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PubMed2003

Comparison of BDPhoenix and VITEK2 automated antimicrobial susceptibility test systems for extended-spectrum beta-lactamase detection in Escherichia coli and Klebsiella species clinical isolates.

The present study compares the ability to detect extended-spectrum beta-lactamases (ESBL) among a collection of 34 ESBL producing clinical isolates belonging to Escherichia coli and Klebsiella species with two new rapid susceptibility and identification instruments-VITEK2 (bioMérieux, Marcy l'Etoile, France) vs. BDPhoenix (BD Biosciences, Sparks, MD). ESBL content in these isolates was previously characterized on the basis of PCR amplification and sequencing results which were used as the reference method in our evaluation. BDPhoenix correctly determined the ESBL outcome for all strains tested (100% detection rate), whereas VITEK2 was not able to detect the ESBL status in 5 isolates (85% detection rate). Detailed analysis revealed that the discrepancies were mainly observed with 'difficult-to-detect' strains. Misidentification was either due to low oximino cephalosporin MIC in these strains or was associated with pronounced 'cefotaximase' or 'ceftazidimase' phenotypes. Klebsiella oxytoca chromosomal beta-lactamase (K1) is phenotypically quite similar to ESBL enzymes. In order to evaluate whether the K1 and ESBL enzymes could be discriminated, we expanded our analysis by 8 clinical K. oxytoca strains with K1 phenotypes. VITEK2 gave excellent identification of these strains whereas 7 out of 8 were falsely labeled ESBL-positive by the BDPhoenix system.

Anti-Bacterial Agents↗
PubMed2003

Comparison of the E-test method with an automated bacterial identification and antimicrobial susceptibility detection system for screening extended-spectrum beta-lactamase producers.

Some automated systems used in clinical microbiology laboratories are able to detect products responsible for antimicrobial resistance. In this study, 626 isolates (436 Escherichia coli, 134 Klebsiella pneumoniae and 56 Klebsiella oxytoca strains) were examined for the presumptive detection of extended-spectrum beta-lactamase (ESBL) production by 2 methods: the Sceptor system (BD, Sparks, MD, USA) and the E-test. ESBL production was detected in 26 E. coli strains (5.96%), 60 K. pneumoniae strains (44.77%) and 15 K. oxytoca strains (26.78%) by ceftazidime/ceftazidime-clavulanate E-test. Using the E-test, ESBL production was detected in 25 of 201 E. coli strains (12.43%), 55 of 75 K. pneumoniae (73.33%) and 14 of 27 K. oxytoca strains (51.85%) that were alerted as ESBL-producing strains by the Sceptor system. ESBL positivity was detected in 1 E. coli, 5 K. pneumoniae and 1 K. oxytoca strains, that were not warned as being ESBL producers by the Sceptor system. These data suggest that clinical microbiology laboratories should not only rely on these rapid automated systems but also use another method for screening ESBL producers, such as the E-test. The rates of these ESBL-producing isolates in this study were lower than those in other studies reported from other parts of Turkey, but higher than those reported from the USA and Europe.

Automation↗
PubMed2006

Laboratory diagnosis of bloodstream infections caused by extended-spectrum beta-lactamase-producing E. coli and Klebsiella species.

Extended-spectrum beta-lactamase (ESBL)-producing organisms are resistant to the third-generation cephalosporins commonly used as empirical therapy for a wide range of serious infections. It is therefore important for laboratories to offer reliable ESBL detection methods. This study compares two combination disc methods (Oxoid and Mast Diagnostics) containing cepodoxime with and without clavulanate with Vitek 2 for routine detection of ESBLs in Escherichia coli and Klebsiella spp. isolated from blood cultures. From December 2003 to April 2005, a total of 58 potential ESBL-producing isolates (resistant to cefotaxime and/or ceftazidime) by BSAC disc susceptibility were tested by the combination discs and Vitek 2. The Advanced Expert System, a feature of Vitek 2 reports possible mechanisms of resistance, based on interpretive reading of MICs. This study detected 7.4% more ESBL-producing isolates by Vitek 2 than by Oxoid disc testing (95% CI: 0.15-14.7%; P < 0.2) and 31.6% more ESBL-producing isolates were detected by Vitek 2 than by Mast disc testing, (95% CI: 16.2-46.96%; P < 0.001). Batch-to-batch variation was evident in disc performance for both disc types. Thus, use of appropriate controls is recommended when testing by the combination disc methods. Although no phenotypic test is 100% sensitive and specific, the Vitek 2 was a reliable system for ESBL detection; however, it is expensive and interpretation of results can be confusing to inexperienced users. Further studies to compare Vitek 2 with cefotaxime and ceftazidime combination discs may reveal disc methodology for ESBL detection to be a more reliable alternative than using cefpodoxime combination discs alone.

