Dissemination of CTX-M-type beta-lactamases among clinical isolates of Enterobacteriaceae in Paris, France

We analyzed 19 clinical isolates of the family Enterobacteriaceae (16 Escherichia coli isolates and 3 Klebsiella pneumoniae isolates) collected from four different hospitals in Paris, France, from 2000 to 2002. These strains had a particular extended-spectrum cephalosporin resistance profile characterized by a higher level of resistance to cefotaxime and aztreonam than to ceftazidime. The bla(CTX-M) genes encoding these beta-lactamases were involved in this resistance, with a predominance of bla(CTX-M-15). Ten of the 19 isolates produced both TEM-1- and CTX-M-type enzymes. One strain (E. coli TN13) expressed CMY-2, TEM-1, and CTX-M-14. bla(CTX-M) genes were found on large plasmids. In 15 cases the same insertion sequence, ISEcp1, was located upstream of the 5' end of the bla(CTX-M) gene. In one case we identified an insertion sequence designated IS26. Examination of the other three bla(CTX-M) genes by cloning, sequencing, and PCR analysis revealed the presence of a complex sul1-type integron that includes open reading frame ORF513, which carries the bla gene and the surrounding DNA. Five isolates had the same plasmid DNA fingerprint, suggesting clonal dissemination of CTX-M-15-producing strains in the Paris area.     

DNA sequence analysis of the genetic environment of various blaCTX-M genes

OBJECTIVES: Over a 3 year period (2000-2003) 21 Escherichia coli, 5 Klebsiella pneumoniae, 1 Serratia marcescens and 1 Proteus mirabilis producing CTX-M-type beta-lactamase were collected from five different hospitals in Paris, France. This study was conducted to analyse the genetic environment of these 28 bla(CTX-M) genes. METHODS: Antimicrobial susceptibility testing was performed by the disc diffusion method and MICs of various beta-lactams were determined by an agar dilution method. PCR was used to detect and sequence alleles encoding CTX-M, TEM, SHV and CMY enzymes. The genetic environment was analysed by amplification and direct sequencing using various set of PCR primers or cloning in pBK-CMV. RESULTS: Sequence analysis revealed that these isolates contained seven different bla(CTX-M) genes: bla(CTX-M-1) (4 strains), bla(CTX-M-2) (2 strains), bla(CTX-M-3) (4 strains), bla(CTX-M-9) (1 strain), bla(CTX-M-14) (5 strains), bla(CTX-M-15) (11 strains), bla(CTX-M-24) (1 strain). TEM-1 was associated with CTX-M-type enzymes in 15 isolates. Two strains produced both CTX-M-15 and SHV-2 or CTX-M-14 and CMY-2. In 25 strains the insertion sequence ISEcp1 was located upstream of the 5' end of the bla(CTX-M) gene. Among these strains, in five isolates, ISEcp1 was disrupted by insertion sequences such as IS26 (in three of them) or IS1 or IS10. Insertion sequence IS903 was found downstream of bla(CTX-M-14) or bla(CTX-M-24). Examination of the other three bla(CTX-M) genes (two bla(CTX-M-2) and one bla(CTX-M-9)) by cloning, sequencing and PCR analysis revealed the presence of complex Class 1 integrons, In35, an integron similar to In60 and a novel integron. CONCLUSIONS: This work further confirmed the predominant role of ISEcp1 in the mobilization of bla(CTX-M) genes of the CTX-M-1 cluster and the presence of In35, of an integron similar to In60 and a novel complex Class 1 integron.     

