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.     

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.     

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.     

Emergence of multidrug-resistant NDM-1-producing Gram-negative bacteria in Bangladesh

The main objective of this study was to investigate the prevalence of bla (NDM-1) in Gram-negative bacteria in Bangladesh. In October 2010 at the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) laboratories, 1,816 consecutive clinical samples were tested for imipenem-resistant Gram-negative organisms. Imipenem-resistant isolates were tested for the bla (NDM-1) gene. Among 403 isolates, 14 (3.5?%) were positive for bla (NDM-1), and the predominant species were Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli. All bla (NDM-1)-positive isolates were resistant to multiple antibiotics. Among β-lactamase genes, bla (CTX-M-1-group) was detected in ten isolates (eight bla (CTX-M-15)), bla (OXA-1-group) in six, bla (TEM) in nine, bla (SHV) in seven, and bla (VIM) and bla (CMY) in two isolates each. The 16S rRNA methylase gene, armA, was detected in five?K. pneumoniae isolates and in one E. coli isolate. rmtB and rmtC were detected in a Citrobacter freundii and two?K. pneumoniae isolates, respectively. qnr genes were detected in two?K. pneumoniae isolates (one qnrB and one qnrS) and in an E. coli isolate (qnrA). Transferable plasmids (60-100?MDa) carrying bla (NDM-1) were detected in 7 of the 11 plasmid-containing isolates. Pulsed-field gel electrophoresis (PFGE) analysis grouped K. pneumoniae isolates into three clusters, while E. coli isolates differed significantly from each other. This study reports that approximately 3.5?% of Gram-negative clinical isolates in Bangladesh are NDM-1-producing.     

广州地区携带blaCTX-M-15流行质粒的分子特征研究

目的 研究广州地区携带blaCTX-M-15质粒的分子特征.方法 以2007年到2008年期间来源于广州9家医院确证产CTX-M-15 ESBL的38株大肠埃希菌和47株肺炎克雷伯菌为研究对象,脉冲场凝胶电泳(PFGE)和多位点序列分型(MLST)法检测blaCTX-M-15阳性株的同源性,MIC法检测菌株对常用抗生素的敏感性.血清凝集试验确定大肠埃希菌血清型.接合试验了解携带blaCTX-M-15质粒的可接合性,限制性内切酶分析质粒的同源性.PCR分析大肠埃希菌的系统发育群,流行质粒的不相容群和耐药基因构成.结果 blaCTX-M-15阳性的大肠埃希菌和肺炎克雷伯菌分别用PFGE确定为28个和30个基因型.携blaCTX-M-15大肠埃希菌的系统发育群以D群(67.8%)和A群(21.4%)为主.大肠埃希菌血清型呈散在分布,未发现O25:H4血清型.大肠埃希菌和肺炎克雷伯菌的ST型无明显集中趋势,大肠埃希菌中发现非O25血清型的ST131,肺炎克雷伯菌中发现2个新的tonB基因和2个新的ST型.58个独立克隆的代表菌株中有40株可将携blaCTX-M-15的质粒传递给受体菌E.coli C600(Rif),其中大肠埃希菌有19株,肺炎克雷伯菌有21株.57.9%(11/19)的大肠埃希菌中携带blaCTX-M-15的质粒大小为65 kb,85.7%(18/21)的肺炎克雷伯菌中携带blaCTX-M-15的质粒大小为92 kb.限制性内切酶分析显示,这2种质粒属流行质粒,分别命名为p15-e和p15-k.通过PCR调查,p15-e存在blaCTX-M-15和ISEcpI;p15-k上存在blaCTX-M-15、ISEcpI、aac(6')-Ⅰ b、aac(3')-Ⅲ、blaOXA-1、qnrB、qnrS、blaDHA-1、blaTEM-1.p15-k同时被证实属于质粒不相容群FⅡ群.结论 广州地区存在携带blaCTX-M-15流行质粒的传播,且流行质粒的大小和基因结构与国外报道不同;无克隆株传播证据.