Anti-Bacterial Agents↗
PubMed2001

Epidemiological investigation of fluoroquinolone resistance in infections due to extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae.

The incidence of infections due to extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae (ESBL-EK) has increased markedly in recent years. Treatment is difficult because of frequent multidrug resistance. Although fluoroquinolones offer effective therapy for ESBL-EK infections, their usefulness is threatened by increasing fluoroquinolone resistance. To identify risk factors for fluoroquinolone resistance in ESBL-EK infections, a case-control study of all patients with ESBL-EK infections from 1 June 1997 through 30 September 1998 was conducted. Of 77 ESBL-EK infections, 43 (55.8%) were resistant to fluoroquinolones. Independent risk factors for fluoroquinolone resistance were fluoroquinolone use (odds ratio [OR], 11.20; 95% confidence interval [CI], 1.99-63.19), aminoglycoside use (OR, 5.83; 95% CI, 1.12-30.43), and long-term care facility residence (OR, 3.39; 95% CI, 1.06-10.83). The genotypes of fluoroquinolone-resistant ESBL-EK isolates were closely related. Efforts should be directed at modification of these risk factors to preserve the utility of fluoroquinolones in the treatment of ESBL-EK infections.

Adolescent↗
PubMed2005

Clinical and molecular epidemiology of extended-spectrum beta-lactamase-producing Escherichia coli as a cause of nosocomial infection or colonization: implications for control.

BACKGROUND: Extended-spectrum beta-lactamase (ESBL)-producing members of the Enterobacteriaceae family are important nosocomial pathogens. Escherichia coli producing a specific family of ESBL (the CTX-M enzymes) are emerging worldwide. The epidemiology of these organisms as causes of nosocomial infection is poorly understood. The aims of this study were to investigate the clinical and molecular epidemiology of nosocomial infection or colonization due to ESBL-producing E. coli in hospitalized patients, consider the specific types of ESBLs produced, and identify the risk factors for infection and colonization with these organisms. METHODS: All patients with nosocomial colonization and/or infection due to ESBL-producing E. coli in 2 centers (a tertiary care hospital and a geriatric care center) identified between January 2001 and May 2002 were included. A double case-control study was performed. The clonal relatedness of the isolates was studied by repetitive extragenic palindromic-polymerase chain reaction and pulsed-field gel electrophoresis. ESBLs were characterized by isoelectric focusing, polymerase chain reaction, and sequencing. RESULTS: Forty-seven case patients were included. CTX-M-producing E. coli were clonally unrelated and more frequently susceptible to nonoxyimino-beta-lactams. Alternately, isolates producing SHV- and TEM-type ESBL were epidemic and multidrug resistant. Urinary catheterization was a risk factor for both CTX-M-producing and SHV-TEM-producing isolates. Previous oxyimino-beta-lactam use, diabetes, and ultimately fatal or nonfatal underlying diseases were independent risk factors for infection or colonization with CTX-M-producing isolates, whereas previous fluoroquinolone use was associated with infection or colonization with SHV-TEM-producing isolates. CONCLUSIONS: The epidemiology of ESBL-producing E. coli as a cause of nosocomial infection is complex. Sporadic CTX-M-producing isolates coexisted with epidemic multidrug-resistant SHV-TEM-producing isolates. These data should be taken into account for the design of control measures.

Adult↗
PubMed2006

Extended-spectrum beta-lactamases and clinical outcomes: current data.

