Outer membrane permeation of beta-lactam antibiotics in Escherichia coli, Proteus mirabilis, and Enterobacter cloacae.

Mutant strains lacking outer membrane protein(s) were isolated from Escherichia coli, Proteus mirabilis, and Enterobacter cloacae. The outer membrane protein(s) of P. mirabilis and E. cloacae corresponding to E. coli porin were identified on the basis of their function, namely, their ability to allow the permeation of glucose as demonstrated by [14C]glucose uptake by intact cells. P. mirabilis has only one outer membrane pore protein (molecular weight, 40,000), but E. cloacae has at least two such proteins (molecular weights, 37,000 and 39,000 to 40,000). When the bacteria lost these proteins or porin, the outer membrane permeation of cefazolin was found to be greatly reduced in these three species. Such a change in the outer membrane permeation closely correlated with a significant decrease in the bacterial susceptibility to cephalosporins, including cefoxitin. These results suggested that the main pathway for cephalosporin permeation is the pore made up of these proteins. The 39,000- to 40,000-molecular-weight protein in E. cloacae was also assumed to play an important role in the outer membrane permeation of tetracycline and chloramphenicol. On the other hand, the permeation route of penicillins was obscure. The susceptibility to penicillins, except in some cases, was little influenced by the absence of the proteins. Ampicillin was found to pass through the outer membrane via the same route as the cephalosporins, but the possibility that ampicillin and other penicillins possess another unknown route for outer membrane permeation could not be ruled out.     

Dissemination of Proteus mirabilis isolates harboring CTX-M-14 and CTX-M-3 beta-lactamases at 2 hospitals in Taiwan

From February to June 2003, 111 clinical isolates of Proteus mirabilis were mainly isolated from patients with respiratory or urinary tract infections hospitalized at 3 district hospitals (A, B, C) in central Taiwan. Among them, 34 (30.6%) strains, isolated within 2 hospitals (A and B), exhibited nonsusceptibility to cefotaxime with significant reduction of MIC (> or = 3 log2 dilution) by the effect of clavulanic acid, which confirmed the phenotype of extended-spectrum beta-lactamases (ESBLs). These ESBL producers were coresistant to gentamicin, isepamicin, and amikacin, but remained susceptible to ceftazidime (MIC, < or = 0.5 microg/mL) and meropenem (MIC, <0.5 microg/mL). By isoelectric focusing analysis, polymerase chain reaction, and nucleotide sequencing, we detected the presence of CTX-M-14 in 33 strains and CTX-M-3 in 6 strains (5 strains harboring both CTX-M-14 and CTX-M-3 enzymes). These beta-lactamase genes can be successfully transferred by the conjugative plasmid. Molecular epidemiology of the 34 ESBL-producing P. mirabilis strains by pulsed-field gel electrophoresis using SfiI restriction enzyme revealed 9 different genotypes, suggesting epidemic clones with intra- and interhospital spread. In conclusion, the broadly extended clonal spreading of CTX-M-type P. mirabilis was first discovered at the district hospitals in Taiwan.     

产超广谱β-内酰胺酶大肠埃希菌和肺炎克雷伯菌的检测

目的 选择和适用于检测产超广谱β－内酰安酶（ＥＳＢＬｓ）临床大肠埃希菌、肺炎克雷伯耐药菌株的最佳方案。方法 临床分离的１１０株大肠埃希菌和８４株肺炎克雷伯菌经初筛试验,用浓度梯度法（Ｅｔｅｓｔ）、单纸片加抑制剂舒巴坦扩散法、阿莫西林及氨苄西林双纸片协同试验,比较５种方法的检出率极度其差异。结果 Ｅｔｅｓｔ法对产ＥＳＢＬｓ大肠埃希菌和肺炎克雷伯菌的检出存在明显的差异（Ｐ〈０．０１）,肺炎克雷伯菌的检出率（４／２０）有显低于大肠埃希菌（１６／２６）;单纸片扩散法加克拉维酸对两种菌的检出均较高（１６／２６、１２／２０）。检测产ＥＳＢＬｓ大肠埃希菌时,除氨 苄西林双纸片协同法（８／２６）检出率低以外,其他４种方法的检出率无明显差异（Ｐ〉０．０５）,５种方法有较好的一致性。检测产ＥＳＢＬｓ肺炎克雷伯菌时,单纤片扩散法加克     

Evaluation of screening methods to detect plasmid-mediated AmpC in Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis

