 29886 and S. aureus ATCC 29887 were included as negative and positive control strains, respectively.
β-Lactamase Testing (Cloverleaf Method)
The assay to determine the production of β-lactamase by staphylococci was described previously by [12]. Briefly, the non-β-lactamase-producing S. aureus Oxford strain 209 is used as indicator. This strain is inoculated on PDM II agar plates (AB Biodisk, Solna, Sweden) to yield an almost confluent growth on the agar surface. In the centre of the agar plate a disk containing 10 μg of penicillin G (PDM Antibiotics Sensitive II, AB Biodisk) is placed in order to induce β-lactamase production in the studied strain. The staphylococci to be tested were streaked in a line from the edge of the plate towards the centre of the penicillin disk. When the investigated strain was positive β-lactamase producer, the indicator strain grew alongside this strain towards the penicillin disk, into the inhibited one. The S. aureus strains ATCC 29213 and ATCC 25923 were included as positive and negative control respectively.
Statistics analyses
The Z-test [21] was performed to compare the proportions of resistant strains to each antimicrobial agents obtained by means of both test.
Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesResults
All values obtained with control strains in both tests were within the expected ranges for all antimicrobial agents analysed. The ranges of MIC of each of the antimicrobial agents tested, MIC50 and MIC90 of the tested strains, and the percentage of resistance obtained by both micro-dilutions and ADDM are presented here for S. aureus (Table 1), CNS (Table 2), Str. agalactiae (Table 3), Streptococcus dysgalactiae (Table 4), Streptococcus uberis (Table 5) and Enterococcus sp (Table 6). Table 1
In vitro susceptibility of 336 strains of Staphylococcus aureus obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
Table 2
In vitro susceptibility of 41 strains of Coagulase Negative Staphylococcus obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
Table 3
In vitro susceptibility of 60 strains of Streptococcus agalactiae obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
Table 4
In vitro susceptibility of 9 strains of Streptococcus dysgalactiae obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
Table 5
In vitro susceptibility of 33 strains of Streptococcus uberis obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
Table 6
In vitro susceptibility of 43 strains of Enterococcus sp. obtained from clinical and sub-clinical bovine mastitis cases from the West Littoral Region of Uruguay.
The differences found between both tests corresponding to each antimicrobial agent were not significant (p > 0.05). Of 336 strains of S. aureus, 215 (64%) were resistant to one or more antimicrobial agents in both tests. There was no resistance to oxacillin, cephalotin, gentamicin, enrofloxacin, clindamycin, and the combination of amoxicillin-clavulanic acid, whereas 160 (47.6%), 157 (46.7%), 45 (13.4%), 10 (3%), 2 (0.6%) and 1 strain (0.3%) were resistant to penicillin, ampicillin, tetracycline, erythromycin, neomycin and trimethoprim-sulphametoxazole, respectively. One hundred and fifty-six S. aureus isolates (46.4%) were β-lactamase producers. While of 41 CNS strains, 10 (27%) presented resistance to penicillin and 9 strains (22.5%) were β-lactamase producers. Seven suspected oxacillin resistant strains of S. aureus on the ADDM were susceptible in the confirmatory test.All isolates of Str. agalactiae, Str. dysgalactiae and Str. uberis were susceptible to penicillin and ampicillin, while 3 (7%) of 43 strains of Enterococcus sp. were resistant to penicillin. Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesDiscussion
The β-lactams (penicillins and cephalosporins) have become the first line of antimicrobial agents used for treatment of bovine mastitis in Uruguay. Within this class, penicillin, amoxicillin, cloxacillin and ampicillin are the mostly used agents. In the Nordic countries penicillin is used as the first-line antibiotic treatment of bovine mastitis, because of a low resistance rate and narrow spectrum. This is an important tool to limit the development of antibiotic resistance as much as possible [1]. In our study 47.6% of S. aureus were classified as penicillin resistant, MIC ≥ 0.25 μg/ml (Table 2), 96% of which produced β-lactamase. This was the same comparing the proportion of resistance (47%) as obtained by [9] in the southern dairy area of Uruguay. The comparison between these results obtained in Uruguay over the years demonstrated that the situation in general has not changed during the last 25 years in relation to penicillin resistance. Whereas, the prevalence of resistance to penicillin was similar in Argentina (40%) [13] and Finland (50.7%) [22]. However, it was higher than in Norway, 4.2% from clinical cases and 18% from sub-clinical cases [16], and Sweden, 6% [11].In relation to CNS, 27% of 41 isolates were penicillin resistant (Table 1). Results from Finland 37% [22] and Norway 26% [16] agree with our findings. The MIC90 of penicillin was 4 μg/ml for our survey, and another study determined 0.5 μg/ml in New Zealand [31].The detection of β-lactamase production in staphylococci is a useful and rapid method to detect penicillin resistance. At the National Veterinary Institute, Uppsala, β-lactamase results are used as rapid screen to indicate penicillin resistance [25]. In this study 96% and 90% of penicillin resistant strains of S.aureus and CNS were positive as indicated by the cloverleaf method. Test for β-lactamase producing should always be done to obtain the true picture of resistance to penicillin in staphylococci.The streptococci and enterococci showed high susceptibility (streptococci 100%) to penicillin in our study (Tables 3, 4, 5 and 6). This agree with the results from monitoring studies done in the Scandinavian countries, where the streptococci populations isolated from mastitis were highly susceptible to penicillin [28]. Only 7% of the Enterococcus sp. strains were classified as resistant against penicillin (> 8 μg/ml). The MIC90 of penicillin for Str. agalactiae, Str. dysgalactiae, and Str. uberis was 0.12 μg/ml in each case and for enterococci 4 μg/ml.Oxacillin was included here as recommended by the [24] to detect methicillin-resistant strains of S. aureus and CNS. In our study oxacillin resistance was not found in staphylococci. However, CNS strains with higher MIC than > 0.5 *g/ml of oxacillin should be tested for possible carriage of the mecA gene, in order to verify the occurrence of this gene [18].Cephalotin was included to determine the resistance against the first-generation cephalosporin class for all bacterial species except Enterococcus sp. All tested microorganisms were 100% sensitive to cephalotin and MIC90 was ≤ 4 μg/ml.Despite their structural differences macrolide and lincosamides antimicrobials have similar biological properties, including their mechanism of action against the 50S subunit of the bacterial ribosome. These common properties easily allow the development of cross-resistance [29].Erythromycin and clindamycin were included here to evaluate the resistance against these groups. Clindamycin was used in our survey to test resistance againts lincosamides in staphylococci and no strains were resistant. For our strains the MIC90 value was < 1 μg/ml, a result remarkably different as compared with 8 μg/ml obtained by [10] for S. aureus strains isolated in the United States.For erythromycin our findings (Tables 1 and 2) showed scarce resistance in S. aureus (3%) and in CNS (0%), similar to the result (2.4%) reported by [9]. The results were lower than reported by [13] for S. aureus in Argentina (11.6%). In Finland, [22] found 2.6% and 11.5% resistance among S. aureus and CNS, respectively, while in Sweden, [11] reported 1% resistance in S. aureus. The MIC90 of erythromycin