Molecular characterisation of extended-spectrum β-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 β-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.     

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).     

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.     

Development of a multiplex PCR and SHV melting-curve mutation detection system for detection of some SHV and CTX-M beta-lactamases of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae in Taiwan

Infection by extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae has been increasing in Taiwan. Accurate identification of the ESBL genes is necessary for surveillance and for epidemiological studies of the mode of transmission in the hospital setting. We describe herein the development of a novel system, which consists of a multiplex PCR to identify bla(SHV), bla(CTX-M-3)-like, and bla(CTX-M-14)-like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalent bla(SHV) genes (bla(SHV-1), bla(SHV-2), bla(SHV-2a), bla(SHV-5), bla(SHV-11), and bla(SHV-12)) in Taiwan. Sixty-five clinical isolates, which had been characterized by nucleotide sequencing of the bla(SHV) and bla(CTX-M) genes, were identified by the system. The system was then used to genotype the ESBLs from 199 clinical isolates, including 40 Enterobacter cloacae, 68 Escherichia coli, and 91 Klebsiella pneumoniae, collected between August 2002 and March 2003. SHV-12 (80 isolates) was the most prevalent type of ESBL identified, followed in order of frequency by CTX-M-3 (65 isolates) and CTX-M-14 (36 isolates). Seventeen (9%) of the 199 clinical isolates harbored both SHV- and CTX-M-type ESBLs. In contrast to Enterobacter cloacae, the majority of which produced SHV-type ESBLs, E. coli and K. pneumoniae were more likely to possess CTX-M-type ESBLs. Three rare CTX-M types were identified through sequencing of the bla(CTX-M-3)-like (CTX-M-15) and bla(CTX-M-14)-like (CTX-M-9 and CTX-M-13) genes. The system appears to provide an efficient differentiation of ESBLs among E. coli, K. pneumoniae, and Enterobacter cloacae in Taiwan. Moreover, the design of the system can be easily adapted for similar purposes in areas where different ESBLs are prevalent.     

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.     

Prevalence of the plasmid-mediated quinolone resistance genes, aac(6')-Ib-cr, qepA, and oqxAB in clinical isolates of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae in Korea

In this study, we sought to determine the prevalence of the plasmid-mediated quinolone resistance (PMQR) genes aac(6')-Ib-cr, qepA, and oqxAB in extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae clinical isolates in South Korea. In total, 104 isolates (63 E. coli and 41 K. pneumoniae) were collected. We found that 23 of the 63 (36.5%) E. coli and nine of the 41 (22.0%) K. pneumoniae isolates were positive for aac(6')-Ib-cr. No isolate was positive for qepA, while transferable oqxAB was detected only in 10 (24.4%) K. pneumoniae isolates. Among the 32 aac(6')-Ib-cr-positive isolates, 30 (93.8%) were positive for both aac(6')-Ib-cr and bla(CTX-M) (CTX-M-15, -14, and -57). Our results suggest that PMQR determinants are highly prevalent in ESBL-producing E. coli and K. pneumoniae isolates in Korea.     

Molecular characterization and antimicrobial susceptibility testing of Escherichia coli isolates from patients with urinary tract infections in 20 Chinese hospitals

