Effects of phenotype and genotype on methods for detection of extended-spectrum-beta-lactamase-producing clinical isolates of Escherichia coli and Klebsiella pneumoniae in Norway

Consecutive clinical isolates of Escherichia coli (n = 87) and Klebsiella pneumoniae (n = 25) with reduced susceptibilities to oxyimino-cephalosporins (MICs > 1 mg/liter) from 18 Norwegian laboratories during March through October 2003 were examined for bla(TEM/SHV/CTX-M) extended-spectrum-beta-lactamase (ESBL) genes, oxyimino-cephalosporin MIC profiles, ESBL phenotypes (determined by the ESBL Etest and the combined disk and double-disk synergy [DDS] methods), and susceptibility to non-beta-lactam antibiotics. Multidrug-resistant CTX-M-15-like (n = 23) and CTX-M-9-like (n = 15) ESBLs dominated among the 50 ESBL-positive E. coli isolates. SHV-5-like (n = 9) and SHV-2-like (n = 4) ESBLs were the most prevalent in 19 ESBL-positive K. pneumoniae isolates. Discrepant ESBL phenotype test results were observed for one major (CTX-M-9) and several minor (TEM-128 and SHV-2/-28) ESBL groups and in SHV-1/-11-hyperproducing isolates. Negative or borderline ESBL results were observed when low-MIC oxyimino-cephalosporin substrates were used to detect clavulanic acid (CLA) synergy. CLA synergy was detected by the ESBL Etest and the DDS method but not by the combined disk method in SHV-1/-11-hyperproducing strains. The DDS method revealed unexplained CLA synergy in combination with aztreonam and cefpirome in three E. coli strains. The relatively high proportion of ESBL-producing E. coli organisms with a low ceftazidime MIC in Norway emphasizes that cefpodoxime alone or both cefotaxime and ceftazidime should be used as substrates for ESBL detection.     

Molecular and phenotypic characterization of Escherichia coli and Klebsiella pneumoniae producing extended-spectrum β-lactamases with focus on CTX-M in a low-endemic area in Sweden

During the last decade increasing prevalence of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae has been detected worldwide, mainly due to dissemination of Escherichia coli and Klebsiella pneumoniae producing CTX-M-type ESBLs. CTX-M-15 is the most widespread CTX-M type, and the predominant type in various countries. Dissemination of ESBL-producing organisms is caused not only by horizontal transfer of plasmids, but also by clonal spread of ESBL-producing strains. In this study, the molecular epidemiology of class A ESBL (ESBL(A))-producing E. coli and K. pneumoniae isolated in ?rebro County, Sweden, was investigated. Out of 200 ESBL(A) -producing E. coli and K. pneumoniae isolates, collected over a 10-year period, 87% were producing CTX-M, belonging to subgroup CTX-M-1 (64%), CTX-M-9 (34%), or CTX-M-2 (2%). The remaining isolates were producing variants of SHV and TEM. Sequencing of the bla(CTX-M) genes revealed 10 different CTX-M types, with a dominance of CTX-M-15 (E. coli 54%, K. pneumoniae 50%) followed by CTX-M-14 (E. coli 28%, K. pneumoniae 27%). Phenotypic characterization of the CTX-M-producing isolates was performed using the PhenePlate system. Although a few minor clusters of CTX-M-15 and CTX-M-14 producers were identified, the majority of the isolates did not appear to be clonally related.     

Extended-spectrum beta-lactamases in Taiwan: epidemiology, detection, treatment and infection control.