Characterization of CTX-M and SHV extended-spectrum beta-lactamases and associated resistance genes in Escherichia coli strains of food samples in Tunisia

OBJECTIVES: To assess the occurrence of extended-spectrum beta-lactamases (ESBLs) in Escherichia coli isolates of faecal samples of animals (n = 40) and food samples (n = 38) obtained in Tunisia in 2006, and to characterize the type of ESBLs, their genetic environments and the associated resistance genes. METHODS: Samples were inoculated in supplemented media (2 mg/L cefotaxime) for isolation of broad-spectrum cephalosporin-resistant E. coli isolates (one isolate/sample). ESBLs and their genetic environments as well as integrons and their gene cassette composition were characterized by PCR and sequencing. RESULTS: ESBL-producing E. coli isolates were detected in 10 of the 38 food samples analysed (26%) and in none of the tested animal faecal samples. Genes found were as follows (number of isolates): bla(CTX-M-1) (5), bla(CTX-M-1) + bla(TEM-1b) (1), bla(CTX-M-14) + bla(TEM-1b) (2), bla(CTX-M-8) (1) and bla(SHV-5) (1). All ESBL-positive isolates showed unrelated PFGE patterns. ISEcp1 and IS903 were detected surrounding bla(CTX-M-14), and ISEcp1/IS26 and orf477 surrounding some of the bla(CTX-M-1) genes. Four of the ESBL-positive strains harboured class 1 integrons including different gene cassette combinations. CONCLUSIONS: ESBLs, mainly of the CTX-M class, are detected in E. coli of food origin in Tunisia, being the first time that this mechanism has been detected in food E. coli strains in Africa.     

Incidence of class A extended-spectrum beta-lactamases in Champagne-Ardenne (France): a 1 year prospective study

OBJECTIVES: To assess the frequency and diversity of extended spectrum beta-lactamases (ESBLs) in the Champagne-Ardenne region France, and to identify genetic elements associated with the bla(CTX-M) genes. METHODS: During 2004, all the non-duplicate isolates of Pseudomonas aeruginosa and Acinetobacter baumannii resistant to ceftazidime and of Enterobacteriaceae intermediate or resistant to ceftazidime and/or cefotaxime, screening samples excluded, were collected in 10 public hospitals and 3 private clinics. bla genes were sequenced and bla(CTX-M) environment characterized by PCR mapping. RESULTS: In Enterobacteriaceae (138/21 861; 0.6%), ESBLs were predominantly TEM-24 (n = 52; 37.7%) and CTX-M-15 (n = 37; 26.8%). Three new enzymes were identified, CTX-M-61 (CTX-M-1 group), TEM- and SHV-type. A. baumannii (n = 5) produced VEB-1 and P. aeruginosa (n = 2) SHV-2a. ISEcp1 was detected in 22/27 strains, disrupted in 7 of them. The IS903-like element was downstream of bla(CTX-M-14) and bla(CTX-M-16). ISCR1 was found upstream of bla(CTX-M-2) and bla(CTX-M-9), and ISCR1 and bla(CTX-M-2) were located on a sul1-type class 1 integron. In comparison with 2001-02, ESBL distribution among Enterobacteriaceae showed an increase in CTX-M-type (44.9% vs 3.7% P < 10(-7)) due to Escherichia coli CTX-M-15 and to the almost total disappearance of TEM-3 (0.9% vs 51.2%). E. coli was the most frequent species (50.0% vs 5.1% in 1998) despite a similar prevalence to that in 1998 (0.5% vs 0.2%). CONCLUSIONS: A careful detection of bla(CTX-M)-type spread to other species would help to anticipate clonal endemics such as those observed in Enterobacter aerogenes TEM-24.     

A novel ceftazidime-hydrolysing extended-spectrum beta-lactamase, CTX-M-54, with a single amino acid substitution at position 167 in the omega loop

OBJECTIVES: To characterize a novel ceftazidime-hydrolysing CTX-M mutant, designated CTX-M-54, produced by Klebsiella pneumoniae clinical isolate BDK0419 and to investigate its genetic environment. METHODS: Antimicrobial susceptibilities were determined by disc diffusion and agar dilution methods, and the double-disc synergy test was carried out. Detection of genes encoding class A beta-lactamases was performed by PCR amplification, and the genetic organization of the blaCTX-M-54 gene was investigated by PCR and sequencing of the regions surrounding this gene. Kinetic parameters were determined from purified CTX-M-54. RESULTS: The strain BDK0419 contained a transferable plasmid with a molecular size of approximately 21 kbp that carries both blaSHV-2a and blaCTX-M-54 beta-lactamase genes, along with two other plasmids. The blaCTX-M-54 gene was flanked upstream by an ISEcp1 insertion sequence and downstream by an IS903-like element. CTX-M-54 had a P167Q substitution within the omega loop region of class A beta-lactamases compared with the sequence of CTX-M-3. The MIC of ceftazidime for K. pneumoniae BDK0419 was 16-fold higher than that of cefotaxime; however, the kinetic parameter of CTX-M-54 against ceftazidime revealed a low catalytic efficiency. CONCLUSIONS: This work shows once again that novel CTX-M enzymes with an expanded activity towards ceftazidime through a single amino acid substitution can be identified from clinical isolates. Thus, detection of CTX-M enzymes can no longer be based solely on the resistance phenotypes of clinical isolates towards ceftazidime and cefotaxime.     