in our study was ≤ 0.5 μg/ml to staphylococci.Streptococci showed high erythromycin susceptibility, only 3.4% of Str. agalactiae and 4.6% of Enterococcus sp. were resistant in our study. Substantial differences were found in relation to results obtained in Finland (17%) for enterococci [22], but no differences with respect to the erythromycin susceptibility result in streptococci (2,8%) obtained by [9]. Our MIC90 value of erythromycin for streptococci was ≤ 0.25 μg/ml except for Str. uberis (0.5 μg/ml), while for enterococci was ≤ 0.5 μg/ml.Aminoglycosides are used with precaution in dairy animals in order to avoid the risk of prolonged residues in milk. However, products for direct infusion into mammary gland containing neomycin are used because of the limited systemic effect caused by this way of administration [30]. The MIC90 (2 μg/ml) of neomycin (Table 1) in our survey for S. aureus was slightly different compared to the results obtained with S. aureus from different countries [10].The S. aureus and CNS bacteria were not gentamicin-resistant and the MIC90 values were ≤ 1 μg/ml for both. This was similar to the results obtained for S. aureus in Argentina [13]. As expected we found high MICs of gentamicin in Str. agalactiae and Str. uberis (Tables 3 and 5), while Str. uberis and Enterococcus sp. had lower MICs (Tables 4 and 6). Aminoglycosides are not the antimicrobials agents of choice for streptococcal mastitis because streptococci have inherited resistance to this class [28].Our results regarding tetracycline-resistance for S. aureus (13.4%) and CNS (13.9%) were similar to those in Finland [22], but higher than the results obtained in Norway for S. aureus (0.2 %) and CNS (3%) [16]. The results were twofold higher than the 6% reported by [9] in Uruguay. A possible explanation for this phenomenon could be that for many years tetracyclines have been the most widely antimicrobial class used by the farmers to treat any infection.In general the streptococci and enterococci were susceptible to oxytetracycline, with the exception of Str. dysgalactiae (Table 4). [28] stated that Str. dysgalactiae strains are less susceptible to tetracyclyne than Str. uberis strains, as also reported by [7]Staphylococci and streptococci were susceptible to trimethoprim-sulfamethoxazole, whereas enterococci were resistant (Table 6).Enrofloxacin is approved for systemic administration to treat bovine mastitis in some Scandinavian countries. We found a high susceptibility in staphylococci (Tables 1 and 2) and a similar situation was found by [22].Both antimicrobial susceptibility tests, ADDM and broth micro-dilution, used in this survey were performed according to the approved standard for bacteria isolated from animals and the interpretative criteria for veterinary use according to [24]. The ADDM is most commonly used in the veterinary laboratories in Uruguay and many other countries. There were no significant differences between the methods when classifying bacterial isolates as susceptible or resistant according to NLCCS (Tables 1, 2, 3, 4, 5 and 6). The results from ADDM could be influenced by several factors, such as: compositions of agar medium, pH, inoculum density, agar depth, timing of drug applications, incubation time, etc [2]. However, [23] have obtained high correlation coefficient (0.875 to 0.975) between both methods in agreement with our results. [19] considered ADDM as a useful tool when the level of compliance with NCCLS guidelines was evaluated periodically. Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesConclusion
This study did not show changes with respect to the penicillin and erythromycin resistance level of udder pathogens (staphylococci and streptococci) during the last 25 years in Uruguay, while a clear increase in tetracycline resistance was found for S. aureus.The Agar Disk Diffusion Method was a good tool, inexpensive, and readily available for regional veterinary laboratories. However, considering the necessity to maintain the surveillance over antimicrobial resistance in a country, it is important to periodically evaluate the compliance with guidelines such as National Committee for Clinical Laboratory Standards guidelines. It is also important to monitor regularly the minimum inhibitory concentrations for the isolated strains from different regions of the country. A responsible antibiotic policy would be highly relevant in a future programme for mastitis control and udder health in Uruguay.Acknowledgements
The authors thank Margareta Horn af Rantzein for her generous support of this work. The authors also acknowledge the staff of the mastitis laboratory, Department of Mastitis and Diagnostical Products, National Veterinary Institute, Uppsala, Sweden, where the work was carried out. R. E. Gianneechini was awarded a scholarship by the Swedish Foundation for International Co-operation in Research and Higher Education (STINT) and a grant from Instituto Nacional de Investigaciones Agropecuarias (INIA), Uruguay.
Other Sections▼
AbstractIntroductionMaterials and methodsResultsDiscussionConclusionReferencesReferences
1.Aarestrup FM, Jensen NE. Development of penicillin resistance among Staphylococcus aureus isolated from bovine mastitis in Denmark and other countries. Microbial Drug Resistance. 1998;4:247–256. [PubMed]
2.Acar JF, Goldstain FW. Antibiotics in Laboratory Medicine. 4. V. Lorian, Williams and Wilkins, Baltimore, MD, USA; 1996. Disk susceptibility test; pp. 1–51.
3.Amsterdam D. Antibiotics in Laboratory Medicine. 4. V. Lorian, Williams and Wilkins, Baltimore, MD, USA; 1996. Susceptibility testing of antimicrobials in liquid media; pp. 52–111.
4.Baker CN, Huang MB, Tenover FC. Optimizing testing of methicillin-resistant Staphylococcus species. Diagn Microbiol Infect Dis. 1994;19:167–170. doi: 10.1016/0732-8893(94)90061-2. [PubMed]
5.Bauer AW, Kirby WMM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J of Clinical Path. 1966;45:493–496. [PubMed]
6.Bouman M, Irigoyen D, Bertón A. Analisis de los resultados de 427 muestras remitidas para aislamiento de bacterias de mastitis y antibiograma. (Study of results from 427 milk samples remitted for bacteriologic cultures and susceptibility testing against antimicrobial agents). Jornadas de Salud de Ubre, Nva Helvecia, Uruguay. 1999. pp. 59–68. (In Spanish).
7.Brown MB, Roberts MC. Tetracycline resistance determinants in streptococcal species isolated from the bovine mammary gland. Vet Microbiol. 1991;29:173–180. doi: 10.1016/0378-1135(91)90124-X. [PubMed]
8.Davies J. Inactivation of antibiotics and the dissemination of resistance genes. Science. 1994;264:375–382. doi: 10.1126/science.8153624. [PubMed]
9.Del Baglivi L, Bonilla M, Laborde M. Investigación sobre mastitis subclinica en rodeos lecheros del Uruguay. (Subclinical mastitis research in dairy herds of Uruguay). Medicina-Veterinaria Montevideo, Uruguay. 1976;12:61,69–77. (In Spanish).
10.De Oliveira AP, Watts JL, Salmon SA, Aarestrup FM. Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Europe and United States. J Dairy Sci. 2000;83:855–862. [PubMed]
11.Franklin A. Antibiotic policy and ocurrence of resistance in Sweden. Proceedings of 25th International Dairy Congress, Aarhus, Denmark. 1998. pp. 229–234.
12.Franklin A, Wierup M. Evaluation of the sensititre method adapted for antimicrobial drug susceptibility testing in veterinary medicine. Vet Microbiol. 1982;7:447–454. doi: 10.1016/0378-1135(82)90061-X. [PubMed]
13.Gentilini E, Denamiel G, Llorente P, Godaly S, Rebuelto M, De Gregorio O. Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Argentina. J Dairy Sci. 2000;83:1224–1227. [PubMed]
14.Gentilini E, Denamiel G, Tirante L, Chavez C, Godaly MS. Bovine Mastitis: β-lactamase production. Staphylococcus aureus antibiotic resistance evolution. Proceedings of the 3rd International Mastitis Seminar, Tel Aviv, Israel. 1995;session 2:84–85.