Extended-spectrum beta-lactamases (ESBLs) efficiently hydrolyze extended-spectrum beta-lactams such as cefotaxime, ceftriaxone, ceftazidime, and aztreonam. ESBLs are most often plasmid-mediated. In Taiwan, the prevalence of ESBLs in bacteria has risen, ranging from 8.5 to 29.8% in Klebsiella pneumoniae and 1.5 to 16.7% in Escherichia coli isolates. The most prevalent types of ESBLs are SHV-5, SHV-12, CTX-M-3, and CTX-M-14 in isolates of K. pneumoniae and E. coli, with differences between institutions. SHV-12 and CTX-M-3 have been reported as the most common ESBLs in isolates of Enterobacter cloacae and Serratia marcescens, respectively. Molecular epidemiology studies suggest that the ESBL-encoding genes have been disseminated either by proliferation of epidemic strains or by transfer of plasmids carrying the resistance traits. The current ESBL screen guidelines of the Clinical and Laboratory Standards Institute (formerly National Committee for Clinical Laboratory Standards) are issued for E. coli, Klebsiella spp., and Proteus mirabilis. Owing to the lack of standard methods, it remains difficult to assure the presence of ESBL in an isolate co-harboring an AmpC beta-lactamase, particularly in cases where the latter is produced in larger amounts than the former. Empirical therapy with piperacillin-tazobactam to replace third-generation cephalosporins may help to reduce the occurrence of ESBLs in an institution with a high prevalence of ESBL producers. Carbapenems remain the drugs of choice for serious infections caused by ESBL-producing organisms. To retard the selection for carbapenem-resistant bacteria, 7-alpha-methoxy beta-lactams or fourth-generation cephalosporins can be therapeutic alternatives for mild-to-moderate infections provided that the pharmacokinetic and pharmacodynamic target can be easily achieved.     

Prevalence and characterization of plasmid-mediated quinolone resistance genes in extended-spectrum β-lactamase-producing Enterobacteriaceae isolates in Mexico

The objectives of this study were to investigate the prevalence of plasmid-mediated quinolone resistance genes in a collection of 226 extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolates and characterize the qnr-positive isolates. The rate of qnr-positive isolates was 21.6% (49/226), 49.5% for aac(6')-Ib-cr (112/226), and 1.7% for qepA1 (4/226). Those isolates carried qnr genes corresponding to types qnrB (71.4%), qnrS1 (24.4%), and qnrA1 (18.3%). The distribution among bacterial species was as follows: 55.8% (19/34) to Enterobacter cloacae, 50% (28/56) to Klebsiella pneumoniae, and 1.4% (2/136) to Escherichia coli. The characterization of qnr-positive isolates indicated the ESBL SHV-types as the most prevalent (81.6%), including the ESBLs SHV-12, SHV-5, and SHV-2a, followed by CTX-M-15 (44.9%) and TLA-1 (8.1%). In addition, for qnr-positive isolates, the prevalence of aac(6')-Ib-cr was 55.1%, but qepA was not identified. Alterations at codons Ser-83 and Asp-87 in GyrA and at codons Ser-80 in ParC were observed in 69% and 80% of the qnr-positive isolates, respectively. The analysis of the transconjugants revealed a cotransmission of bla(CTX-M-15) with qepA1 or aac(6')-Ib-cr and/or qnrA1 and bla(SHV-type) with qnrB5 and qnrB6 genes. To conclude, these findings indicate a high prevalence of qnr and aac(6')-Ib-cr among ESBL-producing isolates from Mexican hospitals and point to the wide spread of qnr-like determinants associated to ESBLs SHV- and CTX-M-type in Mexican clinical isolates.     

Using nucleic acid microarrays to perform molecular epidemiology and detect novel β-lactamases: a snapshot of extended-spectrum β-lactamases throughout the world