Molecular characterization of extended-spectrum-β-lactamase-producing and plasmid-mediated AmpC β-lactamase-producing Escherichia coli isolated from stray dogs in South Korea

A total of 47 extended-spectrum-cephalosporin-resistant Escherichia coli strains isolated from stray dogs in 2006 and 2007 in the Republic of Korea were investigated using molecular methods. Extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase phenotypes were identified in 12 and 23 E. coli isolates, respectively. All 12 ESBL-producing isolates carried bla(CTX-M) genes. The most common CTX-M types were CTX-M-14 (n = 5) and CTX-M-24 (n = 3). Isolates producing CTX-M-3, CTX-M-55, CTX-M-27, and CTX-M-65 were also identified. Twenty-one of 23 AmpC β-lactamase-producing isolates were found to carry bla(CMY-2) genes. TEM-1 was associated with CTX-M and CMY-2 β-lactamases in 4 and 15 isolates, respectively. In addition to bla(TEM-1), two isolates carried bla(DHA-1), and one of them cocarried bla(CMY-2). Both CTX-M and CMY-2 genes were located on large (40 to 170 kb) conjugative plasmids that contained the insertion sequence ISEcp1 upstream of the bla genes. Only in the case of CTX-M genes was there an IS903 sequence downstream of the gene. The spread of ESBLs and AmpC β-lactamases occurred via both horizontal gene transfer, accounting for much of the CTX-M gene dissemination, and clonal spread, accounting for CMY-2 gene dissemination. The horizontal dissemination of bla(CTX-M) and bla(CMY-2) genes was mediated by IncF and IncI1-Iγ plasmids, respectively. The clonal spread of bla(CMY-2) was driven mainly by E. coli strains of virulent phylogroup D lineage ST648. To our knowledge, this is the first report of bla(DHA-1) in E. coli strains isolated from companion animals. This study also represents the first report of CMY-2 β-lactamase-producing E. coli isolates from dogs in the Republic of Korea.     

Distribution of extended-spectrum beta-lactamases in clinical isolates of Enterobacteriaceae in Vietnam

Among 730 Escherichia coli, 438 Klebsiella pneumoniae, and 141 Proteus mirabilis isolates obtained between September 2000 and September 2001 in seven hospitals in Ho Chi Minh City, Vietnam, 26.6% were resistant to ceftazidime, 30% were resistant to cefotaxime, 31.5% were resistant to ceftriaxone, 15.9% were resistant to cefoperazone, and 6% were resistant to cefepime. Resistance to imipenem was found in 5.6% of the isolates. In 55 strains producing extended-spectrum beta-lactamases (32 E. coli isolates, 13 K. pneumoniae isolates, and 10 P. mirabilis isolates), structural genes for VEB-1 (25.5%), CTX-M (25.5%), SHV (38.1%), and TEM (76.3%) enzymes were detected alone or in combination. Sequencing of the PCR products obtained from the K. pneumoniae isolates revealed the presence of bla(VEB-1), bla(CTX-M-14), bla(CTX-M-17), bla(SHV-2), and bla(TEM-1). Molecular typing of the strains with a similar resistance phenotype to broad-spectrum cephalosporins indicated polyclonal spread. ISEcp1 was presumably responsible for dissemination of the bla(CTX-M-like) gene.     