15.Gianneechini R. Thesis. International Master of Science Programme, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala, Sweden; 2001. Occurrence and aetiology of clinical and subclinical mastitis, and antimicrobial susceptibility of udder pathogens in dairy herds in a region of Uruguay; pp. 31–43. ISSN 1403-2201, Report no. 25.
16.Hofshager M, Kruse H, Lassen J, Stavnes T-L, Essun K, Holstad G, Mørk T, Schau J, Grave K. Resistance in bacteria from infections in animals. In: Hilde Kruse, editor. Usage of antimicrobial agents in animals and occurrence of antimicrobal resistance in bacteria from animals, feed, and food in Norway 1999, NORM-VET, Oslo, Norway. 1999. pp. 16–18. ISSN-1502-4695.
17.Herrera B. Etiología de las mastitis subclínicas y estudio de la cuenca lechera de Tacuarembó. (Ethiology of subclinical mastitis and study of the dairy area of Tacuarembó). III Congreso Nacional de Veterinaria, Montevideo, Uruguay. 1982. pp. 495–505. In Spanish.
18.Hussain Z, Stoakes L, Massey V, Diagre D, Fitzgerald V, El Sayed S, Lannigan R. Correlation of oxacillin MIC with mecA gene carriage in Coagulase-negative staphylococci. J Clin Microbiol. 2000;38:752–754. [PubMed]
19.Kiehlbauch JA, Hannett GE, Salfinger M, Archinal W, Monserrat C, Carlyn C. Use of the National Committee for Clinical Laboratory Standards guidelines for disk diffusion susceptibility testing in New York State Laboratories. J Clin Microbiol. 2000;38:3341–3348. [PubMed]
20.Lingaas E. The use of antimicrobials in animal production – a threat to humans? NKVet Symposium, Helsinki, Finland. 1998. pp. 26–27.
21.Milton S. Statistical methods in the biological and health sciences. 2. chapter 8. New York, McGraw-Hill; 1992.
22.Myllys V, Asplund K, Brofeldt E, Hirvelä-Koski V, Honkanen-Buzalske T, Junttila J, Kulkas L, Myllykangas O, Niskanen M, Saloniemi H, Sandholm M, Saranpää T. Bovine mastitis in Finland in 1988 and 1995 – Changes in prevalence and antimicrobial resistance. Acta vet Scand. 1998;39:119–126. [PubMed]
23.Myllys V, Louhi M, Ali-Vehmas T. Comparison of penicillin-G susceptibility testing methods of Staphylococci isolated from bovine mastitis. J Vet Med B. 1992;39:723–731.
24.National Committee of Clinical Laboratories Standards. Performance Standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; approved standard. NCCLS. 1999;19:11,60. Document M31-A.
25.National Veterinary Institute. Department of Mastitis, Accreditation Certificate, Uppsala, Sweden. pp. 1–29.
26.Neu HC. The crisis in antibiotic resistance. Science. 1992;257:1064–1073. doi: 10.1126/science.257.5073.1064. [PubMed]
27.OPYPA. Oficina de Planeamiento y Producción Agropecuaria. Ministerio de Ganadería Agricultura y Pesca. Montevideo-Uruguay. 2000.
28.Pyörälä S, Myllys V. Resistance of bacteria to antimicrobials. The bovine udder and mastitis, University of Helsinki, Faculty of Veterinary Medicine, Helsinki, Finland. 1995. pp. 194–200.
29.Prescott JF. Lincosamides, Macrolides, and Pleuromutilins. In: Prescott JF, Baggot JD, Walker RD, editor. Antimicrobial therapy in veterinary medicine. 3. Iowa State University Press, Ames, Iowa, USA; 2000. pp. 229–262.
30.Prescott JF. Aminoglycosides and Aminocyclitols. In: Prescott JF, Bagott JD, Walker RD, editor. Antimicrobial therapy in veterinary medicine. 3. Iowa State University Press, Ames, Iowa.USA; 2000. pp. 191–228.
31.Salmon SA, Watts JL, Aarestrup JW, Yancey RJ., Jr Minimum Inhibitory Concentrations for selected antimicrobial agents against organisms isolated from the mammary glands of dairy heifers in New Zealand and Denmark. J Dairy Sci. 1998;81:570–578. [PubMed]
--------------------------------------------------------------------------------
Articles from Acta Veterinaria Scandinavica are provided here courtesy of
BioMed Central
Saturday, March 13, 2010
Monday, March 8, 2010
Enterobacter cloaceae
Show latest papers in Rev Esp Anestesiol Reanim
FP7 Partner
Add BioInfo.PL bioinformatics lab to Your FP7 application
bioinfo.pl
View
Help
Login
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
FP7 Partner
Add BioInfo.PL bioinformatics lab to Your FP7 application
bioinfo.pl
View
Help
Login
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
Sunday, March 7, 2010
Klebsiella isolation from Blood
J Clin Microbiol. 2004 March; 42(3): 1337–1340.
doi: 10.1128/JCM.42.3.1337-1340.2004.
PMCID: PMC356840
Copyright © 2004, American Society for Microbiology
Rapid Detection of Klebsiella pneumoniae from Blood Culture Bottles by Real-Time PCR
Prathiba Kurupati,1 Carol Chow,2 Gamini Kumarasinghe,2 and Chit Laa Poh1*
Department of Microbiology, National University of Singapore, Singapore 117597,1 Department of Laboratory Medicine, National University Hospital, Singapore 119074, Republic of Singapore2
*Corresponding author. Mailing address: Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Republic of Singapore. Phone: (65) 687-43674. Fax: (65) 677-66872. E-mail: micpohcl@nus.edu.sg.
Received July 14, 2003; Revised August 27, 2003; Accepted November 17, 2003.
This article has been cited by other articles in PMC.
ABSTRACT
A LightCycler real-time PCR hybridization probe-based assay which detects a partial Klebsiella pneumoniae 16S rRNA gene was developed for the rapid identification of K. pneumoniae directly from growth-positive blood culture bottles (BACTEC 9240 system) within 2 h. No cross-reactivity was observed with 65 negative-control blood cultures that grew bacteria other than K. pneumoniae and 48 negative blood cultures from double-blind experiments, thus demonstrating 100% specificity when compared to results of conventional biochemical characterization. The assay also showed 100% sensitivity, as it correctly identified all 142 positive-control blood cultures and 4 from double-blind trials.
Other Sections▼
Klebsiella pneumoniae is an important hospital-acquired pathogen that causes severe morbidity and mortality among the newborn, the elderly, and immunocompromised patients (3, 4, 15). K. pneumoniae infections acquired in hospitals can be difficult to treat, as many strains are highly resistant to treatment with broad-spectrum cephalosporins and aminoglycosides (1, 7, 8, 10, 13). The isolation of bacteria from blood culture bottles is usually indicative of a serious invasive infection (bacteremia) which requires urgent antimicrobial therapy. Microorganisms isolated from patients usually have different antimicrobial susceptibilities, and successful treatment is dependent on the prompt administration of the appropriate antibiotic (6, 9, 11, 14). K. pneumoniae is identified by various automated and manual products such as Vitek (BioMérieux, Inc.) and the Analytab Products (API) system (BioMérieux, Inc.) or by traditional biochemical tests, and the whole process requires at least 24 to 48 h.