The worldwide dissemination of extended-spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae is a major concern in both hospital and community settings. Rapid identification of these resistant pathogens and the genetic determinants they possess is needed to assist in clinical practice and epidemiological studies. A collection of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis isolates, including phenotypically ESBL-positive (n = 1,093) and ESBL-negative isolates (n = 59), obtained in 2008-2009 from a longitudinal surveillance study (SMART) was examined using an in vitro nucleic acid-based microarray. This approach was used to detect and identify bla(ESBL) (bla(SHV), bla(TEM), and bla(CTX-M) genes of groups 1, 2, 9, and 8/25) and bla(KPC) genes and was combined with selective PCR amplification and DNA sequencing for complete characterization of the bla(ESBL) and bla(KPC) genes. Of the 1,093 phenotypically ESBL-positive isolates, 1,041 were identified as possessing at least one bla(ESBL) gene (95.2% concordance), and 59 phenotypically ESBL-negative isolates, used as negative controls, were negative. Several ESBL variants of bla(TEM) (n = 5), bla(SHV) (n = 11), bla(CTX-M) (n = 19), and bla(KPC) (n = 3) were detected. A new bla(SHV) variant, bla(SHV-129), and a new bla(KPC) variant, bla(KPC-11), were also identified. The most common bla genes found in this study were bla(CTX-M-15), bla(CTX-M-14), and bla(SHV-12). Using nucleic acid microarrays, we obtained a "molecular snapshot" of bla(ESBL) genes in a current global population; we report that CTX-M-15 is still the dominant ESBL and provide the first report of the new β-lactamase variants bla(SHV-129) and bla(KPC-11).     

Outbreak of infection caused by Enterobacter cloacae producing the novel VEB-3 beta-lactamase in China

Over a 4-month period from November 2002 to February 2003, 27 ceftazidime-resistant or cefotaxime-resistant nonrepetitive Enterobacter cloacae isolates were collected from 27 patients hospitalized at HuaShan Hospital, Shanghai, People's Republic of China. The Etest did not detect extended-spectrum beta-lactamases (ESBLs) in those 27 isolates; however, screening by the NCCLS ESBL disk test and confirmatory tests detected ESBLs in 4 of 27 isolates and PCR detected ESBLs in 23 of 27 isolates. The majority of ESBL producers exhibited the same repetitive extragenic palindromic PCR pattern but harbored different ESBL genes. CTX-M-3 was the most prevalent ESBL in our study. Interestingly, 12 clonally related E. cloacae isolates possessed a novel bla(VEB)-type beta-lactamase, bla(VEB-3). Bla(VEB-3) was encoded by the chromosome and was located in an integron. Nine of the 12 isolates harbored both the bla(VEB-3) and the bla(CTX-M-3)-like ESBLs. This is the first report of a VEB-1-like ESBL in China and the first report of the simultaneous presence of VEB-1 and CTX-M-3-like ESBLs in an isolate.     

VIM and IMP metallo-β-lactamases and other extended-spectrum β-lactamases in Escherichia coli and Klebsiella pneumoniae from environmental samples in a Tunisian hospital

An extremely drug-resistant Enterobacteriaceae species emerged in Kasserine Hospital, Tunisia between 2009 and 2010 causing a local outbreak. We aimed to characterize extended-spectrum β-lactamase (ESBL) and metallo-β-lactamase (MBL)-producing Enterobacteriaceae from the hospital environment. Swabs were collected from ten different wards from Kasserine Hospital, Tunisia. A total of 46 isolates were cultured onto MacConkey agar supplemented with ceftazidime to select for ESBL-producing Enterobacteriaceae. Identification and susceptibility patterns were performed using Phoenix-automated phenotypic identification criteria. Extended spectrum β-lactamases (ESBLs) were detected using cefepime ESBL E-test. Colony blotting was first used to detect the occurrence of bla(SHV) , bla(CTX-M) , bla(CMY) , bla(IMP) , and bla(VIM) genes. PCR was used to amplify these genes, and the amplicons were sequenced and analyzed. Total DNA was digested with XbaI, and PFGE was used to type the major isolates that produced IMP-1. Among the 46 isolates, 63% were Klebsiella pneumoniae, 13% were Escherichia coli, 8.7% were Proteus mirabilis, 6% were Enterobacter cloaceae, 4.3% were Providencia rettgeri, 2.5% were Serratia marcescens, and 2.5% were Pantoea agglomerans. PCR amplification and DNA sequencing showed that hospital environment isolates produced SHV-125, CTX-M-15, CMY-2 ESBLs, and IMP-1 and VIM-2 MBLs. PFGE typing showed the emergence of IMP-1 MBL-producing K. pneumoniae isolates that were not clonal. In this study, we report the first characterization of IMP-1 and VIM-2 MBL-producing K. pneumoniae and E. coli isolates collected from Kasserine Hospital, Tunisia.     