Abstract：

Objective To study the molecular characteristic of the epidemic plasmids carrying blaCTX-M-15 in Guangzhou. Methods A total of 38 strains of E. coli and 47 strains of K. pneumoniae both producing CTX-M-15 ESBLs were collected from nine hospitals in Guangzhou from 2007 to 2008. The clonal relationship of isolates carrying blaCTX-M-15 was determined by PFGE and MLST. Antimicrobial susceptibility was determined by microdilution test for all isolates. Conjugative plasmids carrying blaCTX-M-15 were obtained by mating and were subject to restriction analysis. PCR was used to determine phylogenetic groups of E. coli,and to study replicon type and the genetic contexts of the plasmids harboring blaCTX-M-15. Serum agglutination test was used to detect the serotype of E. coli. Results The 37 strains of E. coli were classified into 28 genotypes, while the 47 strains of K. pneumoniae were divided into 30 genotypes. ST131 was found in E. coli but not O25 serotype. Two novel-alleles of tonB and new ST were determined in K. pneumoniae. Forty out of 58 isolates represented independent genotypes have been succeeded to transfer the plasmid carrying blaCTX-M-15 to the E. coli C600(Rif) by conjugation. The sizes of plasmids carrying blaCTX-M-15 are 65 kb in 57.9% isolates of E. coli and 92 kb in 87.5% isolates of K. pneumoniae. Two epidemic plasmids were detected in E.coli and K. pneumoniae by restriction enzyme, designated p15-e and p15-k respectively. The blaCTX-M-15 and ISEcpI were identified on p15-e, and the blaCTX-M-15 ,ISEcpI,aac(6')- Ⅰ b,aac(3')-Ⅲ ,blaOXA-1 ,qnrB,qnrS,blaDHA-1 , blaTEM-1 were determined on p15-k. The p15-k also was identified to belong to the incompatible group FⅡ. Conclusion The local dissemination of blaCTX-M-15 appears to be due to the spread of epidemic plasmids harboring blaCTX-M-15. No evidence supports the dissemination of clone strains which carried blaCTX-M-15.     

Characterization of CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli strains isolated from hospital environments in Algeria

For detecting extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in the hospital environment, sedimentation plates were placed in the rooms of two hospitals. Three environmental isolates, two Klebsiella pneumoniae, and one Escherichia coli resistant to extended-spectrum cephalosporins with a phenotype indicating CTX-M enzymes production (the minimum inhibitory concentration [MIC] of cefotaxime was higher than the MIC of ceftazidime) were recovered. By PCR and sequencing, the three isolates were found to produce CTX-M-15. The bla(CTX-M-15) genes in the three isolates were transferred by conjugation. One K. pneumoniae environmental isolate showed an identical and unique RAPD profile with two other K. pneumoniae clinical isolates recovered from urinary tract infection from patients hospitalized in two different wards of another hospital.     

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.     

Nosocomial outbreak of CTX-M-15-producing E. coli in Norway

Seven E. coli isolates expressing resistance to 3rd generation cephalosporins were recovered from blood (n=2), kidney and lung tissue (n=1), and urinary tract (n=4) samples from seven patients hospitalised or recently discharged from the Divisions of Geriatrics and Pulmonary Medicine, Central Hospital of Rogaland, between July and September 2004. All isolates expressed a typical ESBL-cefotaximase profile (cefotaxime MIC>ceftazidime MIC) with clavulanic acid synergy. A bla(CTX-M-15) genotype was confirmed in six strains that were coresistant to gentamicin, nitrofurantoin, trimethoprim-sulfamethoxazole and ciprofloxacin. A bla(CTX-M-3) genotype was detected in the last strain. XbaI-PFGE patterns of the six bla(CTX-M-15) isolates revealed a clonal relationship. Bla(CTX-M-15) strains were also positive for the ISEcp1-like insertion sequences that have been shown to be involved in the mobilization of bla(CTX-M.) Further analyses revealed two bla(CTX-M-15)-positive E. coli urinary isolates clonally related to the outbreak strain from two different patients at the same divisions in January and February 2004. These patients were later re-hospitalised and one had E. coli with an ESBL-cefotaximase profile in sputum and nasopharyngeal specimen during the outbreak period. Clinical evaluation suggests that the CTX-M-producing E. coli strains contributed to death in three patients due to delayed efficient antimicrobial therapy. The outbreak emphasises the epidemic potential of multiple-antibiotic-resistant CTX-M-15-producing E. coli also in a country with low antibiotic usage and low prevalence of antimicrobial resistance.     

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.     