A total of 222 urinary Escherichia coli isolates from 20 tertiary hospitals in 15 different provinces and 4 municipalities in mainland China were characterized by antimicrobial susceptibility, phylogrouping, and the presence of plasmid-mediated quinolone resistance genes. A subset of 138 suspected extended-spectrum cephalosporinase (ESC) producers were examined for genes encoding cephalosporin resistance. Forty-three isolates harboring bla(CTX-M-14) or bla(CTX-M-15) were analyzed by pulsed-field gel electrophoresis (PFGE), and plasmids containing these genes were typed using PCR-based replicon typing (PBRT). Thirteen phylogroup B2 bla(CTX-M-14)- and bla(CTX-M-15)-positive isolates were analyzed by multilocus sequence typing (MLST). A frequent occurrence of resistance (>46%) was observed toward cephalosporins, gentamicin, and fluoroquinolones. Among the 222 isolates, 4 qnrS1, 4 qepA, and 16 aac(6')-Ib-cr genes were confirmed. Four major phylogroups (A, B1, B2, and D) and nontypeable isolates (NTs) were found among the isolates, with phylogroup D (54%) being the most common phylogroup. A total of 110 (80%) of the 138 screened isolates harbored bla(CTX-M) genes, with bla(CTX-M-14) (71%) and bla(CTX-M-15) (24%) being the most prevalent of these genes. Nine of the 13 CTX-M-15- or CTX-M-14-containing B2 isolates belonged to ST131. PFGE typing showed a high level of diversity, and plasmid analysis indicated a very large pool of different resistance plasmids mediating the spread of bla(CTX-M) genes in mainland China. An equally very high frequency of resistance and equally high levels of diversity in phylogroups, PFGE types, and plasmids were observed among community- and hospital-acquired E. coli isolates, indicating the presence of a large reservoir in the community and a long-term spread of cephalosporin resistance in China.     

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.     

Prevalence of plasmid-mediated quinolone resistance genes in uropathogenic Escherichia coli isolated in a teaching hospital of northern Italy

A retrospective study was conducted to determine the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants in uropathogenic Escherichia coli isolated from inpatients and outpatients in a teaching hospital of northern Italy. The presence of qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA was evaluated in 76 and 72 nalidixic acid-resistant E. coli, isolated in 2004 and 2006, respectively. Positivity for the aac(6')-Ib-cr gene was demonstrated in 3 of 76 (3.9%) and 8 of 72 (11%) isolates, respectively; no other PMQR determinant was found. All aac(6')-Ib-cr-positive strains also showed two point mutations in the gyrA and parC genes. Most aac(6')-Ib-cr-positive isolates demonstrated the contemporary presence of bla(CTX-M-15), bla(OXA-1/30), and bla(TEM-1) genes and 4/11 harbored a class 1 integron with a dfrA17-aadA5 gene cassette arrangement. Interestingly, all aac(6')-Ib-cr-positive isolates belonged to B2 phylogenetic group, O25b antigen type, multi locus sequence type 131, and to a cluster of approximately 70% similarity level by pulsed-field gel electrophoresis (PFGE). These findings suggest the circulation of the previously described intercontinentally spreading E. coli O25:H4-ST131 clone in our geographical area since 2004. Hybridization studies of the PFGE profiles showed the aac(6')-Ib-cr gene to be associated with different molecular weight bands (40-350 kb) and interestingly aac(6')-Ib-cr chromosomal integration was demonstrated in one strain by I-Ceu I method. This represents the first report to investigate the presence and diffusion of PMQR determinants in northern Italy and to describe aac(6')-Ib-cr chromosomal integration in E. coli.     

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.     

Identification of SHV-type extended spectrum beta-lactamase genes in Pseudomonas aeruginosa by PCR-restriction fragment length polymorphism and insertion site restriction-PCR

We propose a simple and rapid method to discriminate SHV-type extended spectrum beta-lactamase (ESBL) genes in P. aeruginosa based on PCR techniques (PCR-RFLP and RSI-PCR). We studied 22 producing ESBL P. aeruginosa strains isolated from seven immunocompromised patients (19 isolates) and from environmental swabs (three isolates) at the Bone Marrow Transplantation Center of Tunis. Screening PCR with primer pairs designed to detect gene encoding TEM, SHV, OXA group I, OXA group II, OXA-18 and PER-1 ESBL was positive for bla(OXA18) and bla(SHV) genes in all isolates. Pulsed field gel electrophoresis using SpeI endonuclease defined five genotypic groups. For at least one isolate corresponding to each genotype observed, restriction of PCR products by DdeI and BsrI revealed the same restriction pattern that the bla(SHV-1) negative control; in the same way, RSI-PCR products digestion by NruI, thus excluding 35, 238 and 240 mutations characterizing reported ESBL in P. aeruginosa (SHV-2a, SHV5 et SHV12), and suggesting that studied bla(SHV) genes were not ESBL ones. Genomic DNA hybridization by southern blot with probe consisting in bla(SHV-1) gene was positive in these isolates. Sequencing the full-length open reading frame revealed nucleotide sequence of the bla(SHV-1). PCR-RFLP and RSI-PCR results were then confirmed. This approach is effective for screening P. aeruginosa for ESBL genes carriage in epidemiological studies and for detecting new variants.     

