ESBL Testing Research Articles

Risk Factors for Community-onset Urinary Tract Infections due to Extended-spectrum β-lactamase Producing Bacteria in Children

β the community. The aim of this study was to identify risk factors for community-onset urinary tract infections (UTIs) by ESBL producers in children. Materials and Methods : We analyzed 854 episodes of community-onset UTIs in children diagnosed at CHA Bundang Medical Center from January, 2004 to April, 2009. The presence of ESBL in Escherichia coli and Klebsiella spp. was screened and confirmed by VITEK ® -2 ESBL test. Controls were matched in a 2:1 ratio to case patients by age and sex. The clinical characteristics, risk factors, antimicrobial resistance, and treatment efficacy were compared with controls. Results : The most common pathogen was Escherichia coli (681 isolates), followed by Klebsiella pneumoniae (60 isolates), Enterococcus faecalis (23 isolates), etc. The number of isolated ESBL producers among the pathogens E. coli and K. pneumoniae was 25 (3.7%) and 7 isolates (11.7%), respectively. No significant differences were noted as to the UTI history prior to the present treatment, underlying urogenital anomaly, and clinical characteris - tics. Use of any antibiotics within a month ( P=0.012, Odds ratio, 6.341, 95% confidence interval, 1.492 to 26.955) was related to the increased risk of community-onset UTI by ESBL producers. Conclusions : ESBL-producing bacteria has been identified in pediatric community-onset UTI, and the use of any antibiotics within the previous month was related to the increased risk of UTI by ESBL producers. Therefore, careful selection and use of antibiotics should be recommended for community-onset UTI in children.

Evaluation of a New Selective Chromogenic Agar Medium for Detection of Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae

A novel chromogenic agar medium (ESBL-Bx; bioMérieux, Marcy l'Etoile, France) was compared to MacConkey agar supplemented with 2 mg ceftazidime/liter (MCKC) for the selective isolation and presumptive identification of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae directly from clinical samples. Of a total of 644 clinical specimens (including 551 fecal samples), 496 yielded no growth and 148 yielded growth on one or both media. Overall, 44 ESBL-producing Enterobacteriaceae strains (Escherichia coli [n=17], Enterobacter aerogenes [n=17], Klebsiella spp. [n=5], and Citrobacter freundii [n=5]) were isolated from 37 specimens by a combination of both methods after 18 to 24 h of incubation. The sensitivities were 97.7 and 84.1% for ESBL-Bx and MCKC, respectively, with 43 ESBL-positive strains isolated as colored colonies from 36 specimens on ESBL-Bx versus 37 ESBL-positive organisms isolated from 32 specimens on MCKC. The specificities by specimens were 89 and 91% for ESBL-Bx and MCKC, respectively. On either one of the two media, natural AmpC-hyperproducing Enterobacter spp. (n=25) and Citrobacter spp. (n=14) were the most common false positives as well as non-ESBL-producing Klebsiella oxytoca (n=18) on ESBL-Bx and Morganella morganii (n=10) on MCKC. We conclude that ESBL-Bx is a sensitive and specific medium for the isolation of ESBL-producing Enterobacteriaceae from clinical samples. The main advantages of ESBL-Bx over MCKC reside in its chromogenic character and its sensitivity and selectivity, which enabled the recovery and presumptive identification of most ESBL-producing Enterobacteriaceae within 24 h and reduced by 27% the need for unnecessary identification and confirmation of ESBL testing when disregarding all colorless colonies growing on this medium.

Occurrence of plasmidic AmpC type β-lactamase-mediated resistance in Escherichia coli: report from the SENTRY Antimicrobial Surveillance Program (North America, 2004)

Among 1429 Escherichia coli isolates collected as part of the SENTRY Antimicrobial Surveillance Program (2004) from 30 North American medical centres, 65 (4.5%) were screen-positive for an extended-spectrum β-lactamase (ESBL). Among the strains with a negative ESBL confirmatory test ( n = 26; clavulanic acid inhibition), a CMY-2 enzyme was detected in 13 isolates (50.0%), FOX-5 in 3 isolates (11.5%) and DHA-1 in 1 isolate (3.8%). These AmpC-producing E. coli were cephamycin (cefoxitin)-resistant but susceptible to cefepime (minimum inhibitory concentration (MIC) ≤0.12–4 mg/L). Clearly, the ESBL tests recommended by the Clinical and Laboratory Standards Institute identify only a fraction of E. coli with elevated β-lactam MIC values as ESBL-producing strains; the majority of the remaining strains would be potentially responsive to some other β-lactams, directed by accurately performed and interpreted susceptibility methods.