In recent years, real-time PCR has emerged as a valuable tool for the rapid testing of various biological specimens and body fluids for the presence of microorganisms (2, 5, 12, 17). The development of highly sensitive and specific PCR assays has alleviated the problems typically associated with microorganisms that are found in low numbers in tissues or body fluids, that are difficult to culture, or that are serologically similar. The LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) offers two different fluorescence detection formats. The first employs SYBR Green I, which is a dye that binds nonspecifically to double-stranded DNA (16); the second uses hybridization probes which allow sequence-specific detection by using fluorescence energy transfer (FRET) between two fluorophores. This result is achieved by attaching the two fluorophores to two oligonucleotide probes designed to hybridize to a complementary region of the target gene, leaving a 1-nucleotide-wide gap. The LightCycler instrument has been developed for fast cycling and real-time monitoring of the amplification process and thus eliminates the need to carry out gel electrophoresis of the samples after PCR.
In this study, we describe a real-time PCR assay with specific primers and hybridization probes targeting the 16S rRNA gene for the direct detection of K. pneumoniae from positive blood culture bottles. The assay also provides specific and sensitive quantification of K. pneumoniae DNA.
We studied 142 blood cultures that were known to be positive controls for K. pneumoniae, 65 that were negative controls for K. pneumoniae, and 52 double-blind samples (Table 1) that showed positive growth in BACTEC Plus aerobic medium in the BACTEC 9240 system. DNA extractions were performed in all experiments by using the QIAamp blood and tissue kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions. DNA extracted from type strains which acted as positive and negative controls was stored at −20°C until use. Oligonucleotide primers were designed to amplify a 126-bp target sequence of the 16S rRNA gene spanning nucleotide positions 44 to 170 of the K. pneumoniae strain with EMBL databank accession no. X93214. The forward primer K16SF (15-mer; position 44 to 58) and reverse primer K16SR (16-mer; position 155 to 170) were used. Within the 16S rRNA gene, fluorescence-labeled probes were designed to hybridize to the species-specific region of K. pneumoniae. The sensor probe used was the K. pneumoniae sensor (19-mer; melting temperature [Tm] = 62.7°C) oligonucleotide labeled with LightCycler Red 640 at the 5′ end and phosphorylated at the 3′ end to block extension. The corresponding fluorescein isothiocyanate-labeled anchor probe at the 3′ end was the K. pneumoniae anchor (30-mer, Tm = 69.9°C) oligonucleotide that binds to the template strand at a distance of one base to the bound sensor probe.
TABLE 1.
Real-time PCR hybridization probe-based testing of microorganisms
REAL-TIME PCR SAMPLE PREPARATION.
The master mixture contained 2 μl of 10× LightCycler-FastStart DNA master hybridization probes (Roche Diagnostics), 3 mM MgCl2 (final concentration), 0.5 μM final concentration of K. pneumonia-specific primers, and 0.2 μM final concentration of hybridization probes. To complete the PCR mixtures, 18 μl of master mixture and 2 μl of a DNA preparation were added into the LightCycler glass capillaries. After a short centrifugation (700 × g for 10 s), the sealed capillaries were placed into the LightCycler.
Other Sections▼
REAL-TIME PCR AMPLIFICATION AND MELTING-CURVE ANALYSIS.
Thermocycling conditions were optimized to one cycle of denaturation at 95°C for 10 min, followed by 30 amplification cycles (temperature transition rate of 20°C/s), each comprising denaturation (95°C for 10 s), annealing (55°C for 15 s), and extension (74°C for 10 s). PCR amplification was followed by melting-curve analysis from 40 to 95°C (temperature transition rate of 0.1°C/s) with continuous fluorescence readings. Fluorescence was measured continuously during the slow temperature rise to monitor the dissociation of the LightCycler Red 640-labeled sensor probe at the F2 channel. Fluorescence signals from F2 were plotted automatically in real time versus temperature (T) to produce melting curves for amplicons. Melting curves were then converted into melting peaks by plotting the negative derivative of fluorescence versus T (−dF2/dT versus T and −dF3/dT versus T). Water was used as the negative control and was included in each set of melting peak determinations. The entire process took approximately 40 min. The presence of the 126-bp amplicon was verified by agarose gel electrophoresis.
Other Sections▼
SENSITIVITY OF REAL-TIME PCR ASSAY FOR K. PNEUMONIAE.
The sensitivity of the real-time PCR assay was optimized to 10 fg of purified K. pneumoniae DNA. Agarose gel electrophoresis of the amplicons showed that the band intensity correlated well with the calculated concentrations of amplicons (Fig. (Fig.11 A), and the sequence was analyzed (data not shown). Real-time PCR sensitivity optimization performed by using the K. pneumoniae type strain (ATCC 13883) resulted in a linear-regression curve across 107 to 1 genomic equivalents (10 ng to 1 fg of DNA) with an error rate of <1% and a correlation coefficient at −1 (Fig. (Fig.1B).1B). The intraassay variation of the threshold/cycle (CT) values was evaluated by using K. pneumoniae DNA dilutions containing 107, 106, 105, 104, 103, 102, and 10 copies. CT values were established between 10 and 27. The CT values, which are inversely related to the quantity of organisms, ranged between 10 and 27 corresponded to 107 to 10 organisms (Fig. (Fig.1B1B).
FIG. 1.
Standard curve for K. pneumoniae DNA quantification. (A) Agarose gel electrophoresis of a 126-bp PCR product. Lane W represents the negative control (water). Lane M represents the 100-bp ladder DNA marker; lanes 1 to 8 represent the 10-fold-dilution series (more ...)
Other Sections▼
SPECIFICITY OF THE REAL-TIME PCR ASSAY.
The specificity of the real-time PCR assay for K. pneumoniae with primers specific for the 16S rRNA gene was investigated by testing 1 ng of purified DNA from each of the 65 negative-control organisms (Table 1). Amplification of Pseudomonas aeruginosa, Streptococcus spp., Staphylococcus aureus, Enterococcus faecalis, Haemophilus influenzae, and Neisseria meningitidis remained negative when tested at all concentrations (10 ng to 1 fg), whereas the CT values for Klebsiella oxytoca, Escherichia coli, and Enterococcus faecium were greater than 29 at a concentration of 1 ng/μl. By comparison, a CT value of ≤13 was achieved for K. pneumoniae at the same concentration of 1 ng/μl (Fig. (Fig.2).2). Upon agarose gel electrophoresis of PCR mixtures derived from negative-control organisms, no amplicon was ever observed. The target sequence was shown to be highly specific for K. pneumoniae, as it failed to detect any other bacteria that are known to cause bacteremia (Fig. (Fig.22).
FIG. 2.
Specificity of the real-time PCR assay for K. pneumoniae. Different bacterial species were subjected to DNA extraction and quantitation by the real-time PCR assay. Data for fluorescence versus cycle number plots for clinical samples are shown.
Other Sections▼
MELTING-CURVE ANALYSIS OF REAL-TIME PCR ASSAY FOR K. PNEUMONIAE.
When the temperature reached the Tm of the probes, a rapid loss of fluorescence was observed as the two adjacently bound probes dissociated from their complementary targets and FRET stopped. This was plotted as the negative derivative of fluorescence versus temperature to define the template-specific melting curves (Fig. (Fig.3).3). Without changing the MgCl2 concentration, we could still maintain a sensitivity of 10 organisms per sample, as shown by a 10-fold dilution series from 107 to 1 genome equivalent(s) of our reference strain. With the hybridization probes, the PCR products were found to reproducibly yield a melting peak of 66°C (Fig. (Fig.33).
