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.     

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.     

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.     

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.     

Dramatic increase in prevalence of fecal carriage of extended-spectrum beta-lactamase-producing Enterobacteriaceae during nonoutbreak situations in Spain

The occurrence of extended-spectrum beta-lactamase (ESBL)-producing isolates has increased worldwide. Fecal carriage of ESBL-producing isolates has mainly been detected in nosocomial outbreaks, and few studies have evaluated fecal carriage during nonoutbreak situations and among patients in the community. We have studied the prevalence of ESBLs in 1,239 fecal samples from 849 patients (64.1% of whom were ambulatory) in 1991 and have compared the prevalence data with those obtained in 2003 for 400 fecal samples from 386 patients (75.9% of whom were ambulatory) and 108 samples from independent healthy volunteers. Samples were diluted in saline and cultured in two MacConkey agar plates supplemented with ceftazidime (1 microg/ml) and cefotaxime (1 microg/ml), respectively. Colonies were screened (by the double-disk synergy test) for ESBL production. The clonal relatedness of all ESBL-producing isolates was determined by pulsed-field gel electrophoresis with XbaI digestion; and the ESBLs of all ESBL-producing isolates were characterized by isoelectric focusing, PCR, and sequencing. The rates of fecal carriage of ESBL-producing isolates increased significantly (P < 0.001) in both hospitalized patients and outpatients, from 0.3 and 0.7%, respectively, in 1991, to 11.8 and 5.5%, respectively, in 2003. The rate of occurrence of ESBL-producing isolates among healthy volunteers was 3.7%. All ESBL-producing isolates recovered in 2003 were nonepidemic clones of Escherichia coli. ESBL characterization revealed an increasing diversity of ESBL types: TEM-4 and CTX-M-10 were the only enzymes detected in 1991, whereas TEM-4, TEM-52, SHV-12, CTX-M-9, CTX-M-10, CTX-M-14, and a CTX-M-2-like enzyme were recovered in 2003. The ESBL-producing isolates recovered from outpatients in 2003 corresponded to a CTX-M-9-type cluster (62.5%) and SHV-12 (31.2%), whereas TEM-4 was detected only in hospitalized patients. The frequencies of coresistance in isolates recovered in 2003 were as follows: sulfonamide, 75%; tetracycline, 64.3%; streptomycin, 57.1%; quinolones, 53.5%; and trimethoprim, 50%. The increased prevalence of fecal carriage of ESBL-producing isolates during nonoutbreak situations in hospitalized patients and the establishment of these isolates in the community with coresistance to non-beta-lactam antibiotics, including quinolones, represent an opportunity for these isolates to become endemic.     

Dissemination of CTX-M-3 and CMY-2 beta-lactamases among clinical isolates of Escherichia coli in southern Taiwan

A total of 1,210 clinical isolates of Escherichia coli collected from a university hospital in southern Taiwan were screened for production of extended-spectrum beta-lactamases (ESBLs). Expression of classical ESBLs (resistant to extended-spectrum beta-lactam agents and susceptible to beta-lactam inhibitors) was inferred in 18 isolates by the phenotypic confirmatory test. These included 10 isolates producing CTX-M-3, 2 strains carrying SHV-12, 1 strain harboring SHV-5, 1 strain expressing TEM-10, and 4 strains producing unidentifiable ESBLs with a pI of 8.05, 8.0, or 7.4. Eighteen isolates that showed decreased susceptibilities to ceftazidime and/or cefotaxime, negative results for the confirmatory test, and high-level resistance to cefoxitin (MICs of >/=128 microg/ml) were also investigated. Five isolates were found to produce CMY-2 AmpC enzymes, one isolate carried both CTX-M-3 and CMY-2, and the remaining three and nine isolates expressed putative AmpC beta-lactamases with pIs of >9.0 and 8.9, respectively. Thus, together with the isolate producing CTX-M-3 and CMY-2, 19 (1.6%) isolates produced classical ESBLs. Pulsed-field gel electrophoresis revealed that all isolates carrying CTX-M-3 and/or CMY-2 were genetically unrelated, indicating that dissemination of resistance plasmids was responsible for the spread of these two enzymes among E. coli in this area. Among the 16 isolates expressing CTX-M-3 and/or CMY-2, 5 might have colonized outside the hospital environment. Our data indicate that CTX-M-3 and CMY-2, two beta-lactamases initially identified in Europe, have been disseminated to and are prevalent in Taiwan.     

High prevalence of CTX-M-15-producing O25b-ST131 Escherichia coli clone in Bulgarian hospitals

According to the European Antimicrobial Resistance Surveillance System project results, Bulgaria has become one of the European countries with dramatically increasing rates of extended-spectrum beta-lactamase (ESBL) producers. The aim of this work was to investigate the epidemiology of ESBL-producing Escherichia coli clinical isolates in Bulgaria, collected from seven clinical centers in three towns, during two study periods: 2002-2003 and 2006-2009. For 193 ESBL-producing E. coli isolates random amplified polymorphic DNA (RAPD) analyses, phylogenetic typing, and screening for O25b-ST131 isolates were carried out. Antimicrobial susceptibility, ESBL-type and transferability of resistance determinants were analyzed. Four different ESBL-types, namely TEM-139, SHV-12, CTX-M-3, and CTX-M-15 were found. CTX-M-15 dominated, being found in 88% of the isolates. RAPD-typing revealed 35 types, among which type A dominated, comprising 65% of the isolates. Sixty-eight percent of the 193 isolates belonged to the O25b-ST131 clone, to the phylogenetic group B2, mostly showed RAPD-type A (92%) and were found in all participating hospitals. O25b-ST131 isolates predominantly produced CTX-M-15 (96%), and less SHV-12 (n=3) or TEM-139 (n=2). In conclusion, this study demonstrated for the first time the country-wide dissemination of a highly resistant B2 O25b-ST131 CTX-M-15 producing E. coli clone in Bulgaria.     