Evaluation of the New VITEK 2 Extended-Spectrum Beta-Lactamase (ESBL) Test for Rapid Detection of ESBL Production in Enterobacteriaceae Isolates

Extended-spectrum beta-lactamases (ESBLs) are a large, rapidly evolving group of enzymes that confer resistance to oxyimino cephalosporins and monobactams and are inhibited by clavulanate. Rapid reliable detection of ESBL production is a prerequisite for successful infection management and for monitoring resistance trends and implementation of intervention strategies. We evaluated the performance of the new VITEK 2 ESBL test system (bioMérieux, Inc, Hazelwood, Mo.) in the identification of ESBL-producing Enterobacteriaceae isolates. We examined a total of 1,129 clinically relevant Enterobacteriaceae isolates (including 218 that had been previously characterized). The ESBL classification furnished by the VITEK 2 ESBL test system was concordant with that of the comparison method (molecular identification of beta-lactamase genes) for 1,121 (99.3%) of the 1,129 isolates evaluated. ESBL production was correctly detected in 306 of the 312 ESBL-producing organisms (sensitivity, 98.1%; positive predictive value, 99.3%). False-positive results emerged for 2 of the 817 ESBL-negative isolates (specificity, 99.7%; negative predictive value, 99.3%). VITEK 2 ESBL testing took 6 to 13 h (median, 7.5 h; mean +/- SD, 8.2 +/- 2.39 h). This automated short-incubation system appears to be a rapid and reliable tool for routine identification of ESBL-producing isolates of Enterobacteriaceae.

Prevalence of Newer β-Lactamases in Gram-Negative Clinical Isolates Collected in the United States from 2001 to 2002

Newer beta-lactamases such as extended-spectrum beta-lactamases (ESBLs), transferable AmpC beta-lactamases, and carbapenemases are associated with laboratory testing problems of false susceptibility that can lead to inappropriate therapy for infected patients. Because there appears to be a lack of awareness of these enzymes, a study was conducted during 2001 to 2002 in which 6,421 consecutive, nonduplicate clinical isolates of aerobically growing gram-negative bacilli from patients at 42 intensive care unit (ICU) and 21 non-ICU sites across the United States were tested on-site for antibiotic susceptibility. From these isolates, 746 screen-positive isolates (11.6%) were referred to a research facility and investigated to determine the prevalence of ESBLs in all gram-negative isolates, transferable AmpC beta-lactamases in Klebsiella pneumoniae, and carbapenemases in Enterobacteriaceae. The investigations involved phenotypic tests, isoelectric focusing, beta-lactamase inhibitor studies, spectrophotometric assays, induction assays, and molecular analyses. ESBLs were detected only in Enterobacteriaceae (4.9% of all Enterobacteriaceae) and were found in species other than those currently recommended for ESBL testing by the CLSI (formerly NCCLS). These isolates occurred at 74% of the ICU sites and 43% of the non-ICU sites. Transferable AmpC beta-lactamases were detected in 3.3% of K. pneumoniae isolates and at 16 of the 63 sites (25%) with no difference between ICU and non-ICU sites. Three sites submitted isolates that produced class A carbapenemases. No class B or D carbapenemases were detected. In conclusion, organisms producing ESBLs and transferable AmpC beta-lactamases were widespread. Clinical laboratories must be able to detect important beta-lactamases to ensure optimal patient care and infection control.