CTX-M-93, a CTX-M variant lacking penicillin hydrolytic activity

Extended-spectrum β-lactamases (ESBLs) of the CTX-M type are increasingly being reported worldwide, with more than 90 known variants. Clinical Escherichia coli isolate Bre-1 was isolated in 2009 and displayed an unusual ESBL phenotype, made of a synergy image between expanded cephalosporins and clavulanic acid discs and susceptibility to penicillins. E. coli Bre-1 harbored a novel CTX-M-encoding gene, designated bla(CTX-M-93). CTX-M-93 differed from CTX-M-27 by only a single L169Q substitution. Compared to CTX-M-27, CTX-M-93 conferred higher MICs of ceftazidime for E. coli (MIC of 8 versus 1.5 μg/ml) and decreased MICs of other expanded-cephalosporins (MIC of cefotaxime of 1 versus 32 μg/ml) and penicillins (MIC of ticarcillin of 0.5 versus >256 μg/ml). A comparison of enzymatic properties revealed that the L169Q substitution led to a decreased Km for ceftazidime (25.5 versus 330 μM) but decreased hydrolytic activity against good substrates, such as cefotaxime (kcat of 0.6 versus 113 s(-1)), probably owing to the alteration of the omega loop positioning during the catalytic process. The blaCTX-M-93 gene was surrounded by the ISEcp1 and IS903 elements and inserted onto a 150-kb non-self-transferrable IncF-type plasmid. E. coli Bre-1 belongs to phylogroup D and is of multilocus sequence type (MLST) 624, a sequence type found only in rare Spanish CTX-M-14-producing E. coli isolates. We have characterized a novel CTX-M variant, CTX-M-93, lacking significant penicillin hydrolysis but with increased ceftazidime hydrolysis.     

Predominance and genetic diversity of community- and hospital-acquired CTX-M extended-spectrum beta-lactamases in York, UK

OBJECTIVES: This study was conducted to detect the presence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae within the faecal flora of both community- and hospital-based patients in York and to characterize the bla(TEM), bla(SHV) and bla(CTX-M) genes present in these isolates. METHODS: One thousand faeces samples were collected and screened at York Hospital during October-December 2003. Ninety-five non-duplicate Enterobacteriaceae isolates resistant to third-generation cephalosporins were recovered; 22 isolates were selected for further study on the basis of a positive double disc diffusion test for ESBL production. Antibiotic susceptibility testing was performed to a range of antibiotics. The TEM, SHV and CTX-M genes were detected by PCR and the DNA sequenced. RESULTS: The distribution of ESBL-positive isolates from the hospital and community was 1.4:1. These included nine Escherichia coli, seven Enterobacter cloacae, four Citrobacter freundii and a single isolate each of Klebsiella spp. and Salmonella spp. A total of 17 isolates contained bla(CTX-M) (five bla(CTX-M-15), three bla(CTX-M-14) and nine bla(CTX-M-9)). ISEcp1 was present in isolates expressing CTX-M-14 and -15, but was absent upstream of In60-associated bla(CTX-M-9). E. coli isolates also contained either a bla(TEM-1) or bla(TEM-2), whereas six of the E. cloacae carried bla(SHV-12) and the Klebsiella spp. bla(SHV-36) in addition to bla(CTX-M-9). The single Salmonella spp. carried bla(SHV-12). CONCLUSIONS: The overall prevalence of ESBL in isolates of Enterobacteriaceae from York was 1.9%. ESBL-producing isolates were found in both the community and hospital, with the CTX-M type most common. This is also the first report of an ESBL-producing Salmonella in the UK.     

Ceftazidime-hydrolysing CTX-M-15 extended-spectrum beta-lactamase (ESBL) in Poland

The CTX-M-15 extended-spectrum beta-lactamase (ESBL) was recently identified in Enterobacteriaceae isolates in India, and demonstrated significant hydrolytic activity against ceftazidime, in contrast to the majority of CTX-M enzymes. CTX-M-15 differs from CTX-M-3, which is one of the most prevalent ESBLs in Poland, by only a single amino acid change (Asp-240-->Gly). Three cefotaxime- and ceftazidime-resistant Enterobacteriaceae isolates, recovered during 1998-2000 in two Polish hospitals, were found to produce CTX-M-15. Similar to those from India, the isolates contained the ISEcp1 insertion sequence located upstream of the bla(CTX-M-15) gene, which has been recently demonstrated to mobilize 3'-adjacent genes to transfer between DNA replicons. However, its different position with respect to the beta-lactamase gene indicated the independent selection of the ESBL gene in the two countries.     