FIG. 3.
Melting curve for the LightCycler KPS and KPA FRET hybridization probes. The specific melting peak can be observed with the quantified number of copies of K. pneumoniae DNA.
Other Sections▼
DOUBLE-BLIND TESTING OF BLOOD CULTURE BOTTLES.
Four of 52 double-blind samples tested positive for K. pneumoniae, with a CT value of <20, and 48 samples displayed no detectable fluorescence above the background level. Double-blind PCR-based testing results were concordant with those of biochemical identification.
PCR-based testing of all 259 blood samples by real-time PCR demonstrated a sensitivity of 100% (146 of 146) and a specificity of 100% (113 of 113). The molecular assay was completed within 2 h upon receiving BACTEC 9240 positive blood culture bottles. No contamination was observed at any time during the study. No false-positive signal was detected when the negative control (which comprised sterile water, primers, and probes) was amplified in the reaction capillaries. In contrast to conventional PCR methods, they are less prone to contamination because postamplification handling is eliminated (12). Our real-time PCR-based approach for identification of K. pneumoniae resulted in significant time saving in comparison to traditional biochemical identification of K. pneumoniae from BACTEC 9240 positive blood cultures.
In conclusion, real-time PCR offers a fast tool with high sensitivity and specificity for the identification of K. pneumoniae from positive blood cultures. If in the future it becomes possible to detect more pathogens by multiplex real-time PCR, this method could become a suitable technology for implementation in routine diagnostic laboratories. It is also suitable for laboratories which encounter high isolation rates for K. pneumoniae or in outbreak situations.
Other Sections▼
REFERENCES
1. Arlet, G., M. J. Sanson-Le Pors, M. Rouveau, G. Fournier, O. Marie, B. Schlemmer, and A. Philippon. 1990. Outbreak of nosocomial infection due to Klebsiella pneumoniae producing SHV-4 β-lactamase. Eur. J. Clin. Microbiol. Infect. Dis. 9:797-803. [PubMed]
2. Bellin, T., M. Pulz, A. Matussek, H. G. Hempen, and F. Gunzer. 2001. Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes. J. Clin. Microbiol. 39:370-374. [PubMed]
3. Bingen, E. H., P. Desjardins, G. Arlet, F. Bourgeois, P. Marianikurkdjian, N. Y. Lambertzechovsky, E. Denamur, A. Philippon, and J. Elion. 1993. Molecular epidemiology of plasmid spread among extended broad-spectrum β-lactamase-producing Klebsiella pneumoniae isolates in a pediatric hospital. J. Clin. Microbiol. 31:179-184. [PubMed]
4. Coovadia, Y. M., A. P. Johnson, R. H. Bhana, G. R. Hutchinson, R. C. George, and I. E. Hafferjee. 1992. Multiresistant Klebsiella pneumoniae in a neonatal nursery: the importance of maintenance of infection control policies and procedures in the prevention of outbreaks. J. Hosp. Infect. 22:197-205. [PubMed]
5. Corless, C. E., M. Guiver, R. Borrow, V. Edwards-Jones, A. J. Fox, and E. B. Kaczmarski. 2001. Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J. Clin. Microbiol. 39:1553-1558. [PubMed]
6. Cunney, R. J., E. B. McNamara, N. Alansari, B. Loo, and E. G. Smyth. 1997. The impact of blood culture reporting and clinical liaison on the empiric treatment of bacteraemia. J. Clin. Pathol. 50:1010-1012. [PubMed]
7. De Champs, C., D. Sirot, C. Chanal, M. C. Poupart, M. P. Dumas, and J. Sirot. 1991. Concomitant dissemination of three extended-spectrum β-lactamases among different Enterobacteriaceae isolated in a French hospital. J. Antimicrob. Chemother. 27:441-457. [PubMed]
8. Jacoby, G. A. 1996. Antimicrobial-resistant pathogens in the 1990s. Annu. Rev. Med. 47:169-179. [PubMed]
9. Jamulitrat, S., U. Meknavin, and S. Thongpiyapoon. 1994. Factors affecting mortality outcome and risk of developing nosocomial bloodstream infection. Infect. Control Hosp. Epidemiol. 15:163-170. [PubMed]
10. Johnson, A. P., M. J. Weinren, B. Ayling-Smith, S. K. Du Bois, S. G. B. Amyes, and R. C. George. 1992. Outbreak of infection in two U.K. hospitals caused by a strain of Klebsiella pneumoniae resistant to cefotaxime and ceftazidime. J. Hosp. Infect. 20:97-103. [PubMed]
11. Lebovici, L. S., I. Shraga, M. Drucker, H. Konigsberger, Z. Samra, and S. D. Pitlik. 1998. The benefit of appropriate empirical antibiotic treatment in patients with bloodstream infection. J. Intern. Med. 244:379-386. [PubMed]
12. Logan, J. M. J., K. J. Edwards, N. A. Saunders, and J. Stanley. 2001. Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR. J. Clin. Microbiol. 39:2227-2232. [PubMed]
13. Nouvellon, M., J. L. Pons, D. Sirot, M. L. Combe, and J. F. Lemeland. 1994. Clonal outbreaks of extended-spectrum β-lactamase-producing strains of Klebsiella pneumoniae demonstrated by antibiotic susceptibility testing, β-lactamase typing, and multilocus enzyme electrophoresis. J. Clin. Microbiol. 32:2625-2627. [PubMed]
14. Pedersen, G., H. C. Schonheyder, and H. T. Sorensen. 1997. Antibiotic therapy and outcome of monomicrobial gram-negative bacteremia: a 3-year population-based study. Scand. J. Infect. Dis. 29:601-606. [PubMed]
15. Rice, L. B., S. H. Willey, G. A. Papanicolaou, A. A. Medeiros, G. M. Eliopoulos, R. C. Moellering, Jr., and G. A. Jacoby. 1990. Outbreak of ceftazidime resistance caused by extended-spectrum β-lactamases at a Massachusetts chronic-care facility. Antimicrob. Agents Chemother. 34:2193-2199. [PubMed]
16. Skeidsvoll, J., and P. M. Ueland. 1995. Analysis of double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection using the monomeric dye SYBR Green I. Anal. Biochem. 231:359-365. [PubMed]
17. Tyagi, S., and F. R. Kramer. 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14:303-308. [PubMed]
Articles from Journal of Clinical Microbiology are provided here courtesy of
American Society for Microbiology (ASM)
PubMed articles by these authors
Kurupati, P.
Chow, C.
Kumarasinghe, G.
Poh, C.
PubMed related articles
Direct identification of bacteria from positive blood cultures by amplification and sequencing of the 16S rRNA gene: evaluation of BACTEC 9240 instrument true-positive and false-positive results. [J Clin Microbiol. 2001]
PCR detection of Klebsiella pneumoniae in infant formula based on 16S-23S internal transcribed spacer. [Int J Food Microbiol. 2008]
Peptide nucleic acid fluorescence in situ hybridization for rapid detection of Klebsiella pneumoniae from positive blood cultures. [J Med Microbiol. 2007]
Development of a real-time fluorescence resonance energy transfer PCR to detect arcobacter species. [J Clin Microbiol. 2007]
ReviewReal-time polymerase chain reaction in transfusion medicine: applications for detection of bacterial contamination in blood products. [Transfus Med Rev. 2007]
» See reviews... | » See all...