Nosocomial infections caused by extended-spectrum beta-lactamase (ESBL)-producing gram-negative bacteria complicate therapy and limit treatment options. However, the clinical significance of infections caused by ESBL-producing bacteria remains unclear. A critical examination of the literature provides divergent views of the effect of ESBL carriage on morbidity and mortality and suggests that ESBL production may have its most marked effect on ceftazidime. Effective strategies for the empirical and directed treatment of infections caused by ESBL-producing pathogens include the use of carbapenems and, possibly, the fourth-generation cephalosporin cefepime. Studies indicate that the use of cefepime to treat serious nosocomial infections (e.g., bacteremia, pneumonia, and urinary tract infections) is associated with high rates of microbiological and clinical success. The probability of attaining time above the minimum inhibitory concentration targets of at least 70% of the dosing interval, an important pharmacodynamic indicator of clinical success, is higher with cefepime than with other antimicrobials against Escherichia coli and Klebsiella pneumoniae strains exhibiting ESBL phenotypes. However, for non-ESBL-producing strains, there is no difference in the time above the minimum inhibitory concentration between ceftazidime and cefepime. When used appropriately in institutional settings, cefepime reduces the overall use of cephalosporins, thereby decreasing selection pressure for presumptive ESBL-producing pathogens.

Anti-Bacterial Agents↗
PubMed1998

Epidemiological typing of klebsiellae with extended-spectrum beta-lactamases from European intensive care units.

Extended-spectrum beta-lactamases (ESBLs) are an increasing cause of resistance to oxyimino-aminothiazolyl cephalosporins, especially in klebsiellae. In a recent survey we detected ESBLs in 220 (23%) of 966 consecutive klebsiellae from 35 intensive care units (ICUs) in southern and western Europe. The present study examined the extent to which this distribution reflected epidemic strain spread, as against the distribution of ESBL genes into unrelated strains. All 220 ESBL producers were subjected to capsular serotyping and pulsed-field gel DNA electrophoresis (PFGE). Beta-Lactamases were typed for strains isolated on three or more occasions, with the emphasis on SHV enzymes, as these were commoner than TEM variants. Serotyping and PFGE typing defined 85 distinct strains, from 23 of the 35 participating centres. Of 14 centres that contributed five or more ESBL producers, all sent representatives of more than one strain, and two centres sent members of ten or more different strains in contributions of 17-21 ESBL-producing isolates. Nevertheless, epidemic strains-defined as those represented by three or more isolates-accounted for a majority (61%) of the collection. Fifty-two isolates of the same serotype K25 (occasionally acapsular) strain with SHV-4 beta-lactamase were recovered at two French hospitals and one in Belgium. This strain has been found by others in France, and has become particularly widespread. Another single strain was found in two separate Portuguese centres, and many individual hospitals had one or more epidemic strain(s), as well as a scatter of diverse ESBL producers. Major variation in antibiogram and plasmid profile was apparent within strains, with some intra-strain variation in beta-lactamase type. These data imply a fluid situation, with resistance determinants being gained, modified or lost. The endemicity of ESBL producers is disturbing since it limits the potential for control by blocking strain spread, while the diversity within strains is disturbing because it complicates the design of antibiotic policies even during 'single strain' outbreaks.

Anti-Bacterial Agents↗
PubMed2004

Frequency and diversity of Class A extended-spectrum beta-lactamases in hospitals of the Auvergne, France: a 2 year prospective study.

OBJECTIVES: To evaluate the frequency and diversity of extended-spectrum beta-lactamases (ESBLs) produced by Enterobacteriaceae and Pseudomonas aeruginosa in one French region. METHODS: During 2001-2002, all the non-duplicate isolates of P. aeruginosa resistant to ceftazidime and of Enterobacteriaceae intermediate or resistant to ceftazidime and/or cefotaxime and/or aminoglycosides with an AAC(6') I phenotype were collected in nine hospitals of the area. ESBL isoelectric points were determined, bla genes were amplified and sequenced and epidemic isolates were genotyped with ERIC2-PCR. RESULTS: ESBLs were observed in 297 Enterobacteriaceae (0.8%). The most frequent were TEM-3 like (n=152; 51.2%) and TEM-24 (n=115; 38.7%). Four new enzymes were observed, TEM-112 (pI 5.4), TEM-113 (pI 6.3), TEM-114 (pI 5.9) and TEM-126 (pI 5.4). Other TEMs were TEM-8, TEM-12, TEM-16, TEM-19, TEM-20, TEM-21, TEM-29 and TEM-71. The other ESBLs were SHV-4, SHV-5 and SHV-12, CTX-M-1, CTX-M-3, CTX-M-14 and CTX-M-15. In 37 P. aeruginosa (0.7%) only one ESBL was observed, PER-1. Five epidemic strains were detected, Serratia marcescens TEM-3 and four observed in several hospitals, Enterobacter aerogenes TEM-24, Citrobacter koseri TEM-3, Proteus mirabilis TEM-3 and P. aeruginosa PER-1. CONCLUSION: ESBL frequency was lower than in 1998, and CTX-M-type frequency higher (2.1% of ESBLs in 2001, 4.9% in 2002). This long-term survey detected new sporadic enzymes (TEM-112, TEM-113, TEM-114 and TEM-126) and interhospital epidemic strains while avoiding any overestimation of ESBL frequency that may otherwise have occurred because of acute epidemics.