WITHDRAWN: “An audit of ESBL and/or AMPC B-lactamase testing methodology, associated infection management, control measures and prevalence in the merseyside and cumbria/lancashire hospitals”

WITHDRAWN: “An audit of ESBL and/or AMPC B-lactamase testing methodology, associated infection management, control measures and prevalence in the merseyside and cumbria/lancashire hospitals”

Characterization of extended-spectrum β-lactamase (ESBL)-producing Kuwait and UK strains identified by the Vitek system, and subsequent comparison of the Vitek system with other commercial ESBL-testing systems using these strains

Two hundred and fifty-one unique patient isolates of Klebsiella pneumoniae (123), Escherichia coli (114), Klebsiella oxytoca (7), Enterobacter cloacae (5) and Citrobacter freundii (2), flagged as extended-spectrum beta-lactamase (ESBL) positive by the Vitek system (GNS-526 card), were collected. These strains were isolated from a variety of clinical specimens submitted to the clinical bacteriology laboratories of the Royal Infirmary of Edinburgh (RIE), Edinburgh, UK (and associated GP practices), Hairmyers Hospital, Glasgow, UK, and the Amiri and Farwania Hospitals, Kuwait. Of the 101 RIE strains tested, 15 E. coli strains were found to be ESBL negative by Etest ESBL strips. On retesting the 15 E. coli strains with the Vitek GNS-532 card, 14 were found to be ESBL negative, despite being originally flagged as ESBL positive. The remaining 236 ESBL-producing strains were also subjected to the double disc-diffusion (DDD) technique for the detection of ESBLs. Of these, two were false negatives by Etest ESBL test strips (using both cefotaxime and ceftazidime strips), and 38 were false negatives by the DDD method. The Etest false-negative ESBL-producing strains of K. pneumoniae were positive by DDD. Technically, the Vitek method was the least demanding method to perform, as it was an integral part of the routine susceptibility test card. Etest strips were reliable, but were the most expensive of all the techniques used. The DDD test, while relatively inexpensive, was technically subjective, and in our hands, seven of the ESBL-positive strains that were confirmed by the other two techniques were not detected. Despite the false-positive ESBL-producing E. coli strains, the Vitek susceptibility card with its integral ESBL test offers the clinical laboratory a valuable and quick option to screen for ESBL-producing Klebsiella spp. and E. coli as part of the routine laboratory methodology.

Study on plasmid-mediated AmpC β-lactamases and their resistance phenotypes in K.pneumonia

目的 证实临床分离的部分肺炎克雷伯菌(Kp)除产超广谱β内酰胺酶(ESBLs)外,还同时产AmpC酶,并探讨了AmpC酶编码基因存在方式.方法对临床分离的可疑同时产ESBLs和其他高活性广谱β内酰胺酶的8株Kp进行接合试验,并对供体菌和接合子同时采用三维试验、ESBLs试验、AmpC酶诱导试验和β内酰胺类抗生素最低抑菌浓度(MIC)试验测定,并进行AmpC基因携带状态、表达方式和酶活性分析.结果 8株Kp菌通过接合试验共得到10株接合子,其中有6株供体菌均只得到同时表达AmpC和ESBLs的1种接合子;另2株供体菌,各得2株接合子,1株同时表达AmpC和ESBLs,1株仅表达ESBLs;8株供体菌均未得到单独表达AmpC的接合子.表达AmpC接合子的耐药表型与受体菌非常接近,酶的活性也必须用克拉维酸(CA)和邻氯西林(CLO)两种酶抑制剂才能完全抑制,其中有5株可被头孢西丁(FOX)诱导使某些指示药出现截平现象.结论在Kp中,已出现质粒携带的AmpC基因,有可能与编码ESBLs的基因存在于同一质粒上,并具有诱导型和非诱导型两种表达形式。

Investigations of multiresistance to antibiotics and chemotherapeutics and extended spectrum beta: Lactamase effect (ESBL test) in strains E.coli and salmonella originating from domestic animals