Recent Activity
Clear Turn Off
Rapid Detection of Klebsiella pneumoniae from Blood Culture Bottles by Real-Time PCR
Links
Nucleotide
PubMed
Taxonomy
Taxonomy Tree
Molecular epidemiology of plasmid spread among extended broad-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a pediatric hospital.
[J Clin Microbiol. 1993]
Multiresistant Klebsiella pneumoniae in a neonatal nursery: the importance of maintenance of infection control policies and procedures in the prevention of outbreaks.
[J Hosp Infect. 1992]
Outbreak of ceftazidime resistance caused by extended-spectrum beta-lactamases at a Massachusetts chronic-care facility.
[Antimicrob Agents Chemother. 1990]
Outbreak of nosocomial infections due to Klebsiella pneumoniae producing SHV-4 beta-lactamase.
[Eur J Clin Microbiol Infect Dis. 1990]
See more articles cited in this paragraph
Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes.
[J Clin Microbiol. 2001]
Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR.
[J Clin Microbiol. 2001]
Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR.
[J Clin Microbiol. 2001]
Molecular beacons: probes that fluoresce upon hybridization.
[Nat Biotechnol. 1996]
Analysis of double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection using the monomeric dye SYBR green I.
[Anal Biochem. 1995]
Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR.
[J Clin Microbiol. 2001]
You are here: NCBI > Literature > PubMed CentralWrite to the Help Desk
Simple NCBI Directory
GETTING STARTED
NCBI Help Manual
NCBI Handbook
Training & Tutorials
RESOURCES
Literature
DNA & RNA
Proteins
Sequence Analysis
Genes & Expression
Genomes & Maps
Domains & Structures
Genetics & Medicine
Taxonomy
Data & Software
Training & Tutorials
Homology
Small Molecules
Variation
POPULAR
PubMed
PubMed Central
Bookshelf
BLAST
Gene
Nucleotide
Protein
GEO
Conserved Domains
Structure
PubChem
FEATURED
GenBank
Reference Sequences
Map Viewer
Genome Projects
Human Genome
Mouse Genome
Influenza Virus
Primer-BLAST
Sequence Read Archive
NCBI INFORMATION
About NCBI
Research at NCBI
NCBI Newsletter
NCBI FTP Site
NIHDHHSUSA.gov
Copyright | Disclaimer | Privacy | Accessibility | Contact
doi: 10.1128/JCM.42.3.1337-1340.2004.
PMCID: PMC356840
Copyright © 2004, American Society for Microbiology
Rapid Detection of Klebsiella pneumoniae from Blood Culture Bottles by Real-Time PCR
Prathiba Kurupati,1 Carol Chow,2 Gamini Kumarasinghe,2 and Chit Laa Poh1*
Department of Microbiology, National University of Singapore, Singapore 117597,1 Department of Laboratory Medicine, National University Hospital, Singapore 119074, Republic of Singapore2
*Corresponding author. Mailing address: Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117597, Republic of Singapore. Phone: (65) 687-43674. Fax: (65) 677-66872. E-mail: micpohcl@nus.edu.sg.
Received July 14, 2003; Revised August 27, 2003; Accepted November 17, 2003.
This article has been cited by other articles in PMC.
ABSTRACT
A LightCycler real-time PCR hybridization probe-based assay which detects a partial Klebsiella pneumoniae 16S rRNA gene was developed for the rapid identification of K. pneumoniae directly from growth-positive blood culture bottles (BACTEC 9240 system) within 2 h. No cross-reactivity was observed with 65 negative-control blood cultures that grew bacteria other than K. pneumoniae and 48 negative blood cultures from double-blind experiments, thus demonstrating 100% specificity when compared to results of conventional biochemical characterization. The assay also showed 100% sensitivity, as it correctly identified all 142 positive-control blood cultures and 4 from double-blind trials.
Other Sections▼
Klebsiella pneumoniae is an important hospital-acquired pathogen that causes severe morbidity and mortality among the newborn, the elderly, and immunocompromised patients (3, 4, 15). K. pneumoniae infections acquired in hospitals can be difficult to treat, as many strains are highly resistant to treatment with broad-spectrum cephalosporins and aminoglycosides (1, 7, 8, 10, 13). The isolation of bacteria from blood culture bottles is usually indicative of a serious invasive infection (bacteremia) which requires urgent antimicrobial therapy. Microorganisms isolated from patients usually have different antimicrobial susceptibilities, and successful treatment is dependent on the prompt administration of the appropriate antibiotic (6, 9, 11, 14). K. pneumoniae is identified by various automated and manual products such as Vitek (BioMérieux, Inc.) and the Analytab Products (API) system (BioMérieux, Inc.) or by traditional biochemical tests, and the whole process requires at least 24 to 48 h.
In recent years, real-time PCR has emerged as a valuable tool for the rapid testing of various biological specimens and body fluids for the presence of microorganisms (2, 5, 12, 17). The development of highly sensitive and specific PCR assays has alleviated the problems typically associated with microorganisms that are found in low numbers in tissues or body fluids, that are difficult to culture, or that are serologically similar. The LightCycler system (Roche Molecular Biochemicals, Mannheim, Germany) offers two different fluorescence detection formats. The first employs SYBR Green I, which is a dye that binds nonspecifically to double-stranded DNA (16); the second uses hybridization probes which allow sequence-specific detection by using fluorescence energy transfer (FRET) between two fluorophores. This result is achieved by attaching the two fluorophores to two oligonucleotide probes designed to hybridize to a complementary region of the target gene, leaving a 1-nucleotide-wide gap. The LightCycler instrument has been developed for fast cycling and real-time monitoring of the amplification process and thus eliminates the need to carry out gel electrophoresis of the samples after PCR.
In this study, we describe a real-time PCR assay with specific primers and hybridization probes targeting the 16S rRNA gene for the direct detection of K. pneumoniae from positive blood culture bottles. The assay also provides specific and sensitive quantification of K. pneumoniae DNA.
We studied 142 blood cultures that were known to be positive controls for K. pneumoniae, 65 that were negative controls for K. pneumoniae, and 52 double-blind samples (Table 1) that showed positive growth in BACTEC Plus aerobic medium in the BACTEC 9240 system. DNA extractions were performed in all experiments by using the QIAamp blood and tissue kit (QIAGEN, Valencia, Calif.) according to the manufacturer's instructions. DNA extracted from type strains which acted as positive and negative controls was stored at −20°C until use. Oligonucleotide primers were designed to amplify a 126-bp target sequence of the 16S rRNA gene spanning nucleotide positions 44 to 170 of the K. pneumoniae strain with EMBL databank accession no. X93214. The forward primer K16SF (15-mer; position 44 to 58) and reverse primer K16SR (16-mer; position 155 to 170) were used. Within the 16S rRNA gene, fluorescence-labeled probes were designed to hybridize to the species-specific region of K. pneumoniae. The sensor probe used was the K. pneumoniae sensor (19-mer; melting temperature [Tm] = 62.7°C) oligonucleotide labeled with LightCycler Red 640 at the 5′ end and phosphorylated at the 3′ end to block extension. The corresponding fluorescein isothiocyanate-labeled anchor probe at the 3′ end was the K. pneumoniae anchor (30-mer, Tm = 69.9°C) oligonucleotide that binds to the template strand at a distance of one base to the bound sensor probe.
TABLE 1.
Real-time PCR hybridization probe-based testing of microorganisms
REAL-TIME PCR SAMPLE PREPARATION.