Amino Acid Substitution↗
PubMed2006

Identification and characterization of ceftriaxone resistance and extended-spectrum beta-lactamases in Malawian bacteraemic Enterobacteriaceae.

OBJECTIVES: To enumerate and characterize extended-spectrum beta-lactamases (ESBLs) amongst ceftriaxone-resistant coliforms in Blantyre, Malawi, where third-generation cephalosporin use is currently highly restricted. METHODS: Over the period April 2004-March 2005 all ceftriaxone-resistant isolates from blood cultures were examined for the presence of ESBLs. Isoelectric focusing was performed on enzyme extracts. PCR and DNA sequencing of amplicons were used to identify the underlying genetic determinants responsible for the ESBL phenotypes. Transferability of the ESBL phenotypes was tested by conjugation to a susceptible Escherichia coli J53. RESULTS: Enterobacteriaceae were isolated from 1191 blood cultures, of which 19 (1.6%) were ceftriaxone resistant. Ten isolates (0.7% of all isolates) demonstrated an ESBL phenotype but only eight were characterized as three isolates were from the same patient. Genotypes SHV-11 (n = 1), SHV-12 (n = 3), SHV-27 (n = 1), TEM-63 (n = 2) and CTX-M-15 (n = 1) were detected. Plasmid transfer of the ESBL resistance phenotype was successful for all the isolates. CONCLUSIONS: In a clinical setting of minimal cephalosporin usage there is already a diversity of ESBL genotypes. Increased use of cephalosporins in this setting is likely to result in a rapid expansion of ESBLs and their prevalence will need to be carefully monitored.

Adult↗
PubMed2006

Combination of altered PBPs and expression of cloned extended-spectrum beta-lactamases confers cefotaxime resistance in Haemophilus influenzae.

BACKGROUND: Resistance to beta-lactams in Haemophilus influenzae is mostly due to the presence of TEM beta-lactamases. beta-Lactamase-negative ampicillin resistance (BLNAR) also occurs as a result of PBP3 modifications. BLNAR strains are particularly common in Japan and France, and strains with combined mechanisms of altered PBP3 and beta-lactamase (BLPACR) are emerging. Although the prevalence of beta-lactamase-positive strains is high, TEM-derived extended-spectrum beta-lactamase (ESBL)-mediated cephalosporin resistance, which is common in Enterobacteriaceae, has not been reported in H. influenzae. In this study, the ability of ESBLs TEM-3, -4 and -5 to confer beta-lactam resistance in H. influenzae strains with or without modified PBP3 was tested. METHODS: The genes encoding TEM-1, -3, -4 and -5 were cloned into plasmid pLS88 and were used to transform H. influenzae Rd and BLNAR H. influenzae Rd strains with PBP3 modifications. MICs were determined using CLSI macrobroth dilution. The ability of amoxicillin/clavulanate pre-diffusion disc tests to discriminate between ESBL-producing strains and other strains was also tested. RESULTS: The presence of ESBLs in BLNAR strains conferred cefotaxime resistance, but H. influenzae Rd strains expressing ESBLs and BLNAR strains expressing TEM-1 remained cefotaxime susceptible. The amoxicillin/clavulanate pre-diffusion tests were able to discriminate the ESBL-producing strains from the other strains. CONCLUSIONS: The presence of altered PBP3 increases the effect of ESBLs in recombinant H. influenzae strains to produce cefotaxime resistance. Amoxicillin/clavulanate pre-diffusion disc tests can discriminate ESBL-producing strains from other strains.