There are currently no standardized phenotypic methods for the screening and detection of AmpC enzymes. This study aimed to evaluate different methods to detect AmpC enzymes in Escherichia coli, Klebsiella spp., and Proteus spp., comparing the results from two disk-based methods and an agar dilution method. AmpC activity was determined for 255 clinical isolates by use of a three-dimensional enzyme assay combined with a multiplex PCR assay for plasmid-borne ampC genes. These results were compared against a disk-based inhibitor assay using various combinations of cefpodoxime and cefoxitin as antibiotic substrates and boronic acid or cloxacillin as an AmpC inhibitor. The presence of enzyme induction by disk approximation was evaluated using imipenem, cefoxitin, and amoxicillin-clavulanate as inducing agents against ceftazidime. Finally, an agar dilution assay was performed, using cefoxitin with and without added cloxacillin. AmpC activity was present in 49.8% of test isolates, 93.7% of which were positive for plasmid-borne ampC genes. CIT-like enzymes were predominant in E. coli, and DHA-like enzymes were predominant in Klebsiella spp. The disk-based inhibitor tests performed better than the agar dilution assay, while detection of AmpC by disk induction had a poor sensitivity. The cefoxitin-cloxacillin disk combination provided the best overall performance, with a sensitivity and specificity of 95%. This study confirmed the accuracy of disk-based inhibitor screening for AmpC enzymes, which proved reliable at detecting CIT- and DHA-like plasmid-borne ampC genes. The methods are simple enough for introduction into clinical microbiology laboratories.     

CTX-M-2 and a new CTX-M-39 enzyme are the major extended-spectrum beta-lactamases in multiple Escherichia coli clones isolated in Tel Aviv, Israel

The rate of occurrence of the extended-spectrum beta-lactamase (ESBL)-producing phenotype among Escherichia coli isolates in Tel Aviv is 12% (22). The aim of this study was to understand the molecular epidemiology of E. coli ESBL producers and to identify the ESBL genes carried by them. We studied 20 single-patient ESBL-producing E. coli clinical isolates. They comprised 11 distinct nonrelated pulsed-field gel electrophoresis (PFGE) genotypes: six isolates belonged to the same PFGE clone, four other clones included two isolates each, and six unrelated clones included only one isolate. All isolates produced various beta-lactamases with pIs ranging from 5.2 to 8.2, varying within similar PFGE clones. The most prevalent ESBL gene was bla(CTX-M); 16 isolates carried bla(CTX-M-2) and three carried a new ESBL gene designated bla(CTX-M-39). Three strains carried bla(SHV) (two bla(SHV-12) and one bla(SHV-5)), and two strains carried inhibitor-resistant ESBL genes, bla(TEM-33) and bla(TEM-30); 18 strains carried bla(TEM-1) and eight strains carried bla(OXA-2). Plasmid mapping and Southern blot analysis with a CTX-M-2 probe demonstrated that bla(CTX-M-2) is plasmid borne. The wide dissemination of ESBLs among E. coli isolates in our institution is partly related to clonal spread, but more notably to various plasmid-associated ESBL genes, occurring in multiple clones, wherein the CTX-M gene family appears almost uniformly. We report here a new CTX-M gene, designated bla(CTX-M-39), which revealed 99% homology with bla(CTX-M-26), with a substitution of arginine for glutamine at position 225.     

Prevalence of extended-spectrum beta-lactamases in Proteus mirabilis in a Taiwanese university hospital, 1999 to 2005: identification of a novel CTX-M enzyme (CTX-M-66)

A total of 1574 nonduplicate Proteus mirabilis isolates collected at a Taiwanese hospital during 1999 to 2005 were analyzed for production of extended-spectrum beta-lactamases (ESBLs). Forty-four ESBL-producing isolates including 22 CTX-M-14, 18 CTX-M-3, 2 CTX-M-24, and 2 CTX-M-66 producers were detected, and the proportion of ESBL producers increased from 0.7% in 1999 to approximately 6% after 2002. CTX-M-66 is a novel variant of CTX-M ESBLs that differs from CTX-M-3 by a Ser to Asn change at amino acid position 23. Coresistances to aminoglycosides and ciprofloxacin were very common in the CTX-M-3 producers. The presence of ArmA-type or RmtB-type 16S rRNA methylase that confers high-level aminoglycoside resistance was detected in 12 CTX-M-3 producers and 4 CTX-M-14 producers. Twenty-four clones including an endemic CTX-M-14-producing clone were observed among the 44 ESBL producers by pulsed-field gel electrophoresis, suggesting that both horizontal transfer and clonal spread contributed to the increased prevalence of bla(CTX-M) in P. mirabilis.     