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.     

Comparative epidemiology of CTX-M-14 and CTX-M-15 producing Escherichia coli: Association with distinct demographic groups in the community in New Zealand

Extended spectrum beta-lactamase producing E. coli (ESBL-EC) are an emerging public health issue. In New Zealand (NZ), bla (CTX-M-14) and bla (CTX-M-15) are the most common ESBL genes. Although many studies describe risk factors for ESBL-EC, few describe risk factors for specific ESBL genes. Between January 2006 and December 2007, we characterized 108 consecutive, non-duplicate isolates of ESBL-EC at the Auckland Hospital laboratory. Demographic and clinical data were recorded. Of the 108, 54.6% (59) were CTX-M-15-EC, 26.9% (29) were CTX-M-14-EC and 12.09% were CTX-M-9 (13). The remaining seven isolates carried CTX-M-3 (3; 2.7%), CTX-M-65 (2; 1.8%), CTX-M-27 (1; 0.9%) and CTX-M-57 (1; 0.9%). CTX-M-15-EC were more likely than CTX-M-14-EC to be fluoroquinolone-resistant (86.4% versus 32.4%; p=0.006) and to be non-susceptible to amoxicillin-clavulanate (84.7% versus 41.4%; p=0.0001). Patients with CTX-M-15-EC were more likely to be of Indian ethnicity (34.5% versus 0%; p=0.0012) and to have travelled recently (31.6% versus 4%; p=0.0088). Patients with CTX-M-14-EC were more likely to have Chinese or South-East Asian ethnicity (48.1% versus 5.2%; p<0.0001) and to have no history of either travel or prior hospital admission (44% versus 8.9%; p=0.0006). These data imply that CTX-M-15 and CTX-M-14 producing E. coli are associated with distinct demographic subgroups in NZ.     

Molecular characterization and occurrence of extended-spectrum beta-lactamase resistance genes among Salmonella enterica serovar Corvallis from Thailand, Bulgaria, and Denmark

Fifty nine Salmonella Corvallis isolates from humans and food products in Bulgaria, Denmark, and Thailand were examined for antimicrobial susceptibility and characterized by pulsed-field gel electrophoresis (PFGE). Cephalosporin-resistant isolates were examined for the presence of genes encoding beta-lactamases by PCR and sequencing. Ten different PFGE types were observed. One type (30 isolates) was recovered in all three countries; three types were found only in Bulgaria, two only in Denmark, two only in Thailand, and two both in Denmark and Thailand. Ten isolates were susceptible to all antimicrobial agents tested, whereas 41 were resistant to three or more antimicrobials. Most resistance was observed among the isolates from Bulgaria. Of the 25 isolates from Bulgaria, 20 displayed resistance to ampicillin and the cephalosporins ceftiofur and cephalothin. All 20 isolates tested negative for bla (CMY-1), bla (CMY-2), and bla (ACC), but positive for bla (SHV), of which five were sequenced to bla (SHV-2). Plasmid profiling and hybridization revealed that the bla (SHV) gene was located on plasmids of approximately 70 kb. Five plasmid profiles were found among these 20 isolates. The plasmid profiling confirmed the PFGE-type and was able to further subdivide the strains. Seventeen of these 20 isolates contained also bla (TEM), of which nine representatives were sequenced to bla (TEM-1B), or bla (TEM-1H). One isolate contained bla (CTX-M-15), bla (SHV-2), and bla (TEM-1H), with the bla (CTX-M-15), and bla (TEM-1H) genes located on a 63-kb transferable plasmid. This study showed a high frequency of resistance among S. Corvallis isolated from humans and food products in Bulgaria, with a lower frequency in Thailand and Denmark. The clonal relatedness among the isolates from three countries could indicate a recent spread of this serovar.     