Dissemination of extended-spectrum beta-lactamase-producing Enterobacteriaceae in intensive care units of a medical center in Taiwan

The prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in a tertiary hospital in Taiwan was assessed over a 16-month period. A total of 125 nonrepetitive ESBL-producing isolates of Enterobacter cloacae, Escherichia coli, and Klebsiella pneumoniae were available for investigation using molecular methods. Four predominant intensive care units (ICUs) were identified, and SHV-12 (59%), CTX-M- 3 (36%), and CTX-M-14 (14%) were the three most frequent ESBLs. SHV-12 was predominant among E. cloacae in the burn unit and K. pneumoniae in the other three chest medicine-related ICUs. CTX-M-3 was predominant among E. coli and K. pneumoniae in three other ICUs. The dissemination of ESBL-producing Enterobacteriaceae in four ICUs of a medical center in Taiwan is a consequence of the clonal dissemination of a few epidemic strains along with the horizontal transmission of resistance genes-carrying plasmids among bacterial organisms.     

Characterization of clinical isolates of Enterobacteriaceae from Italy by the BD Phoenix extended-spectrum beta-lactamase detection method

Production of extended-spectrum beta-lactamases (ESBLs) is an important mechanism of beta-lactam resistance in Enterobacteriaceae: Identification of ESBLs based on phenotypic tests is the strategy most commonly used in clinical microbiology laboratories. The Phoenix ESBL test (BD Diagnostic Systems, Sparks, Md.) is a recently developed automated system for detection of ESBL-producing gram-negative bacteria. An algorithm based on phenotypic responses to a panel of cephalosporins (ceftazidime plus clavulanic acid, ceftazidime, cefotaxime plus clavulanic acid, cefpodoxime, and ceftriaxone plus clavulanic acid) was used to test 510 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Providencia stuartii, Morganella morganii, Enterobacter aerogenes, Enterobacter cloacae, Serratia marcescens, Citrobacter freundii, and Citrobacter koseri. Of these isolates, 319 were identified as ESBL producers, and the remaining 191 were identified as non-ESBL producers based on the results of current phenotypic tests. Combined use of isoelectric focusing, PCR, and/or DNA sequencing demonstrated that 288 isolates possessed bla(TEM-1)- and/or bla(SHV-1)-derived genes, and 28 had a bla(CTX-M) gene. Among the 191 non-ESBL-producing isolates, 77 isolates produced an AmpC-type enzyme, 110 isolates possessed TEM-1, TEM-2, or SHV-1 beta-lactamases, and the remaining four isolates (all K. oxytoca strains) hyperproduced K1 chromosomal beta-lactamase. The Phoenix ESBL test system gave positive results for all the 319 ESBL-producing isolates and also for two of the four K1-hyperproducing isolates of K. oxytoca. Compared with the phenotypic tests and molecular analyses, the Phoenix system displayed 100% sensitivity and 98.9% specificity. These findings suggest that the Phoenix ESBL test can be a rapid and reliable method for laboratory detection of ESBL resistance in gram-negative bacteria.     