The master mixture contained 2 μl of 10× LightCycler-FastStart DNA master hybridization probes (Roche Diagnostics), 3 mM MgCl2 (final concentration), 0.5 μM final concentration of K. pneumonia-specific primers, and 0.2 μM final concentration of hybridization probes. To complete the PCR mixtures, 18 μl of master mixture and 2 μl of a DNA preparation were added into the LightCycler glass capillaries. After a short centrifugation (700 × g for 10 s), the sealed capillaries were placed into the LightCycler.
Other Sections▼
REAL-TIME PCR AMPLIFICATION AND MELTING-CURVE ANALYSIS.
Thermocycling conditions were optimized to one cycle of denaturation at 95°C for 10 min, followed by 30 amplification cycles (temperature transition rate of 20°C/s), each comprising denaturation (95°C for 10 s), annealing (55°C for 15 s), and extension (74°C for 10 s). PCR amplification was followed by melting-curve analysis from 40 to 95°C (temperature transition rate of 0.1°C/s) with continuous fluorescence readings. Fluorescence was measured continuously during the slow temperature rise to monitor the dissociation of the LightCycler Red 640-labeled sensor probe at the F2 channel. Fluorescence signals from F2 were plotted automatically in real time versus temperature (T) to produce melting curves for amplicons. Melting curves were then converted into melting peaks by plotting the negative derivative of fluorescence versus T (−dF2/dT versus T and −dF3/dT versus T). Water was used as the negative control and was included in each set of melting peak determinations. The entire process took approximately 40 min. The presence of the 126-bp amplicon was verified by agarose gel electrophoresis.
Other Sections▼
SENSITIVITY OF REAL-TIME PCR ASSAY FOR K. PNEUMONIAE.
The sensitivity of the real-time PCR assay was optimized to 10 fg of purified K. pneumoniae DNA. Agarose gel electrophoresis of the amplicons showed that the band intensity correlated well with the calculated concentrations of amplicons (Fig. (Fig.11 A), and the sequence was analyzed (data not shown). Real-time PCR sensitivity optimization performed by using the K. pneumoniae type strain (ATCC 13883) resulted in a linear-regression curve across 107 to 1 genomic equivalents (10 ng to 1 fg of DNA) with an error rate of <1% and a correlation coefficient at −1 (Fig. (Fig.1B).1B). The intraassay variation of the threshold/cycle (CT) values was evaluated by using K. pneumoniae DNA dilutions containing 107, 106, 105, 104, 103, 102, and 10 copies. CT values were established between 10 and 27. The CT values, which are inversely related to the quantity of organisms, ranged between 10 and 27 corresponded to 107 to 10 organisms (Fig. (Fig.1B1B).
FIG. 1.
Standard curve for K. pneumoniae DNA quantification. (A) Agarose gel electrophoresis of a 126-bp PCR product. Lane W represents the negative control (water). Lane M represents the 100-bp ladder DNA marker; lanes 1 to 8 represent the 10-fold-dilution series (more ...)
Other Sections▼
SPECIFICITY OF THE REAL-TIME PCR ASSAY.
The specificity of the real-time PCR assay for K. pneumoniae with primers specific for the 16S rRNA gene was investigated by testing 1 ng of purified DNA from each of the 65 negative-control organisms (Table 1). Amplification of Pseudomonas aeruginosa, Streptococcus spp., Staphylococcus aureus, Enterococcus faecalis, Haemophilus influenzae, and Neisseria meningitidis remained negative when tested at all concentrations (10 ng to 1 fg), whereas the CT values for Klebsiella oxytoca, Escherichia coli, and Enterococcus faecium were greater than 29 at a concentration of 1 ng/μl. By comparison, a CT value of ≤13 was achieved for K. pneumoniae at the same concentration of 1 ng/μl (Fig. (Fig.2).2). Upon agarose gel electrophoresis of PCR mixtures derived from negative-control organisms, no amplicon was ever observed. The target sequence was shown to be highly specific for K. pneumoniae, as it failed to detect any other bacteria that are known to cause bacteremia (Fig. (Fig.22).
FIG. 2.
Specificity of the real-time PCR assay for K. pneumoniae. Different bacterial species were subjected to DNA extraction and quantitation by the real-time PCR assay. Data for fluorescence versus cycle number plots for clinical samples are shown.
Other Sections▼
MELTING-CURVE ANALYSIS OF REAL-TIME PCR ASSAY FOR K. PNEUMONIAE.
When the temperature reached the Tm of the probes, a rapid loss of fluorescence was observed as the two adjacently bound probes dissociated from their complementary targets and FRET stopped. This was plotted as the negative derivative of fluorescence versus temperature to define the template-specific melting curves (Fig. (Fig.3).3). Without changing the MgCl2 concentration, we could still maintain a sensitivity of 10 organisms per sample, as shown by a 10-fold dilution series from 107 to 1 genome equivalent(s) of our reference strain. With the hybridization probes, the PCR products were found to reproducibly yield a melting peak of 66°C (Fig. (Fig.33).
FIG. 3.
Melting curve for the LightCycler KPS and KPA FRET hybridization probes. The specific melting peak can be observed with the quantified number of copies of K. pneumoniae DNA.
Other Sections▼
DOUBLE-BLIND TESTING OF BLOOD CULTURE BOTTLES.
Four of 52 double-blind samples tested positive for K. pneumoniae, with a CT value of <20, and 48 samples displayed no detectable fluorescence above the background level. Double-blind PCR-based testing results were concordant with those of biochemical identification.
PCR-based testing of all 259 blood samples by real-time PCR demonstrated a sensitivity of 100% (146 of 146) and a specificity of 100% (113 of 113). The molecular assay was completed within 2 h upon receiving BACTEC 9240 positive blood culture bottles. No contamination was observed at any time during the study. No false-positive signal was detected when the negative control (which comprised sterile water, primers, and probes) was amplified in the reaction capillaries. In contrast to conventional PCR methods, they are less prone to contamination because postamplification handling is eliminated (12). Our real-time PCR-based approach for identification of K. pneumoniae resulted in significant time saving in comparison to traditional biochemical identification of K. pneumoniae from BACTEC 9240 positive blood cultures.
In conclusion, real-time PCR offers a fast tool with high sensitivity and specificity for the identification of K. pneumoniae from positive blood cultures. If in the future it becomes possible to detect more pathogens by multiplex real-time PCR, this method could become a suitable technology for implementation in routine diagnostic laboratories. It is also suitable for laboratories which encounter high isolation rates for K. pneumoniae or in outbreak situations.