CTX-M-15 extended-spectrum (beta)-lactamase from Nigerian Klebsiella pneumoniae

OBJECTIVES: In this study, extended-spectrum beta-lactamases (ESBLs) were characterized from 30 selected multidrug-resistant Klebsiella pneumoniae strains isolated from patients with community-acquired urinary tract infections from Southwest Nigeria. METHODS: The beta-lactamases were phenotypically characterized using isoelectric focusing, genotypically characterized using PCR assays and hybridization of the PCR products. Two of the bla(CTX-M) genes were completely sequenced. The location of the CTX-M-type genes was determined using transformation, DNA-DNA hybridization, PCR assays and hybridization of the PCR products from the Escherichia coli transformants. RESULTS: All 30 isolates produced at least one beta-lactamase. Seventeen of the isolates were resistant to cefotaxime, and had > or =100-fold reduction in susceptibility with cefotaxime plus clavulanic acid (4 mg/L), indicating the presence of an ESBL. The 17 isolates were shown to have bla(CTX-M) genes that were associated with large plasmids (> or =58 kb), which also carried a tetracycline resistance gene, tet(A), and various aminoglycoside resistance genes. Two CTX-M-type genes were sequenced and had amino acid sequences indistinguishable from previously sequenced CTX-M-15 beta-lactamases. The ISEcp1 element was located upstream of bla(CTX-M-15) in the same position as previously described. In addition, 23 of the isolates produced TEM beta-lactamases, 27 produced SHV beta-lactamases and four produced AmpC beta-lactamases. CONCLUSIONS: Thirty K. pneumoniae produced multiple beta-lactamases, with 57% producing CTX-M enzymes. This is the first characterization of CTX-M-15-positive K. pneumoniae in Western Africa.     

Molecular characterization of resistance to extended-spectrum cephalosporins in clinical Escherichia coli isolates from companion animals in the United States

Resistance to extended-spectrum cephalosporins (ESC) among members of the family Enterobacteriaceae occurs worldwide; however, little is known about ESC resistance in Escherichia coli strains from companion animals. Clinical isolates of E. coli were collected from veterinary diagnostic laboratories throughout the United States from 2008 to 2009. E. coli isolates (n = 54) with reduced susceptibility to ceftazidime or cefotaxime (MIC ≥ 16 μg/ml) and extended-spectrum-β-lactamase (ESBL) phenotypes were analyzed. PCR and sequencing were used to detect mutations in ESBL-encoding genes and the regulatory region of the chromosomal gene ampC. Conjugation experiments and plasmid identification were conducted to examine the transferability of resistance to ESCs. All isolates carried the bla(CTX-M-1)-group β-lactamase genes in addition to one or more of the following β-lactamase genes: bla(TEM), bla(SHV-3), bla(CMY-2), bla(CTX-M-14-like), and bla(OXA-1.) Different bla(TEM) sequence variants were detected in some isolates (n = 40). Three isolates harbored a bla(TEM-181) gene with a novel mutation resulting in an Ala184Val substitution. Approximately 78% of the isolates had mutations in promoter/attenuator regions of the chromosomal gene ampC, one of which was a novel insertion of adenine between bases -28 and -29. Plasmids ranging in size from 11 to 233 kbp were detected in the isolates, with a common plasmid size of 93 kbp identified in 60% of isolates. Plasmid-mediated transfer of β-lactamase genes increased the MICs (≥ 16-fold) of ESCs for transconjugants. Replicon typing among isolates revealed the predominance of IncI and IncFIA plasmids, followed by IncFIB plasmids. This study shows the emergence of conjugative plasmid-borne ESBLs among E. coli strains from companion animals in the United States, which may compromise the effective therapeutic use of ESCs in veterinary medicine.     