The presence of multiresistance to the effects of antibiotics and chemotherapeutics and extended spectrum beta-lactamase were examined in 45 strains of E. coli and 35 strains of Salmonella. The strains of E. coli originated from several species of domestic animals: dogs, cats, poultry, and cattle, and 30 strains of Salmonella originated from poultry, 4 strains from cattle, and 1 strain from swine. The presence of the following serovarieties was established using serological examinations: Salmonella Enteritidis 17 strains, Salmonella Gallinarum 1 strain, Salmonella Hartford 5 strains, Salmonella Anatum 1 strain, Salmonella Typhimurium 4 strains, Salmonella Agona 1 strain, Salmonella Infantis 1 strain, Salmonella Thompson var. Berlin 1 strain, Salmonella Tennessee 1 strain, Salmonella Senftenberg 1 strain, Salmonella Glostrup 1 strain, and Salmonella Hadar 1 strain. In the examinations of the listed strains we used antibiogram discs of ampicillin, amoxicillin with clavulanic acid, cephalexin, cephtriaxon, cephotaxim, cephtazidime, aztreonam, gentamycin, chloramphenicol, tetracycline, cyprofloxacine, and a combination of sulphamethoxasole and trimethoprim. The lowest prevalence of multiresistance in E. Coli strains to 3 or more antibiotics was established in dogs 20%, and the highest in 60% strains originating from swine. In 62.88% strains of Salmonella we established sensitivity to all applied antibiotics. Resistance was also established in a small number of the examined strains to ampicillin (11 strains), to tetracycline (5 strains), to amoxicillin with clavulanic acid (5 strains), to sulphamethoxasole with trimethoprim (5 strains), to gentamycin (3 strains), and to cloramphenicol (1 strain). Of all the examined strains of Salmonella, 6 strains originating from poultry exhibited multiresistence. The presence of extended spectrum beta-lactamase effects examined using the ESBL test, was not established in strains of E. coli and Salmonella strains.

Evaluation of an Accelerated Protocol for Detection of Extended-Spectrum β-Lactamase-Producing Gram-Negative Bacilli from Positive Blood Cultures

We evaluated a protocol for the accelerated detection of extended-spectrum beta-lactamases (ESBLs) in gram-negative bloodstream pathogens. Two hundred eighty-three blood culture bottles were subjected to direct ESBL testing by inoculating samples directly from blood culture bottles onto agar plates containing cefotaxime and ceftazidime disks, with and without clavulanate. Standard ESBL testing in accordance with the NCCLS guidelines after subculturing on agar plates was performed in parallel. Results of the direct ESBL testing were reported 2.3 days sooner and were comparable to those of the standard NCCLS method with sensitivity, specificity, and positive and negative predictive values of 100, 98, 94, and 100%, respectively.

Phenotypic and Molecular Detection of CTX-M-β-Lactamases Produced by Escherichia coli and Klebsiella spp

Organisms producing CTX-M-beta-lactamases are emerging around the world as a source of resistance to oxyiminocephalosporins such as cefotaxime (CTX). However, the laboratory detection of these strains is not well defined. In this study, a molecular detection assay for the identification of CTX-M-beta-lactamase genes was developed and used to investigate the prevalence of these enzymes among clinical isolates of Escherichia coli and Klebsiella species in the Calgary Health Region during 2000 to 2002. In addition, National Committee for Clinical Laboratory Standards (NCCLS) recommendations were evaluated for the ability to detect isolates with CTX-M extended-spectrum beta-lactamases (ESBLs). The PCR assay consisted of four primer sets and demonstrated 100% specificity and sensitivity for detecting different groups of CTX-M-beta-lactamases in control strains producing well-characterized ESBLs. Using these primer sets, 175 clinical strains producing ESBLs were examined for the presence of CTX-M enzymes; 24 (14%) were positive for bla(CTX-M-1-like) genes, 95 (54%) were positive for bla(CTX-M-14-like) genes, and the remaining 56 (32%) were negative for bla(CTX-M) genes. Following the NCCLS recommendations for ESBL testing, all of the control and clinical strains were detected when screened with cefpodoxime and when both cefotaxime and ceftazidime with clavulanate were used as confirmation tests.