Dissemination of SHV-12 and CTX-M-type extended-spectrum beta-lactamases among clinical isolates of Escherichia coli and Klebsiella pneumoniae and emergence of GES-3 in Korea

OBJECTIVES: To assess the prevalence and genotypes of Ambler class A extended-spectrum beta-lactamases (ESBLs) in Korea. METHODS: Clinical isolates of Escherichia coli and Klebsiella pneumoniae collected from 12 Korean hospitals during February-July 2003 were evaluated. Antimicrobial susceptibilities were determined by disc diffusion and agar dilution methods, and the putative ESBL-producing strains were tested by the double-disc synergy method. Detection of genes encoding class A beta-lactamases was performed by PCR amplification, and the PCR products were subjected to direct sequencing. RESULTS: The double-disc synergy test showed positive results in 9.3% (23/246) of E. coli and 23.0% (55/239) of K. pneumoniae isolates. The most prevalent types of Ambler class A ESBLs in E. coli isolates were CTX-M-15 (n = 4) and CTX-M-3 (n = 3), and those in K. pneumoniae isolates were SHV-12 (n = 30) and CTX-M-3 (n = 13). Two isolates produced both SHV-12 and GES-3, simultaneously. CONCLUSIONS: CTX-M-type and/or SHV-12 ESBL-producing E. coli and K. pneumoniae isolates are spreading, and a GES-type ESBL has emerged in Korea.     

Nationwide study of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum beta-lactamases in Spain

Clonal dissemination of extended-spectrum beta-lactamases (ESBL) in 170 Escherichia coli isolates and 70 Klebsiella pneumoniae isolates from a nationwide study of 40 Spanish centers in 2000 was not observed in most centers. The most prevalent ESBL were CTX-M-9 (27.3%), SHV-12 (23.9%), and CTX-M-14 (20.5%) for E. coli and TEM-3 (16.7%) and TEM-4 (25%) for K. pneumoniae. A new ESBL, TEM-133, with mutations L21F, E104K, and R164S, was identified.     

CMY-2-producing Salmonella enterica, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis and Escherichia coli strains isolated in Spain (October 1999-December 2000)

CMY-2 plasmid-mediated AmpC beta-lactamase (CMY-2) was detected in 21 isolates from two hospitals located in different geographical regions of Spain between October 1999 and December 2000. The isolates comprised two Salmonella enterica serovars (Mikawasima and Montevideo), 16 Escherichia coli, one Klebsiella pneumoniae, one Klebsiella oxytoca and one Proteus mirabilis. In addition to the expected resistance to beta-lactams, including extended-spectrum cephalosporins and cefoxitin, all isolates showed a broad spectrum of associated resistance. All were resistant to sulfamethoxazole, chloramphenicol, tetracycline and streptomycin, and all but two were also resistant to gentamicin. Five isolates were studied in detail and all transferred CMY-2 and other resistance determinants by conjugation. Genomic DNA restriction pattern analysis of the E. coli isolates excluded the dissemination of a single clone. To the best of our knowledge this is the first time that CMY-2 has been detected in P. mirabilis, K. oxytoca and S. enterica serovars Mikawasima and Montevideo. It is also the first time that CMY-2 has been described in Spain.     

Molecular and kinetic comparison of the novel extended-spectrum beta-lactamases CTX-M-25 and CTX-M-26

CTX-M-25 is a novel extended-spectrum β-lactamase isolated from a single Canadian Escherichia coli isolate. Susceptibility testing demonstrated that this enzyme confers resistance to both cefotaxime and ceftazidime, but the level of resistance was reduced with the addition of β-lactamase inhibitors. The bla_CTX-M-25 gene was detected on a 111-kb plasmid. It is a member of the CTX-M-8 group and has the closest amino acid identity (99%; three amino acid substitutions) with CTX-M-26. The bla_CTX-M-26 gene was detected on a 100-kb plasmid isolated from a Klebsiella pneumoniae strain from the United Kingdom, and plasmid profiling revealed that it showed some homology to the bla_CTX-M-25-harboring plasmid. Both CTX-M genes were located downstream of ISEcp1, although the copy upstream of bla_CTX-M-25 was disrupted by IS50-A. Comparative kinetic studies of recombinant CTX-M-25 and CTX-M-26 enzymes showed that CTX-M-25 has a higher level of ceftazidime hydrolysis (k_cat values, 33 and 0.005 s⁻¹ for CTX-M-25 and CTX-M-26, respectively).     