Multiple outbreaks of nosocomial salmonellosis in Russia and Belarus caused by a single clone of Salmonella enterica serovar Typhimurium producing an extended-spectrum beta-lactamase

Thirty-four cefotaxime-resistant Salmonella enterica serovar Typhimurium isolates representative of the isolates that caused outbreaks of gastroenteritis in 10 hospitals in seven regions of Russia and Belarus from 1994 to 2003 were analyzed. All isolates produced the CTX-M-5-like extended-spectrum beta-lactamase, which confers high-level resistance to cefotaxime and ceftriaxone and decreased susceptibility to ceftazidime. The bla(CTX-M) genes were located on small (7.4- to 12-kb) non-self-transferable plasmids approximately 20 bp downstream of the ISEcp1 insertion sequences. Some isolates carried additional conjugative plasmids mediating resistance to penicillin-inhibitor combinations and various non-beta-lactam agents, including tetracycline, chloramphenicol, gentamicin, tobramycin, and co-trimoxazole. Despite the minor differences in susceptibility patterns, all isolates were considered clonally related on the basis of arbitrarily primed PCR and pulsed-field gel electrophoresis analysis. The similarities of the restriction profiles of the CTX-M-coding plasmids further supported the clonal origin of these isolates.     

Nosocomial outbreak by Proteus mirabilis producing extended-spectrum beta-lactamase VEB-1 in a Korean university hospital

OBJECTIVES: To examine the molecular mechanisms involved in the beta-lactam resistance of multidrug-resistant Proteus mirabilis isolates that showed an unusual synergy between imipenem and ceftazidime in a Korean hospital. METHODS: Over an 11 month period, a total of 12 P. mirabilis isolates showing resistance to ampicillin, gentamicin, ceftazidime, cefotaxime, cefuroxime, cefalothin, cefepime, piperacillin, trimethoprim/sulfamethoxazole and ciprofloxacin, were recovered from the sputum and urine specimens of nine patients who were hospitalized in the neurosurgery ward. The extended-spectrum beta-lactamases were screened with a double disc synergy test using ceftazidime, cefotaxime, aztreonam, cefepime and clavulanate. The ESBL types were determined by PCR using specific primers for bla(TEM-1), bla(SHV-1), bla(CTX-M-1), bla(CTX-M-2), bla(CTX-M-8), bla(CTX-M-9), bla(PER-1), bla(GES-1), bla(VEB-1), bla(OXA-10) and bla(OXA-13) followed by sequencing. All the isolates underwent molecular typing by PFGE. The transferability was examined by conjugation. RESULTS AND CONCLUSIONS: All the isolates showed a marked synergy between the extended-spectrum cephalosporins and clavulanate together with an unusual synergy between cefoxitin and the cephalosporins (cefalothin, cefuroxime, ceftazidime, cefotaxime) and between imipenem, and ceftazidime and cefotaxime. Isoelectric focusing of the crude bacterial extracts showed a beta-lactamase band with a pI value of 5.4, which was inhibited by clavulanate. PCR and sequencing identified the gene to be bla(VEB-1). In addition, the aadB gene was detected, conferring aminoglycoside resistance. The resistance was not transferred by conjugation. The outbreak was of a clonal origin as shown by PFGE demonstrating an identical banding pattern. This is the first report of VEB-1-producing Enterobacteriaceae in Korea.     

Spread of extended-spectrum beta-lactamase CTX-M-producing escherichia coli clinical isolates in community and nosocomial environments in Portugal