The changing epidemiology of extended spectrum β-lactamase-producing Klebsiella pneumoniae

In the last two decades, Klebsiella pneumoniae demonstrated some characteristics of acquisition of plasmids coding extended spectrum β-lactamases (ESBL). The review data showed an increase in worldwide prevalence of ESBL and a temporal shift in the prevalence of ESBL types in K.?pneumoniae during this last decade. CTX-M-15 seems to be the predominant ESBL type in K.?pneumoniae in some parts of the world. The dissemination of several nosocomial CTX-M-15-producing K.?pneumoniae clones was reported unlike the worldwide dissemination of a single virulent ST131 CTX-M-15 producing Escherichia coli clone. The diversity of plasmids carrying the bla(CTX-M-15) gene in K.?pneumoniae suggested the frequent transfer of this gene between different replicons. The acquisition of the bla(CTX-M-15) gene by K.?pneumoniae was probably occurred via horizontal transfer from E.?coli.     

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.     

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.     

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.     

Raman spectroscopic analysis of the clonal and horizontal spread of CTX-M-15-producing Klebsiella pneumoniae in a neonatal intensive care unit

Nosocomial outbreaks of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae are an increasing concern in neonatal intensive care units (NICUs). We describe an outbreak of ESBL-producing K. pneumoniae that lasted 5?months and affected 23 neonates in our NICU. Proton pump inhibitor and extended-spectrum cephalosporin exposure were significantly associated with the risk of ESBL-producing K. pneumoniae colonisation and/or infection. Thirty isolates recovered from clinical, screening and environmental samples in the NICU were studied by means of Raman spectroscopy, pulsed-field gel electrophoresis and repetitive extragenic palindromic polymerase chain reaction (rep-PCR). The Raman clustering was in good agreement with the results of the other two molecular methods. Fourteen isolates belonged to the Raman clone 1 and 16 to the Raman clone 3. Molecular analysis showed that all the strains expressed SHV-1 chromosomal resistance, plasmid-encoded TEM-1 and CTX-M-15 β-lactamases. Incompatibility groups of plasmid content identified by PCR-based replicon typing indicated that resistance dissemination was due to the clonal spread of K. pneumoniae and horizontal CTX-M-15 gene transfer between the two clones.     

Emergence of extended-spectrum-beta-lactamase (CTX-M-9)-producing multiresistant strains of Salmonella enterica serotype Virchow in poultry and humans in France

During 2002 to 2003, eight Salmonella enterica serotype Virchow poultry and poultry product isolates from various sources (chicken farms, poultry slaughterhouse, or retail store) and one S. enterica rough strain isolated from human feces were found to produce extended-spectrum beta-lactamase CTX-M-9. Poultry and poultry product isolates were recovered from different locations in the southwest of France. The human rough isolate had sequences of flagellin genes (fliC and fljB) typical of serotype Virchow and ribotyping and pulsed-field gel electrophoresis (PFGE) patterns closely similar to those of serotype Virchow strains. PFGE confirmed the clonal relationship between the poultry isolates, while the human isolate displayed a pattern with 94% homology. The bla(CTX-M-9) gene was located on a conjugative plasmid and was shown to be linked to orf513. Plasmid profiling found a very similar EcoRI restriction pattern in six transconjugants studied, including transconjugants obtained from the human isolate. A single hatchery, supplying chicks to the six farms, was identified. Emergence of extended-spectrum beta-lactamase-producing S. enterica strains in food animals is a major concern, as such strains could disseminate on a large scale and lead to antibiotic therapy difficulties.     

A Swordless Knight: Epidemiology and Molecular Characteristics of the blaKPC-Negative Sequence Type 258 Klebsiella pneumoniae Clone

In June 2010, a bla(KPC)-negative, ertapenem-resistant ST-258 Klebsiella pneumoniae strain was isolated from a patient in the Laniado Medical Center (LMC). Our aims were (i) to describe its molecular characteristics and resistance mechanisms and (ii) to assess whether the bla(KPC)-negative ST-258 K. pneumoniae clone spreads as efficiently as its KPC-producing isogenic strain. In a prospective study, surveillance of all ertapenem-resistant, carbapenemase-negative K. pneumoniae (ERCNKP) isolates was conducted from June 2010 to May 2011 at LMC (314 beds) and from July 2008 to December 2010 at the Tel Aviv Sourasky Medical Center (TASMC) (1,200 beds). Molecular typing was done by arbitrarily primed PCR, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). A total of 8 of 42 (19%) ERCNKP isolates in LMC and 1 of 32 (3.1%) in TASMC belonged to the ST-258 clone. These strains carried the bla(CTX-M-2) or the bla(CTX-M-25) extended-spectrum β-lactamase (ESBL) gene. Sequencing of the ompK genes showed a frameshift mutation in the ompK35 gene. The fate of the bla(KPC)-carrying plasmid, pKpQIL, was determined by S1 analysis and by PCR of the Tn4401 transposon, repA, and the truncated bla(OXA-9). Plasmid analysis of the ERCNKP ST-258 isolates showed variability in plasmid composition and absence of the Tn4401 transposon and the pKpQIL plasmid. In addition, the ST-258 clone was identified in 35/35 (100%) of KPC-producing K. pneumoniae isolates but in none of 62 ertapenem-susceptible K. pneumoniae isolates collected in the two centers. Our results suggest that ERCNKP ST-258 evolved by loss of the bla(KPC)-carrying plasmid pKpQIL. ERCNKP ST-258 appears to have low epidemic potential.     