Characterization of CMY-type beta-lactamases in clinical strains of Proteus mirabilis and Klebsiella pneumoniae isolated in four hospitals in the Paris area

We isolated five clinical strains (three Proteus mirabilis and two Klebsiella pneumoniae) with beta-lactam resistance phenotypes consistent with production of an AmpC-type beta-lactamase. The predicted amino acid sequences of the enzymes were typical of class C beta-lactamases. The enzymes were identified as CMY-2, CMY-4 and a new CMY-variant beta-lactamase, CMY-12. The AmpC beta-lactamases from the two K. pneumoniae isolates were found to be encoded on self-transferable plasmids. The genes encoding the AmpC-type beta-lactamase produced by the three P. mirabilis isolates were chromosomal. Four of the five clinical isolates were from patients transferred from Greece, Algeria and Egypt; one of the K. pneumoniae strains was recovered from a French patient. PFGE analysis and rep-PCR fingerprinting showed that the two P. mirabilis isolates from Greek patients were closely related.     

First description of Klebsiella pneumoniae harboring CTX-M beta-lactamases (CTX-M-14 and CTX-M-3) in Taiwan

Klebsiella pneumoniae isolates from Taiwan medical centers (50 strains; 1998 to 2000) with a CTX-M resistance phenotype (ceftazidime susceptible and ceftriaxone or cefotaxime nonsusceptible) were selected for initial isoelectric focusing analysis. beta-Lactamases with pIs of 7.9 (n = 22) and 8.4 (n = 28) in addition to 5.4 and/or 7.6 were detected. DNA gene sequencing identified the beta-lactamases with pIs of 7.9 and 8.4 as CTX-M-14 and CTX-M-3, respectively. Molecular typing suggested inter- and intrahospital clonal dissemination of these Taiwanese CTX-M-producing Klebsiella strains.     

Plasmid-mediated beta-lactamases in clinical isolates of Klebsiella pneumoniae and Escherichia coli resistant to ceftazidime.

Low-level transferable resistance to ceftazidime was detected in seven strains of Klebsiella pneumoniae and one strain of Escherichia coli. Six of the Klebsiella strains and the E. coli strain were shown to produce a novel beta-lactamase (CAZ-lo) with a pI of 5.6 that hydrolyzed broad-spectrum cephalosporins at low but comparable levels. One strain of K. pneumoniae was of a serotype different from that of the other strains and produced a plasmid-encoded cefuroximase (FUR) with a pI of 7.5 that mediated moderate levels of resistance to different broad-spectrum cephalosporins. High-level resistance to ceftazidime was detected in one other strain of K. pneumoniae, which produced a beta-lactamase with a pI of 6.5 (CAZ-hi). Apart from its pI, this enzyme differed from CAZ-lo by a specific and high hydrolytic activity against ceftazidime. The epidemiological context suggested that CAZ-hi may be a mutant of CAZ-lo, and this hypothesis was supported by the isolation of laboratory mutants of CAZ-lo showing properties identical to those of the clinical CAZ-hi enzyme.     