Anti-Bacterial Agents↗
PubMed2006

Prior use of carbapenems may be a significant risk factor for extended-spectrum beta-lactamase-producing Escherichia coli or Klebsiella spp. in patients with bacteraemia.

BACKGROUND: The increasing prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae will probably trigger a rise in the use of carbapenems. The effect of these antibiotics on the risk of involvement of ESBL-producing organisms in serious infections is unclear. METHODS: Retrospective analysis of 2172 episodes of healthcare-associated bacteraemia diagnosed during a 3 year period in a teaching hospital. Putative risk factors included demographics, co-morbidities, previous isolation of an ESBL-producing organism and exposure to antibiotics. Univariate and multivariate analysis of the association of risk factors with ESBL-producing organisms was performed in the entire series of bacteraemic episodes and in those due to Escherichia coli or Klebsiella spp. RESULTS: In the entire series, prior isolation of an ESBL-producing organism [odds ratio (OR) 5.9 (3.02, 11.5)]; an ultimately/finally fatal co-morbidity [OR 2.8 (1.55, 4.95)]; renal transplantation [OR 4.3 (1.96, 9.63)]; a urinary source [OR 4.2 (2.22, 7.84)]; shock [OR 2.4 (1.35, 4.1)] and previous use of cephalosporins [OR 2.6 (1.54, 4.51)], carbapenems [OR 2.5 (1.24, 5.05)] and glycopeptides [OR 0.4 (0.13, 0.93)] were significantly associated with ESBL-producing E. coli or Klebsiella spp. by multivariate analysis. Prior isolation of an ESBL-producing organism, an ultimately/finally fatal co-morbidity, renal transplantation, and previous use of cephalosporins and carbapenems were also significant in the analysis restricted to episodes due to E. coli or Klebsiella spp. CONCLUSIONS: In patients with healthcare-associated bacteraemia, prior use of carbapenems may be only second to cephalosporins as the most significant antibiotic exposure associated with the involvement of ESBL-producing organisms.

Adult↗
PubMed2006

Detection of extended-spectrum beta-lactamases in clinical isolates of Pseudomonas aeruginosa.

With the occurrence of extended-spectrum beta-lactamases (ESBLs) in Pseudomonas aeruginosa being increasingly reported worldwide, there is a need for a reliable test to detect ESBLs in clinical isolates of P. aeruginosa. In our study, a total of 75 clinical isolates of P. aeruginosa were studied. Nitrocefin tests were performed to detect the beta-lactamase enzyme; isoelectric focusing electrophoresis, PCR, and PCR product sequencing were designed to further characterize the contained ESBLs. Various ESBL-screening methods were designed to compare the reliabilities of detecting ESBLs in clinical isolates of P. aeruginosa whose beta-lactamases were well characterized. Thirty-four of 36 multidrug-resistant P. aeruginosa clinical isolates were positive for ESBLs. bla(VEB-3) was the most prevalent ESBL gene in P. aeruginosa in our study. Among the total of 34 isolates that were considered ESBL producers, 20 strains were positive using conventional combined disk tests and 10 strains were positive using a conventional double-disk synergy test (DDST) with amoxicillin-clavulanate, expanded-spectrum cephalosporins, aztreonam, and cefepime. Modifications of the combined disk test and DDST, which consisted of shorter distances between disks (20 mm instead of 30 mm) and the use of three different plates that contained cloxacillin (200 microg/ml) alone, Phe-Arg beta-naphthylamide dihydrochloride (MC-207,110; 20 microg/ml) alone, and both cloxacillin (200 microg/ml) and MC-207,110 (20 microg/ml) increased the sensitivity of the tests to 78.8%, 91.18%, 85.29%, and 97.06%.

Bacterial Proteins↗
PubMed2002

Mechanisms of decreased susceptibility to cefpodoxime in Escherichia coli.