Characterization and molecular epidemiology of extended spectrum beta-lactamase producing Enterobacter cloacae isolated from a Tunisian hospital

In 2009, out of the 66 nonrepetitive Enterobacter cloacae collected at Charles Nicolle hospital in Tunisia, 44 were extended spectrum β-lactamase (ESBL) producers. The aim of the current study was to detect and characterize the genes encoding the ESBLs including blaTEM, blaSHV, and blaCTX-M groups by polymerase chain reaction and sequencing. Pulsed-field gel electrophoresis (PFGE) analysis was used to determine the genetic relatedness between isolates. All strains were susceptible to carbapenems. They were resistant to fluoroquinolones, gentamicin, tobramycin, and trimethoprim+sulfamethoxazole but variably resistant to netilmicin, amikacin, and tetracyclines. Sequence analysis of the polymerase chain reaction products revealed the presence of blaCTX-M-15 (39 strains), blaSHV-12 (6 strains), and blaSHV-27 (1 strain). The coexistence of two ESBLs was observed in two isolates harboring, respectively, SHV-12+CTX-M-15 and SHV-27+CTX-M-15. PFGE revealed 36 unrelated profiles. Diffusion of E. cloacae producing CTX-M-15 ESBL in our hospital is the consequence of dissemination of identical or related plasmids harboring the CTX-M-15 gene.     

Variety of TEM-, SHV-, and CTX-M-type beta-lactamases present in recent clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae from Taiwan

A total of 171 hospitals' isolates of E. coli, K. pneumoniae, and E. cloacae with a minimum inhibitory concentration (MIC) of > or =2 microg/ml for ceftazidime or cefotaxime were evaluated for the production of beta-lactamases. PCR amplification with specific primers for the bla (SHV), bla (TEM), and bla (CTX) genes revealed that a total of 53, 81, and 43 of these genes were amplified, respectively. Sequencing results confirmed that TEM-1, CTX-M-3 and -14, SHV-1, -5, -11, -12, and -33, OXY-1a, and LEN-1 were presented among these isolates. No specific large cluster of isolates carried the same beta-lactamases, indicating the wide diversity of the collected strains. Plasmid spread between E. coli and K. pneumoniae was identified in few isolates. Combinations of TEM, SHV, and CTX beta-lactamase genes, including extended-spectrum beta-lactamase genes, were observed in all three species.     

Phenotypic and molecular detection of CTX-M-beta-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.     

Characterisation and molecular epidemiology of extended-spectrum β-lactamase-producing Enterobacter cloacae isolated from a district teaching hospital in Taiwan

Enterobacter cloacae (n = 110) isolates from a district hospital in Taiwan were screened for extended-spectrum beta-lactamases (ESBLs). In total, 17 ESBL-producers were identified, based on the combination-disk synergy test using cefotaxime and ceftazidime +/- clavulanic acid. Investigation of ESBL genes in 33 ceftazidime-resistant isolates revealed the SHV-12 gene in the same 17 ESBL-producers. In addition, one isolate also carried the CTX-M-3 gene, and two isolates also carried the CTX-M-9 gene. No major epidemic clone of ESBL-producers was identified by pulsed-field gel electrophoresis. Routine screening for the ESBL phenotype, focusing on ceftazidime-resistant E. cloacae, should be undertaken in this area.     

Multiple CTX-M-type extended-spectrum beta-lactamases in nosocomial isolates of Enterobacteriaceae from a hospital in northern Italy

Twelve isolates of Enterobacteriaceae (1 of Klebsiella pneumoniae, 8 of Escherichia coli, 1 of Proteus mirabilis, and 2 of Proteus vulgaris) classified as extended-spectrum beta-lactamase (ESBL) producers according to the ESBL screen flow application of the BD-Phoenix automatic system and for which the cefotaxime MICs were higher than those of ceftazidime were collected between January 2001 and July 2002 at the Laboratory of Clinical Microbiology of the San Matteo University Hospital of Pavia (northern Italy). By PCR and sequencing, a CTX-M-type determinant was detected in six isolates, including three of E. coli (carrying bla(CTX-M-1)), two of P. vulgaris (carrying bla(CTX-M-2)), and one of K. pneumoniae (carrying bla(CTX-M-15)). The three CTX-M-1-producing E. coli isolates were from different wards, and genotyping by pulsed-field gel electrophoresis (PFGE) revealed that they were clonally unrelated to each other. The two CTX-M-2-producing P. vulgaris isolates were from the same ward (although isolated several months apart), and PFGE analysis revealed probable clonal relatedness. The bla(CTX-M-1) and bla(CTX-M-2) determinants were transferable to E. coli by conjugation, while conjugative transfer of the bla(CTX-M-15) determinant from K. pneumoniae was not detectable. Present findings indicate that CTX-M enzymes of various types are present also in Italy and underscore that different CTX-M determinants can be found in a single hospital and can show different dissemination patterns. This is also the first report of CTX-M-2 in P. vulgaris.     