产ESBLs大肠埃希菌和肺炎克雷伯菌的检测及其基因分析

目的了解临床分离菌中产超广谱β内酰胺酶(ESBLs)大肠埃希菌和肺炎克雷伯菌发生率,分析其耐药表型,探讨其ESBLs基因类别。方法采用美国国家临床实验室标准化委员会(NCCLS)推荐的纸片扩散法,对364株大肠埃希菌和71株肺炎克雷伯菌进行产ESBLs菌株初筛试验,并用双纸片确证试验和双纸片协同试验进行产ESBLs菌株确证。按照NCCLS文件标准,用KirbyBauer纸片扩散法进行产ESBLs株21种抗生素药敏试验。用PCR方法扩增168株ESBLs表型阳性菌中blaTEM1、blaSHV1、CTXM1组、TOHO1组等4种基因。结果(1)大肠埃希菌和克雷伯菌中产ESBLs的检出率分别为46.7%和32.4%。(2)产ESBLs菌对青霉素类100%耐药;对第1、2代头孢菌素耐药率达85%～100%;对除头孢他定以外的第3代头孢菌素,产ESBLs大肠埃希菌耐药率为80%以上,产ESBLs肺炎克雷伯菌耐药率为65%～75%以上;产ESBLs菌对复方新诺明的耐药率为75%～85%;产ESBLs大肠埃希菌对环丙沙星耐药率为80%～90%、庆大霉素耐药率为50%～75%;产ESBLs菌对阿米卡星、头孢替坦、亚胺培南有较好的敏感性(80%～90%以上)。(3)产ESBLs大肠埃希菌中,blaTEM1、blaSHV1、CTXM1组等3种基因扩增阳性率分别为93.9%、7.4%和53.4%。51.4%的菌株同时携带2种耐药基因,2.7%的菌株同时携带3种耐药基因。产ESBLs肺炎克雷伯菌中blaTEM1、blaSHV1、CTXM1组等3种基因扩增阳性率分别为50.0%、95.0%和20.0%。同时携带2种耐药基因的菌株占35.0%,同时携带3种耐药基因的菌株占15.0%。两种细菌中均未检出TOHO1组基因。结论大肠埃希菌和肺炎克雷伯菌中产ESBLs的检出率较高,大肠埃希菌中产ESBLs菌比例有逐年上升趋势。产ESBLs菌对头孢噻肟的耐药率远高于头孢他定,且多数菌株对青霉素类,第1、2、3代头孢菌素、喹诺酮类、磺胺类、氨基糖甙类等抗生素呈多重耐药。大肠埃希菌中以TEM型和CTXM型基因为主。肺炎克雷伯菌以SHV型基因为主,同时存在TEM型和CTXM型基因。     

产ESBLs大肠埃希菌和肺炎克雷伯菌的检测及其基因分析

目的了解临术分离菌中产超广谱β内酰胺酶（ESBLs）大肠埃希菌和肺炎克雷伯菌发生率，分析其耐药表型，探讨其ESBLs基因类别。方法采用美国国家临床实验室标准化委员会（NCCLS）推荐的纸片扩散法，对364株大肠埃希菌和71株肺炎克雷伯菌进行产ESBLs菌株初筛试验，并用双纸片确证试验和双纸片协同试验进行产ESBLs菌株确证。按照NCCLS文件标准，用Kirby-Bauer纸片扩散法进行产ESBLs株21种抗生素药敏试验。用PCR方法扩增168株ESBLs表型阳性菌中blaTEM-1、blaSHV-1、CTX-M-1组、TOHO-1组等4种基因。结果（1）大肠埃希菌和克雷伯菌中产ESBLs的检出率分别为46．7％和32．4％。（2）产ESBLs菌对青霉素类100％耐药；对第1、2代头孢菌素耐药率达85％～100％；对除头孢他定以外的第3代头孢菌素，产ESBLs大肠埃希菌耐药率为80％以上，产ESBLs肺炎克雷伯菌耐药率为65％～75％以上；产ESBLs菌对复方新诺明的耐药率为75％～85％；产ESBLs大肠埃希菌对环丙沙星耐药率为80％～90％、庆大霉素耐药率为50％～75％；产ESBLs菌对阿米卡星、头孢替坦、亚胺培南有较好的敏感性（80％～90％以上）。（3）产ES-BLs大肠埃希菌中，blaTEM-1、blasSHV-1、CTX-M-1组等3种基因扩增阳性率分别为93．9％、7．4％和53．4％。51．4％的菌株同时携带2种耐药基因，2．7％的菌株同时携带3种耐药基因。产ESBLs肺炎克雷伯菌中blatTEM-1、blaSHV-1、CTX-M-1组等3种基因扩增阳性率分别为50．0％、95．0％和20．0％。同时携带2种耐药基因的菌株占35．0%，同时携带3种耐药基因的菌株占15．0％。两种细菌中均未检出TOHO-1组基因。结论大肠埃希菌和肺炎克雷伯菌中产ESBLs的检出率较高，大肠埃希菌中产ESBLs菌比例有逐年上升趋势。产ESBLs菌对头孢噻肟的耐药率远高于头孢他定，且多数菌株对青霉素类，第1、2、3代头孢菌素、喹诺酮类、磺胺类、氨基糖甙类等抗生素呈多重耐药。大肠埃希菌中以TEM型和CTX-M型基因为主。肺炎克雷伯菌以SHV型基因为主，同时存在TEM型和CTX-M型基因。     

Prevalence and characterization of extended-spectrum beta-lactamases in Klebsiella pneumoniae and Escherichia coli isolates from Colombian hospitals