Emergence and dominance of CTX-M-15 extended spectrum beta-lactamase among Escherichia coli isolates from children

Of forty-seven extended-spectrum cephalosporin-resistant Escherichia coli isolates, collected from children at the Children's Hospital in 2006 (Tunis, Tunisia), we analyzed 32 isolates that were genotypically different by enterobacterial repetitive intergenic consensus -polymerase chain reaction. For all isolates, the double-disk diffusion test revealed synergy between clavulanate and cefotaxime and/or ceftazidime, suggesting the production of extended-spectrum beta-lactamases. Polymerase chain reaction experiments, performed on plasmid DNA, and sequencing revealed the presence of bla(TEM-1B) (26 isolates, 81%), bla(TEM-34(IRT-6)) (3 isolates, 9%), bla(SHV-12) (2 isolates, 6%), and bla(CTX-M-15) (31 isolates, 97%). Further, the insertion sequence ISEcp1 was found upstream from the bla(CTX-M-15) gene in 11 isolates. The bla genes were found alone or in various combinations in a single isolate. bla(TEM-1B) and bla(CTX-M-15) genes were detected in 26 out of the 32 isolates. Three isolates harbored both bla(TEM-34(IRT-6)) and bla(CTX-M-15). bla(SHV-12) was identified either alone or with bla(CTX-M-15) in a single isolate. Our investigation showed the dominance of CTX-M-type extended-spectrum beta-lactamases, with CTX-M-15 particularly common, and to our best knowledge, this is the first report of the coexistence of CTX-M-15 and IRT-6 in E. coli isolates from children in Tunisia.     

Prevalence and characterization of multidrug-resistant and extended-spectrum beta-lactamase-producing Escherichia coli from pediatric wards of a Malaysian hospital

The emergence of Escherichia coli resistant to extended-spectrum cephalosporins (ESCs) is of concern as ESC is often used to treat infections by Gram-negative bacteria. One-hundred and ten E. coli strains isolated in 2009-2010 from children warded in a Malaysian tertiary hospital were analyzed for their antibiograms, carriage of extended-spectrum beta-lactamase (ESBL) and AmpC genes, possible inclusion of the beta-lactamase genes on an integron platform, and their genetic relatedness. All E. coli strains were sensitive to carbapenems. About 46% of strains were multidrug resistant (MDR; i.e., resistant to ≥3 antibiotic classes) and almost half (45%) were nonsusceptible to ESCs. Among the MDR strains, high resistance rates were observed for ampicillin (98%), tetracycline (75%), and trimethoprim/sulfamethoxazole (73%). Out of 110 strains, bla(TEM-1) (49.1%), bla(CTX-M) (11.8%), and bla(CMY-2) (6.4%) were detected. Twenty-one strains were ESBL producers. CTX-M-15 was the predominant CTX-M variant found and this is the first report of a CTX-M-27-producing E. coli strain from Malaysia. Majority (3.1%) of the strains harbored class 1 integron-encoded integrases with a predominance of aadA and dfr genes within the integron variable region. No gene cassette encoding ESBL genes was found and integrons were not significantly associated with ESBL or non-ESBL producers. Possible clonal expansion was observed for few CTX-M-15-positive strains but the O25-ST131 E. coli clone known to harbor CTX-M-15 was not detected while CMY-2-positive strains were genetically diverse.     

Prevalence and diversity of extended-spectrum β-lactamases in faecal Escherichia coli isolates from healthy humans in Spain

Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates were detected in seven of 105 faecal samples from healthy humans, from two Spanish cities, during 2007. In these isolates, five ESBLs were detected, CTX-M-14 ( n  = 2), CTX-M-1 ( n  = 2), CTX-M-32 ( n  = 1), CTX-M-8 ( n  = 1) and TEM-52 ( n  = 1). Both bla _CTX-M-14a (surrounded by IS Ecp1 -IS 903 ) and bla _CTX-M-14b variants (included in an integron structure) were identified in this study. This is the first time that the bla _CTX-M-8 gene and ESBLs of the CTX-M-8 group have been found in Europe and Spain, respectively. Faecal E. coli of healthy humans therefore constitute a reservoir of bla _CTX-M genes with different surrounding genetic elements.     

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.