Of the 181 unduplicated Escherichia coli strains isolated in nine different hospitals in three Portuguese regions, 119 were extended-spectrum beta-lactamase (ESBL)-CTX-M producers and were selected for phenotype and genotype characterization. CTX-M producer strains were prevalent among community-acquired infections (56%), urinary tract infections (76%), and patients >/=60 years old (76%). In MIC tests, all strains were resistant to cefotaxime, 92% were resistant to ceftazidime, 93% were resistant to quinolones, 89% were resistant to aminoglycoside, and 26% were resistant to trimethoprim-sulfamethoxazole; all strains were sensitive to carbapenems, and 92% of the strains had a multidrug resistance phenotype. Molecular methods identified 109 isolates harboring a bla(CTX-M-15) gene, 1 harboring the bla(CTX-M-32) gene (first identification in the country), and 9 harboring the bla(CTX-M-14) gene. All isolates presented the ISEcp1 element upstream from the bla(CTX-M) genes; one presented the IS903 element (downstream of bla(CTX-M-14) gene), and none had the IS26 element; 85% carried bla(TEM-1B), and 84% also carried a bla(OXA-30). Genetic relatedness analysis based on pulsed-field gel electrophoresis defined five clusters and indicated that 76% of all isolates (from cluster IV) corresponded to a single epidemic strain. Of the 47 strains from one hospital, 41 belonged to cluster IV and were disseminated in three main wards. CTX-M-producing E. coli strains are currently a problem in Portugal, with CTX-M-15 particularly common. This study suggests that the horizontal transfer of bla(CTX-M) genes, mediated by plasmids and/or mobile elements, contributes to the dissemination of CTX-M enzymes to community and hospital environments. The use of extended-spectrum cephalosporins, quinolones, and aminoglycosides is compromised, leaving carbapenems as the therapeutic option for severe infections caused by ESBL producers.     

Occurrence, prevalence and genetic environment of CTX-M beta-lactamases in Enterobacteriaceae from Indian hospitals

OBJECTIVES: To determine occurrence, prevalence and CTX-M genotypes produced by Enterobacteriaceae from clinical samples from three geographically distant Indian hospitals and to detect linkage of IS26 with bla(CTX-M) and map its precise insertion position. METHODS: A total of 130, non-duplicate Escherichia coli and Klebsiella pneumoniae resistant to a third-generation cephalosporin (3GC) from three Indian centres were screened for extended-spectrum beta-lactamase (ESBL) production using phenotypic detection methods. All isolates were screened for bla(CTX-M) using multiplex PCR. Precise CTX-M genotype was identified using reverse-line hybridization. All CTX-M-producing isolates were screened for linkage of IS26 with bla(CTX-M). DNA sequencing was used to map the exact insertion position of this mobile element. RESULTS: Ninety-five of 130 3GC-resistant (73%) (73% of total E. coli, 72% of total K. pneumoniae) isolates were found to carry bla(CTX-M-15). No other CTX-M genotype was detected. IS26 linkage with bla(CTX-M-15) was detected in 31% of isolates carrying bla(CTX-M-15). DNA sequencing revealed variable insertion of this mobile element within tnpA of ISEcp1. RAPD-PCR typing demonstrated great diversity in isolates carrying bla(CTX-M-15); no predominant clone was identified. CONCLUSIONS: In contrast with other studies where greater diversity exists, CTX-M-15 was the only CTX-M ESBL produced in this Indian collection of unrelated E. coli and K. pneumoniae. This is the first systematic survey report from India detecting CTX-M-type beta-lactamases This is also the first report indicating such high mobility/diversity of insertion of IS26 in close association with bla(CTX-M) in a single bacterial collection.     

Detection of CTX-M-type beta-lactamase genes in fecal Escherichia coli isolates from healthy children in Bolivia and Peru

A survey was carried out from August to November 2002 to evaluate the antimicrobial susceptibilities of fecal Escherichia coli isolates from 3,208 healthy children from four different urban areas of Latin America, two in Bolivia (Camiri and Villa Montes) and two in Peru (Yurimaguas and Moyobamba). Ceftriaxone-resistant E. coli isolates were detected in four children, one from each of the areas sampled. The isolates exhibited a multidrug-resistant phenotype, including resistance to oxyimino-cephalosporins and aztreonam, and the MICs of ceftazidime for the isolates were lower than those of cefotaxime. By PCR and sequencing, the bla(CTX-M-2) determinant was detected in three isolates and the bla(CTX-M-15) determinant was detected in one isolate (from Peru). The CTX-M-2-producing isolates belonged to three different phylogenetic groups (groups A, B2, and D), while the CTX-M-15-producing isolate belonged to phylogenetic group D. The bla(CTX-M-2) determinants were transferable to E. coli by conjugation, while conjugative transfer of the bla(CTX-M-15) determinant was not detectable. Plasmids harboring the bla(CTX-M-2) determinant exhibited similar restriction profiles, and in all of them the gene was located on a 2.2-kb PstI fragment, suggesting a genetic environment similar to that present in In35 and InS21. The findings of the present study confirm the widespread distribution of CTX-M-type beta-lactamases and underscore the role that commensal E. coli isolates could play as a potential reservoir of these clinically relevant resistance determinants. This is the first report of CTX-M-type enzymes in Bolivia and Peru and also the first report of the detection of CTX-M-15 in Latin America.     