Gram-negative pathogens harboring extended-spectrum beta-lactamases (ESBL) are widely prevalent in Latin America, but little is known about their prevalence in Colombia. A network of 8 tertiary care hospitals in Bogotá, Medellín, and Cali, Colombia, was formed in January 2002 to determine the prevalence of ESBL-producing Klebsiella pneumoniae and Escherichia coli. We characterized and established the molecular epidemiology of ESBLs from these hospitals. Data from 1074 E. coli and 394 K. pneumoniae isolates were obtained from hospital laboratories during 6 months. Isolates resistant to third-generation cephalosporins or aztreonam were sent to a central laboratory. The prevalence of strains with this phenotype was 32.6% in K. pneumoniae and 11.8% in E. coli from the intensive care units, with slightly lower percentages from wards. Although TEM and SHV enzymes were present, the dominant class was CTX-M. Molecular typing of chromosomal DNA showed that most strains were not clonal.     

β-Lactamases Responsible for Resistance to Expanded-Spectrum Cephalosporins in Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis Isolates Recovered in South Africa

Although resistance to the expanded-spectrum cephalosporins among members of the family Enterobacteriaceae lacking inducible β-lactamases occurs virtually worldwide, little is known about this problem among isolates recovered in South Africa. Isolates of Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis resistant to expanded-spectrum cephalosporins recovered from patients in various parts of South Africa over a 3-month period were investigated for extended-spectrum β-lactamase production. Antibiotic susceptibility was determined by standard disk diffusion and agar dilution procedures. Production of extended-spectrum β-lactamases was evaluated by using the double-disk test, and the β-lactamases were characterized by spectrophotometric hydrolysis assays and an isoelectric focusing overlay technique which simultaneously determined isoelectric points and general substrate or inhibitor characteristics. DNA amplification and sequencing were performed to confirm the identities of these enzymes. The P. mirabilis and E. coli isolates were found to produce TEM-26-type, SHV-2, and SHV-5 extended-spectrum β-lactamases. An AmpC-related enzyme which had a pI of 8.0 and which conferred resistance to cefoxitin as well as the expanded-spectrum cephalosporins was found in a strain of K. pneumoniae. This is the first study which has identified organisms producing different extended-spectrum β-lactamases from South Africa and the first report describing strains of P. mirabilis producing a TEM-26-type enzyme. The variety of extended-spectrum β-lactamases found among members of the family Enterobacteriaceae isolated from major medical centers in South Africa is troubling and adds to the growing list of countries where these enzymes pose a serious problem for antimicrobial therapy.     

Extended-spectrum beta-lactamases and plasmid-mediated AmpC enzymes among clinical isolates of Escherichia coli and Klebsiella pneumoniae from seven medical centers in Taiwan

Production of extended-spectrum beta-lactamases and plasmid-mediated AmpC enzymes was investigated among 291 Escherichia coli and 282 Klebsiella pneumoniae isolates that showed decreased susceptibilities to extended-spectrum cephalosporins from seven Taiwanese medical centers. CTX-M-type and SHV-type enzymes were the most prevalent extended-spectrum beta-lactamases. CMY-2-like and DHA-1-like beta-lactamases were the most prevalent AmpC-type enzymes.     

Screening and confirmatory testing for extended spectrum beta-lactamases (ESBL) in Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca clinical isolates

Escherichia coli and Klebsiella spp. were screened for ESBL based on routine susceptibility testing results. Isolates with intermediate or resistant susceptibilities for extended spectrum cephalosporins or aztreonam were reported as probable ESBL producers. By using the NCCLS proposed ESBL confirmatory method, we tested 61 screen-positive isolates from 42 patients, 30 randomly selected susceptible isolates, and 12 isolates with previously characterized beta-lactamases. Ceftazidime contributed to 97% of screen-positive isolates, whereas aztreonam added a single patient isolate. An ESBL was confirmed in 86% of K. pneumoniae, 100% of K. oxytoca, and 20% of E. coli screen-positive single patient isolates. None of the susceptible isolates were shown to produce ESBL. Based on these findings a comment regarding the presence of ESBL seems sufficient for Klebsiella spp. but confirmatory testing is indicated for E. coli. 0.25 microg/mL was used to indicate the presence of ESBL, the specificity of the assay increased to 100%. The NCCLS ESBL phenotypic confirmatory method was reproducible and accurate enough to be used in the clinical laboratory.