Cefpodoxime is one of five antimicrobial agents recommended by the National Committee for Clinical Laboratory Standards for screening isolates of Klebsiella spp. and Escherichia coli for extended-spectrum beta-lactamase (ESBL) production. In a prior study, we noted that among 131 E. coli isolates for which the MIC of at least one extended-spectrum cephalosporin (ESC) or aztreonam was > or =2 micro g/ml (suggesting the presence of ESBL production), there were 59 isolates (45.0%) for which the MIC of cefpodoxime was 2 to 4 micro g/ml (i.e., a positive ESBL screening test), but the MICs of ceftazidime, cefotaxime, and ceftriaxone were < or =1 micro g/ml (below the ESBL screening breakpoint). Thus, the results appeared to be false-positive ESBL screening tests. These 59 isolates were divided into five phenotypic groups based on the susceptibility patterns of the organisms to a variety of beta-lactam agents and further characterized. The first group (32 isolates) all produced a TEM-1 beta-lactamase, and changes in the major outer membrane proteins were detected in representative strains. The second group (18 isolates) lacked bla(TEM) but showed a number of porin changes; some also showed a modest elevation in production of the AmpC chromosomal beta-lactamase. In the third phenotypic group (seven isolates) all expressed an OXA-30 beta-lactamase. Some also harbored altered porins. The two remaining phenotypes each had a distinct pattern of porin changes with or without beta-lactamase production. These data indicate that several factors are associated with decreased susceptibility to cefpodoxime in E. coli, but none of the mechanisms are related to ESBL production. Current screening methods produced false-positive ESBL results for these isolates. Such isolates should not be classified as containing ESBLs, nor should interpretations of ESCs or aztreonam susceptibility be changed to resistant on test reports for these isolates.

Ceftizoxime↗
PubMed2002

Energy-dependent accumulation of norfloxacin and porin expression in clinical isolates of Klebsiella pneumoniae and relationship to extended-spectrum beta-lactamase production.

The relationships between porin deficiency, active efflux of fluoroquinolones, and extended-spectrum beta-lactamase (ESBL) production were determined for 53 clinical isolates of Klebsiella pneumoniae. Thirty-two ESBL-positive strains (including 22 strains expressing porins and 10 strains lacking porins) and 21 ESBL-negative strains were evaluated. Active efflux of norfloxacin was defined as a >/=50% increase in the accumulation of norfloxacin in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) in comparison with the corresponding basal value in the absence of CCCP. The quinolone resistance-determining regions of both gyrA and parC from 13 strains, representing all isolates with different porin profiles and with or without active efflux, were determined. Porin loss was significantly more common among ESBL-positive strains (10 of 32 [31.2%]) than among ESBL-negative strains (0 of 2 [0%]) (P < 0.01). Active efflux was observed in 7 of 10 (70%) strains lacking porins and in 4 of 43 (9.3%) strains producing porins (P < 0.001). The 11 strains showing active efflux corresponded to 3 of 21 (14.3%) ESBL-negative strains and 8 of 32 (25.5%) ESBL-positive strains (P > 0.05). Basal values of norfloxacin accumulation were higher in strains lacking active efflux than in those that had this mechanism (P < 0.05). In the absence of topoisomerase changes, the contribution of either porin loss or active efflux to fluoroquinolone resistance in K. pneumoniae was negligible. It is concluded that among K. pneumoniae strains of clinical origin, porin loss was observed only in those producing ESBL, and that a significant number of porin-deficient strains also expressed active efflux of norfloxacin. In terms of fluoroquinolone resistance, both mechanisms are significant only in the presence of topoisomerase modifications.

Anti-Infective Agents↗
PubMed2003

Extended-spectrum beta-lactamase-producing Enterobacteriaceae in community and private health care centers.