Development of a sensitive and specific enzyme-linked immunosorbent assay for detecting and quantifying CMY-2 and SHV beta-lactamases

Polyclonal rabbit antibodies against SHV-1 and CMY-2 beta-lactamases were produced and characterized, and enzyme-linked immunosorbent assays (ELISAs) were developed. Immunoblots revealed that the anti-SHV-1 antibody recognized SHV-1 but did not recognize TEM-1, K-1, OXA-1, or any AmpC beta-lactamase tested. The anti-CMY-2 antibody detected Escherichia coli CMY-2, Enterobacter cloacae P99, Klebsiella pneumoniae ACT-1, and the AmpC beta-lactamases of Enterobacter aerogenes, Morganella morganii, and Citrobacter freundii. No cross-reactivity of the anti-CMY-2 antibody was seen against laboratory strains of E. coli possessing TEM-1, SHV-1, K-1, or OXA-1 beta-lactamases. Operating conditions for performing ELISAs were optimized. Both anti-CMY-2 and anti-SHV-1 antibodies detected picogram quantities of purified protein in ELISAs. The reactivity of the anti-CMY-2 antibody was tested against a number of AmpC beta-lactamases by assaying known quantities of purified enzymes in ELISAs (AmpC beta-lactamases of M. morganii, C. freundii, E. coli, and E. cloacae). As the homology to CMY-2 beta-lactamase decreased, the minimum level needed for detection increased (e.g., 94% homology recognized at 1 ng/ml and 71% homology recognized at 10 ng/ml). The ELISAs were used to assay unknown clinical isolates for AmpC and SHV beta-lactamases, and the results were confirmed with PCR amplification of bla(AmpC) and bla(SHV) genes. Overall, we found that our ELISAs were at least 95% sensitive and specific for detecting SHV and AmpC beta-lactamases. The ELISA format can facilitate the identification of AmpC and SHV beta-lactamases and can be used to quantify relative amounts of beta-lactamase enzymes in clinical and laboratory isolates.     

Extended-spectrum beta-lactamase-producing Enterobacteriaceae in Bulgarian hospitals

The aim of the study was to describe the emergence, the spread, and the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in Bulgaria. Over eight years (1996-2003), 442 ESBL-screen-positive isolates were collected in nine medical institutions in four Bulgarian towns. Class A ESBLs of the SHV, TEM, and CTX-M groups were identified in seven species. SHV-type enzymes persisted during the whole study period, TEM-ESBLs appeared first in 1999, and CTX-M-types appeared first in 2001. The rate of CTX-M enzyme producers increased rapidly between 2001 and 2003, while the rate of SHV producers decreased. Six different ESBL-types were identified, namely, SHV-2, -5, and -12, CTX-M-3 and -15, and a new TEM-3-like variant (TEM-139). The most widespread enzymes were SHV-12, CTX-M-15, and CTX-M-3 found in seven centers. TEM-139 was identified mainly in one center. A trend for strains harboring more than one ESBL gene, for example, CTX-M + SHV, was observed since 2002. Plasmid fingerprinting and random amplified polymorphic DNA analysis typing revealed wide dissemination of identical plasmids among different bacterial species and hospitals, as well as clonal spread of ESBL producers. Our data contribute to clarify the dynamics in the prevalence of ESBLs in Bulgaria and demonstrate the importance of molecular procedures for their analysis.     