Antimicrobial susceptibility to parenteral and oral agents in a largely polyclonal collection of CTX-M-14 and CTX-M-15-producing Escherichia coli and Klebsiella pneumoniae

Activity of oral and parenteral antimicrobials against consecutively isolated extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (n = 149) and Klebsiella pneumoniae (n = 20) was determined, and susceptibility test methods were compared for parenteral β-lactams. Polymerase chain reaction (PCR) targeting bla(CTX-M), bla(SHV) and bla(TEM), and DNA sequencing and epidemiological typing with pulsed-field gel electrophoresis were performed. PCR targeting pabB was screened for E. coli O25b-ST131. Minimum inhibitory concentrations (MICs) were determined using Etest and broth microdilution. Disc diffusion was performed according to European Committee on Antimicrobial Susceptibility Testing (EUCAST). Dominating genotypes were bla(CTX-M-15) (75%) and bla(CTX-M-14) (23%). Four E. coli clusters (7-18 isolates) were found. Forty-two per cent of E. coli belonged to O25b-ST131. Ciprofloxacin resistance was 72%, trimethoprim resistance was 70%. Among E. coli, resistance to mecillinam (13%), nitrofurantoin (7%) and fosfomycin (3%) was low, although resistance was high in K. pneumoniae (25%, 60%, 85%). Susceptibility to ertapenem was 99%, piperacillin-tazobactam 91%, tigecycline 96% and temocillin 76%. Susceptibility rates obtained with broth microdilution and Etest were in agreement for cefotaxime (2 vs 1%) and ceftazidime (9 vs 11%), but not for piperacillin-tazobactam (59 vs 91%). With disc diffusion major errors occurred with piperacillin-tazobactam (18/169). Several therapeutic alternatives exist for ESBL-producing E. coli, but few exist for K. pneumoniae. Disc diffusion and Etest can accurately predict susceptibility to cefotaxime and ceftazidime, but not to piperacillin-tazobactam with the present breakpoints.     

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 of plasmid-mediated AmpC beta-lactamases in a Chinese university hospital from 2003 to 2005: first report of CMY-2-Type AmpC beta-lactamase resistance in China

The aim of this study was to investigate the prevalences of plasmid-mediated AmpC beta-lactamases (PABLs) in isolates of Escherichia coli and Klebsiella spp. from a university hospital in China. A total of 1,935 consecutive nonrepeat clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca were collected between January 2003 and July 2005. The isolates with cefoxitin zone diameters less than 18 mm (screen positive) were selected for PCR of the bla(AmpC) genes and sequencing. Fifty-four (2.79%) isolates harbored PABLs, as demonstrated by PCR and isoelectric focusing. Sequence analysis revealed the presence of bla(DHA-1) and bla(CMY-2) genes. The Southern blot hybridization studies confirmed that bla(CMY-2) and bla(DHA-1) were located on plasmids. Based on species, PABLs were detected in 4.29% (29 isolates of DHA-1 and 1 isolate of CMY-2) of K. pneumoniae, 1.91% (11 isolates of DHA-1 and 12 isolates of CMY-2) of E. coli, and 3.03% (1 isolate of DHA-1) of K. oxytoca isolates. In contrast to our anticipation, the occurrence rate of DHA-1-producing K. pneumonia significantly decreased (P < 0.01), from 7.54% in 2003 to 2.72% in 2004. The results of random amplified polymorphic DNA analysis indicate that the prevalences of DHA-1-producing K. pneumoniae and CMY-2-producing E. coli strains were not due to epidemic strains. In conclusion, DHA-1 was the most prevalent acquired AmpC beta-lactamase in this collection of isolates from a medical center in China, and DHA-1-producing K. pneumoniae was the most prevalent bacterium harboring a PABL. To the best of our knowledge, this is the first report of CMY-2-type AmpC beta-lactamases in the Chinese mainland.