In 1999, 39 of 2,599 isolates of the family Enterobacteriaceae (1.5%) collected by eight private laboratories in the Aquitaine region in France produced an extended-spectrum beta-lactamase (ESBL). Among these were 19 Enterobacter aerogenes isolates; 8 Klebsiella pneumoniae isolates; 6 Escherichia coli isolates; 3 Proteus mirabilis isolates; and 1 isolate each of Serratia marcescens, Morganella morganii, and Providencia stuartii. ESBL producers were isolated from 38 patients, including 33 residents of 11 clinics or nursing homes and 5 ambulatory patients. Seven different ESBLs were characterized. These mainly consisted of TEM-24 (25 isolates) and TEM-21 (9 isolates), but TEM-15 (2 isolates) and TEM-3, TEM-19, SHV-4, and CTX-M-1 (1 isolate each) were also characterized. Seven strains showed the coexistence of different TEM- and/or SHV-encoding genes, including a new SHV-1 variant, SHV-44, defined by the substitution R205L previously reported for SHV-3 in association with S238G. The epidemiology of the ESBL producers was investigated by random amplification of polymorphic DNA, typing by enterobacterial repetitive intergenic consensus PCR, analysis of resistance cotransferred with the ESBL, and analysis of the restriction profiles of the ESBL-encoding plasmids. Of the TEM-24-expressing strains, 18 were E. aerogenes isolates, including 9 from the same clinic, that were representatives of the epidemic clone disseminating in France. Of the TEM-21-producing strains that belonged to different species of the family Enterobacteriaceae (E. coli, K. pneumoniae, and P. mirabilis), 8 were isolated in the same nursing home. Outbreaks due to strain and/or plasmid dissemination in these clinic and nursing home were demonstrated. The presence of ESBL producers in five ambulatory patients probably resulted from nosocomial acquisition. Our data highlight the serious need to monitor patients for ESBL-producing Enterobacteriaceae in general practice.

Cloning, Molecular↗
PubMed2002

Epidemiology of extended-spectrum beta-lactamase-producing Enterobacter isolates in a Spanish hospital during a 12-year period.

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.

Bacterial Proteins↗
PubMed2005

SHV-type extended-spectrum beta-lactamase production is associated with Reduced cefepime susceptibility in Enterobacter cloacae.

Cefepime is a potentially useful antibiotic for treatment of infections with Enterobacter cloacae. However, in our institution the MIC(90) for E. cloacae bloodstream isolates is 16 microg/ml. PCR amplification of bla genes revealed that one-third (15/45) of E. cloacae bloodstream isolates produced SHV-type extended-spectrum beta-lactamases (ESBLs) in addition to hyperproduction of AmpC-type beta-lactamases. The majority (11/15) of ESBL producers also produced the TEM-1 beta-lactamase. The SHV types included SHV-2, -5, -7, -12, -14, and -30. All but two of the ESBL-producing E. cloacae isolates, but none of the non-ESBL-producing strains, had MICs of cefepime of >or=2 microg/ml. The MIC(90) for cefepime for ESBL-producing strains was 64 mug/ml, while for non-ESBL producers it was 0.5 microg/ml. Using current Clinical and Laboratory Standards Institute breakpoints for cefepime, two thirds (10/15) of ESBL-producing isolates would have been regarded as susceptible to cefepime. Phenotypic ESBL detection methods were generally unreliable with these E. cloacae isolates. Based on these results, pharmacokinetic, pharmacodynamic, and clinical reevaluation of cefepime breakpoints for E. cloacae may be prudent.

Anti-Bacterial Agents↗
PubMed2005

[Comparison of three microbiological methods for detection of expanded-spectrum betalactamases in Enterobacteriaceae isolated in Santa Fe (Argentina)].

INTRODUCTION: Expanded-spectrum betalactamases (ESBLs) are the main source of resistance to oxyimino cephalosporins and monobactams in Enterobacteriaceae. Most of them derive from TEM or SHV, however the incidence of other families like CTX-M, OXA and PER has increased. In Argentina, the most frequent ESBL in Enterobacteriaceae is CTX-M-2. This specific circumstance, which differs from the situation in the Northern Hemisphere, motivated us to study new diagnostic strategies for the detection of ESBLs in our region. METHOD: Microbiological ESBL detection was performed by double-disk synergy tests, cefotaxime and ceftazidime disks with and without clavulanic acid (NCCLS), and cefotaxime and ceftazidime disks in Müeller-Hinton agar supplemented with lithium clavulanate (MH-cla). Betalactamases were characterized by isoelectric focusing, hydrolysis profile and PCR amplification. RESULTS: Among 575 clinical isolates of Enterobacteriaceae, 14% were oxyimino cephalosporin-resistant. Two different ESBLs were detected in 31 resistant strains: CTX-M-2 (28) and PER-2 groups (3). The double-disk synergy test was the least sensitive method for ESBL detection. ESBLs were detected by the other two methods in all isolates with the use of cefotaxime disks, but not with ceftazidime disks. CONCLUSION: The microbiological method employing MH-cla with cefotaxime disks had a sensitivity and specificity comparable to the referral test using the same antibiotic proposed by the NCCLS for the detection of ESBLs.

Argentina↗