Molecular epidemiology of ciprofloxacin-resistant extended-spectrum β-lactamase-producing Klebsiella pneumoniae in Taiwan

Fluoroquinolone resistance in extended-spectrum β-lactamases (ESBL)-producing isolates results in very few antimicrobial treatment options. In Taiwan's Surveillance of Antimicrobial Resistance (TSAR) III program, 124 (52.8%) cases of ESBL-producing Klebsiella pneumoniae (ESBL-KP) were resistant to ciprofloxacin. The prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and chromosomal quinolone resistance-determining regions (QRDR) of gyrA and parC genes among ESBL-KP isolates was assessed via PCR sequencing. Chromosomal QRDR mutations were present in most of the 123 (96.8%) cases of ciprofloxacin-resistant ESBL-KP isolates. Sixty-six (53.2%) isolates had at least one PMQR gene. qnrB2, qnrB4, and qnrS1 were detected in 26, 19, and 13 isolates, respectively, whereas qnrA, qnrC, and qnrD were not detected. ESBL genes were transferable via conjugation with either aac(6')Ib-cr or qnrB in 63.6% of the isolates carrying PMQR genes. QnrB was associated with either CTX-M-15 or SHV-12, and aac(6')Ib-cr was linked to CTX-M-3 or CTX-M-14 in plasmids. qnrS did not co-transfer with ESBL genes. Clonal spread of PMQR genes harboring ESBL-KP isolates was observed in three hospitals. QnrA, which is common in Asia, was unexpectedly absent in ESBL-KP in Taiwan. Aside from transmission via clonal spread for ciprofloxacin-resistant ESBL-KP, concomitant transference of PMQR genes with either bla(CTX-M) or bla(SHV) via plasmid was common.     

Molecular characterisation of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella spp. isolates at a tertiary-care centre in Lebanon.

The prevalence of bla CTX-M, bla TEM and bla SHV genes among extended-spectrum beta-lactamase (ESBL)-producing clinical isolates of Escherichia coli (n = 50) and Klebsiella spp. (n = 50) from Lebanon was 96%, 57% and 67%, and 40%, 82% and 84%, respectively. Genotyping revealed that the clonal diversity was unrelated to the presence of bla genes. Sequence analysis of 16 selected isolates identified the bla CTX-M-15, bla TEM-1, bla OXA-1 and six bla SHV genes, as well as the gene encoding the quinolone-modifying enzyme AAC(6')-Ib-cr. The genes encoding CTX-M-15 and AAC(6')-Ib-cr were carried on a 90-kb plasmid of the pC15-1a or pCTX-15 type, which transferred both ESBL production and quinolone resistance from donors to transconjugants.     

Multiplex PCR amplification assay for the detection of blaSHV, blaTEM and blaCTX-M genes in Enterobacteriaceae

Extended-spectrum beta-lactamases (ESBLs) are often mediated by (bla-)SHV, (bla)TEM and (bla)CTX-M genes in Enterobacteriaceae and other Gram-negative bacteria. Numerous molecular typing methods, including PCR-based assays, have been developed for their identification. To reduce the number of PCR amplifications needed we have developed a multiplex PCR assay which detects and discriminates between (bla-)SHV, (bla)TEM and (bla)CTX-M PCR amplicons of 747, 445 and 593 bp, respectively. This multiplex PCR assay allowed the identification of (bla-)SHV, (bla)TEM and (bla)CTX-M genes in a series of clinical isolates of Enterobacteriaceae with previously characterised ESBL phenotype. The presence of (bla)SHV, (bla)TEM and (bla)CTX-M genes was confirmed by partial DNA sequence analysis. Apparently, the universal well-established CTX-M primer pair used here to reveal plasmid-encoded (bla)CTX-M genes would also amplify the chromosomally located K-1 enzyme gene in all Klebsiella oxytoca strains included in the study.