Novel Cefotaximase (CTX-M-16) with Increased Catalytic Efficiency Due to Substitution Asp-240→Gly

Three clinical strains (Escherichia coli Rio-6, E. coli Rio-7, and Enterobacter cloacae Rio-9) collected in 1996 and 1999 from hospitals in Rio de Janeiro (Brazil) were resistant to broad-spectrum cephalosporins and gave a positive double-disk synergy test. Two bla(CTX-M) genes encoding beta-lactamases of pl 7.9 and 8.2 were implicated in this resistance: the bla(CTX-M-9) gene observed in E. coli Rio-7 and E. cloacae Rio-9 and a novel CTX-M-encoding gene, designated bla(CTX-M-16), observed in E. coli strain Rio-6. The deduced amino acid sequence of CTX-M-16 differed from CTX-M-9 only by the substitution Asp-240-->Gly. The CTX-M-16-producing E. coli transformant exhibited the same level of resistance to cefotaxime (MIC, 16 microg/ml) but had a higher MIC of ceftazidime (MIC, 8 versus 1 microg/ml) than the CTX-M-9-producing transformant. Enzymatic studies revealed that CTX-M-16 had a 13-fold higher affinity for aztreonam and a 7.5-fold higher k(cat) for ceftazidime than CTX-M-9, thereby showing that the residue in position 240 can modulate the enzymatic properties of CTX-M enzymes. The two bla(CTX-M-9) genes and the bla(CTX-M-16) gene were located on different plasmids, suggesting the presence of mobile elements associated with CTX-M-encoding genes. CTX-M-2 and CTX-M-8 enzymes were found in Brazil in 1996, and two other CTX-M beta-lactamases, CTX-M-9 and CTX-M-16, were subsequently observed. These reports are evidence of the diversity of CTX-M-type extended-spectrum beta-lactamases in Brazil.     

Extended-spectrum-β-lactamase-producing Escherichia coli as a cause of pediatric infections: report of a neonatal intensive care unit outbreak due to a CTX-M-14-producing strain

Little information is available about pediatric infections caused by extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli. We characterized an outbreak caused by a CTX-M-14-producing E. coli isolate in a neonatal intensive care unit (NICU) and studied other infections caused by ESBL-producing E. coli in non-NICU pediatric units. All children ≤4 years old who were infected or colonized by ESBL-producing E. coli isolates between January 2009 and September 2010 were included. Molecular epidemiology was studied by phylogroup analysis, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing. Antibiotic resistance genes were analyzed by PCR and sequencing. Plasmids were studied by PFGE with S1 nuclease digestion and by incompatibility group analysis using a PCR-based replicon-typing scheme. Of the ESBL-producing E. coli isolates colonizing or infecting the 30 newborns, identical PFGE results were observed for 21 (70%) isolates, which were classified as CTX-M-14-producing E. coli of ST23 phylogroup A. bla(CTX-M-14a) was linked to ISEcp1 and was carried on an ～80-bp IncK plasmid. A smaller ongoing outbreak due to SHV-12-producing ST131 E. coli was also identified in the same NICU. Fifteen additional infections with ESBL-producing E. coli were identified in non-NICU pediatric units, but none was caused by the CTX-M-14-producing E. coli epidemic clone. Overall, CTX-M-14 (71.1%), CTX-M-15 (13.3%), and SHV-12 (13.3%) were the most important ESBLs causing pediatric infections in this study. Infections of newborns with CTX-M-14-producing E. coli were caused by both clonal and nonclonal isolates.