Other Sections▼
REFERENCES
1. Arlet, G., M. J. Sanson-Le Pors, M. Rouveau, G. Fournier, O. Marie, B. Schlemmer, and A. Philippon. 1990. Outbreak of nosocomial infection due to Klebsiella pneumoniae producing SHV-4 β-lactamase. Eur. J. Clin. Microbiol. Infect. Dis. 9:797-803. [PubMed]
2. Bellin, T., M. Pulz, A. Matussek, H. G. Hempen, and F. Gunzer. 2001. Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes. J. Clin. Microbiol. 39:370-374. [PubMed]
3. Bingen, E. H., P. Desjardins, G. Arlet, F. Bourgeois, P. Marianikurkdjian, N. Y. Lambertzechovsky, E. Denamur, A. Philippon, and J. Elion. 1993. Molecular epidemiology of plasmid spread among extended broad-spectrum β-lactamase-producing Klebsiella pneumoniae isolates in a pediatric hospital. J. Clin. Microbiol. 31:179-184. [PubMed]
4. Coovadia, Y. M., A. P. Johnson, R. H. Bhana, G. R. Hutchinson, R. C. George, and I. E. Hafferjee. 1992. Multiresistant Klebsiella pneumoniae in a neonatal nursery: the importance of maintenance of infection control policies and procedures in the prevention of outbreaks. J. Hosp. Infect. 22:197-205. [PubMed]
5. Corless, C. E., M. Guiver, R. Borrow, V. Edwards-Jones, A. J. Fox, and E. B. Kaczmarski. 2001. Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR. J. Clin. Microbiol. 39:1553-1558. [PubMed]
6. Cunney, R. J., E. B. McNamara, N. Alansari, B. Loo, and E. G. Smyth. 1997. The impact of blood culture reporting and clinical liaison on the empiric treatment of bacteraemia. J. Clin. Pathol. 50:1010-1012. [PubMed]
7. De Champs, C., D. Sirot, C. Chanal, M. C. Poupart, M. P. Dumas, and J. Sirot. 1991. Concomitant dissemination of three extended-spectrum β-lactamases among different Enterobacteriaceae isolated in a French hospital. J. Antimicrob. Chemother. 27:441-457. [PubMed]
8. Jacoby, G. A. 1996. Antimicrobial-resistant pathogens in the 1990s. Annu. Rev. Med. 47:169-179. [PubMed]
9. Jamulitrat, S., U. Meknavin, and S. Thongpiyapoon. 1994. Factors affecting mortality outcome and risk of developing nosocomial bloodstream infection. Infect. Control Hosp. Epidemiol. 15:163-170. [PubMed]
10. Johnson, A. P., M. J. Weinren, B. Ayling-Smith, S. K. Du Bois, S. G. B. Amyes, and R. C. George. 1992. Outbreak of infection in two U.K. hospitals caused by a strain of Klebsiella pneumoniae resistant to cefotaxime and ceftazidime. J. Hosp. Infect. 20:97-103. [PubMed]
11. Lebovici, L. S., I. Shraga, M. Drucker, H. Konigsberger, Z. Samra, and S. D. Pitlik. 1998. The benefit of appropriate empirical antibiotic treatment in patients with bloodstream infection. J. Intern. Med. 244:379-386. [PubMed]
12. Logan, J. M. J., K. J. Edwards, N. A. Saunders, and J. Stanley. 2001. Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR. J. Clin. Microbiol. 39:2227-2232. [PubMed]
13. Nouvellon, M., J. L. Pons, D. Sirot, M. L. Combe, and J. F. Lemeland. 1994. Clonal outbreaks of extended-spectrum β-lactamase-producing strains of Klebsiella pneumoniae demonstrated by antibiotic susceptibility testing, β-lactamase typing, and multilocus enzyme electrophoresis. J. Clin. Microbiol. 32:2625-2627. [PubMed]
14. Pedersen, G., H. C. Schonheyder, and H. T. Sorensen. 1997. Antibiotic therapy and outcome of monomicrobial gram-negative bacteremia: a 3-year population-based study. Scand. J. Infect. Dis. 29:601-606. [PubMed]
15. Rice, L. B., S. H. Willey, G. A. Papanicolaou, A. A. Medeiros, G. M. Eliopoulos, R. C. Moellering, Jr., and G. A. Jacoby. 1990. Outbreak of ceftazidime resistance caused by extended-spectrum β-lactamases at a Massachusetts chronic-care facility. Antimicrob. Agents Chemother. 34:2193-2199. [PubMed]
16. Skeidsvoll, J., and P. M. Ueland. 1995. Analysis of double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection using the monomeric dye SYBR Green I. Anal. Biochem. 231:359-365. [PubMed]
17. Tyagi, S., and F. R. Kramer. 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14:303-308. [PubMed]
Articles from Journal of Clinical Microbiology are provided here courtesy of
American Society for Microbiology (ASM)
PubMed articles by these authors
Kurupati, P.
Chow, C.
Kumarasinghe, G.
Poh, C.
PubMed related articles
Direct identification of bacteria from positive blood cultures by amplification and sequencing of the 16S rRNA gene: evaluation of BACTEC 9240 instrument true-positive and false-positive results. [J Clin Microbiol. 2001]
PCR detection of Klebsiella pneumoniae in infant formula based on 16S-23S internal transcribed spacer. [Int J Food Microbiol. 2008]
Peptide nucleic acid fluorescence in situ hybridization for rapid detection of Klebsiella pneumoniae from positive blood cultures. [J Med Microbiol. 2007]
Development of a real-time fluorescence resonance energy transfer PCR to detect arcobacter species. [J Clin Microbiol. 2007]
ReviewReal-time polymerase chain reaction in transfusion medicine: applications for detection of bacterial contamination in blood products. [Transfus Med Rev. 2007]
» See reviews... | » See all...
Recent Activity
Clear Turn Off
Rapid Detection of Klebsiella pneumoniae from Blood Culture Bottles by Real-Time PCR
Links
Nucleotide
PubMed
Taxonomy
Taxonomy Tree
Molecular epidemiology of plasmid spread among extended broad-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates in a pediatric hospital.
[J Clin Microbiol. 1993]
Multiresistant Klebsiella pneumoniae in a neonatal nursery: the importance of maintenance of infection control policies and procedures in the prevention of outbreaks.
[J Hosp Infect. 1992]
Outbreak of ceftazidime resistance caused by extended-spectrum beta-lactamases at a Massachusetts chronic-care facility.
[Antimicrob Agents Chemother. 1990]
Outbreak of nosocomial infections due to Klebsiella pneumoniae producing SHV-4 beta-lactamase.
[Eur J Clin Microbiol Infect Dis. 1990]
See more articles cited in this paragraph
Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes.
[J Clin Microbiol. 2001]
Simultaneous detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae in suspected cases of meningitis and septicemia using real-time PCR.
[J Clin Microbiol. 2001]
Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR.
[J Clin Microbiol. 2001]
Molecular beacons: probes that fluoresce upon hybridization.
[Nat Biotechnol. 1996]
Analysis of double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection using the monomeric dye SYBR green I.
[Anal Biochem. 1995]
Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR.
[J Clin Microbiol. 2001]
You are here: NCBI > Literature > PubMed CentralWrite to the Help Desk
Simple NCBI Directory
GETTING STARTED
NCBI Help Manual
NCBI Handbook
Training & Tutorials
RESOURCES
Literature
DNA & RNA
Proteins
Sequence Analysis
Genes & Expression
Genomes & Maps
Domains & Structures
Genetics & Medicine
Taxonomy
Data & Software
Training & Tutorials
Homology
Small Molecules
Variation
POPULAR
PubMed
PubMed Central
Bookshelf
BLAST
Gene
Nucleotide
Protein
GEO
Conserved Domains
Structure
PubChem
FEATURED
GenBank
Reference Sequences
Map Viewer
Genome Projects
Human Genome
Mouse Genome
Influenza Virus
Primer-BLAST
Sequence Read Archive
NCBI INFORMATION
About NCBI
Research at NCBI
NCBI Newsletter
NCBI FTP Site
NIHDHHSUSA.gov
Copyright | Disclaimer | Privacy | Accessibility | Contact
Thursday, March 4, 2010
Tuesday, March 2, 2010
Subscribe to:
Posts (Atom)