Extended-Spectrum β-Lactamases (ESBLs): Characterization, Epidemiology and Detection

β-lactamases of Enterobacteriaceae are the most important mechanism of resistance against β-lactam drugs. Two types of β-lactamases can confer resistance against 3rd generation cephalosporins. Chromosomally mediated β-lactamases are inducible and are not inhibited by clavulanic acid. Resistance due to these enzymes is non-transferable. The 2nd type of enzyme is plasmid-mediated β-lactamases, which are inhibited by clavulanic acid. These enzymes are more important clinically as these can be transferred between various species of Enterobacteria ceae. These enzymes are called extended-spectrum β-lactamases (ESBLs). ESBL-producing Enterobacteriaceae have been responsible for numerous outbreaks of infection throughout the world and pose challenging infection control issues. Antibacterial choice is often complicated by multi-resistance. ESBLs can confer resistance against all β-lactam drugs except carbapenems and cephamycins. Nursing home patients may be an important reservoir of ESBL-containing multiple antibiotic-resistant organisms. Use of broad-spectrum oral antibiotics and probably poor infection control practices may facilitate spread of this plasmid-mediated resistance. In addition to known populations at risk, ambulatory patients with chronic conditions represent another patient population that may harbor ESBL-producing organisms. Various methods can be used for detection of ESBLs in the laboratory. These tests include double disc diffusion test, Vitek ESBL test, E Tests, MIC Determination, genetic method, and isoelectric focusing (IEF).

Evaluation of MicroScan ESBL confirmation panel for Enterobacteriaceae-producing, extended-spectrum β-lactamases isolated in Japan

We assessed use of the MicroScan ESBL confirmation panel (Dade Behring, Tokyo, Japan) for the detection of eight Enterobacteriaceae-producing extended-spectrum β-lactamases (ESBL) species. Of 137 bacterial strains isolated from patients in 32 hospitals in the Kinki area of Japan, 91 produced ESBL and comprised 60 bacteria (of E. coli, K. oxytoca, and K. pneumoniae) targeted by the NCCLS ESBL test and 31 non-target bacteria such as chromosomal AmpC-producing bacteria (e.g., Serratia marcescens, Enterobacter spp.). Sensitivity and specificity of the MicroScan panel for the target bacteria were 92% and 93%, respectively; sensitivity and specificity for non-target bacteria were 52% and 100%, respectively. There were 20 ESBL-positive strains that were not inhibited by clavulanic acid in the MicroScan panel (3 of 32 ESBL-producing E. coli strains, 1 of 24 K. pneumoniae, 1 of 4 K. oxytoca, 8 of 13 E. cloacae, and 7 of 14 S. marcescens), and most of them were bacteria not targeted by the NCCLS test. In 19 of the 20 strains, the synergy effect of clavulanic acid was observed in the modified-double-disk synergy test using only the cefepime-disk. Because these strains had high MICs of ≥ 16 μg/ml for cephamycins such as cefoxitin and cefmetazole, these strains might produce high levels of AmpC in addition to ESBL. The MicroScan ESBL confirmation panel showed excellent performance in detecting target, but not other bacteria. Addition of cefepime and clavulanic acid to the MicroScan panel may significantly improve detection of non-target bacteria.

Further Occurrence of Extended-Spectrum β-Lactamase-Producing Salmonella enteritidis

We describe the further occurrence of Salmonella enteritidis producing an extended spectrum β-lactamase (ESBL) and transferring cefotaxime, ceftazidime and aztreonam resistance. In a previous communication we describe the isolation of a strain of S. enteritidis (No. 35) from a septic neonate with high-level resistance to cefuroxime, cefotaxime, ceftazidime and aztreonam. Six weeks later, a second neonate was found, in the same Pediatric University Hospital, to be infected with apparently the same strain of S. enteritidis (No. 100) which was isolated from blood with similar properties as the strain No. 35. In this strain we were able to study, in more detail, the transferability of its resistance to 3rd generation cephalosporins and its ESBL production. In this report we describe results of transfer experiments, relative rates of hydrolysis (Vmax) and ESBL tests which indicate that the capacity to produce ESBL was transferred from the multiply-resistant strain of S. enteritidis No. 100 to S. typhimurium LT-2 rif+str+.