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.     

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.     

Identification and characterization of OXA-48 producing, carbapenem-resistant Enterobacteriaceae isolates in Turkey

Carbapenem resistance in Enterobacteriaceae isolates has been reported from Turkey and is most often mediated by OXA-48 type carbapenemases. We report the identification and characterization of four carbapenem-resistant isolates (three Klebsiella pneumoniae and one Escherichia coli) among 515 clinical Enterobacteriaceae isolates collected during a 7-month study period in Ankara, Turkey. The four isolates were recovered from blood and urine specimens in patients with varied clinical manifestations. They had distinct pulsed-field gel electrophoresis patterns and harbored a variety of β-lactamases including bla(TEM-1), bla(SHV-12) genes, bla(SHV-11), and/or bla(CTX-M-15). PCR and sequencing analysis revealed that the bla(OXA-48) gene was present in all four isolates. Our data indicated that the OXA-48-type carbapenemase was the only mechanism for carbapenem resistance in our hospital.     

Epidemiology of extended-spectrum beta-lactamase-producing Enterobacter isolates in a Spanish hospital during a 12-year period

Fifteen Enterobacter clinical isolates (11 Enterobacter cloacae isolates, 3 Enterobacter aerogenes isolates, and 1 Enterobacter gergoviae isolate), representing 0.4% of all Enterobacter isolates recovered in our hospital from 1989 to 2000, were suspected of harboring an extended-spectrum beta-lactamase (ESBL). These isolates were recovered from 14 different patients. ESBLs were transferred by conjugation into an Escherichia coli recipient strain. Pulsed-field gel electrophoresis (PFGE) revealed a single clone of E. aerogenes and six different clones of E. cloacae. Four of these E. cloacae clonal types were represented by only one isolate each, but the other two were represented by three and four isolates, respectively. Isoelectric focusing, susceptibility phenotyping, PCR analysis, and sequencing demonstrated the presence of three different ESBLs. The most frequent was the recently characterized CTX-M-10 ESBL, which was found in the E. gergoviae isolate and in all but one of the E. cloacae isolates. The remaining E. cloacae isolate harbored a TEM-27 ESBL, and the three E. aerogenes isolates harbored a TEM-24 ESBL. PFGE revealed that our E. aerogenes strain was indistinguishable from the French TEM-24-producing E. aerogenes endemic clone. Although a low prevalence of ESBL-producing Enterobacter isolates was found in our institution over a 12-year period, a diversity of nonepidemic E. cloacae clones was detected, as was the persistence of the CTX-M-10 beta-lactamase. The presence of the TEM-24-producing E. aerogenes French clone in our institution also demonstrates the intercountry dissemination of ESBL-producing isolates.     

同时产ArmA型16S rRNA甲基化酶及KPC-2型β-内酰胺酶泛耐药阴沟肠杆菌的研究

目的 探讨上海交通大学医学院附属瑞金医院分离的5株泛耐药阴沟肠杆菌产碳青霉烯酶及16S rRNA甲基化酶的情况及两者之间的相关性.方法 用E test法检测5株泛耐药阴沟肠杆菌对10种抗菌药物的MIC值.PCR扩增16S rRNA甲基化酶基因armA、mtA、rmtB、rmtC、rmtD、npmA,β-内酰胺酶基因TEM、SHV、CTX-M-1、CTX-M-2、CTX-M-8、CTX-M-9、CTX-M-25、PER-1、VEB-1.鸟枪克隆法克隆碳青霉烯酶基因,并对克隆片段进行测序.质粒接合试验验证碳青霉烯酶基因及16S rRNA甲基化酶基因是否具有转移性.脉冲场凝胶电泳(PFGE)法对5株菌进行分子分型.等电聚焦电泳法检测β-内酰胺酶等电点.Southern杂交法对耐药决定因子进行定位.结果 5株阴沟肠杆菌对10种抗菌药物的MIC值均＞32 mg/L.克隆的碳青霉稀酶基因为blaKPC-2,酶编码基因上游为一转座酶基因,两侧为复制靶位,下游为ISKpn6的插入序列,该序列包括一个重复序列和tnpA转座子,酶编码基因位于54.2 kb的一个非接合性大质粒上.等电聚焦电泳显示5株菌均产4种β-内酰胺酶(TEM-1,pI5.4;KPC-2,pI6.7;SHV-12,pI8.2;CTX-M-14,pI8.4).16S rRNA甲基化酶基因位于接合性质粒上,而blaKPC-2基因则位于非接合性质粒上,5株菌PFGE型别一致.结论 5株泛耐药阴沟肠杆菌对碳青霉烯类耐药由产碳青霉烯酶KPC-2所介导.blaKPC-2与armA型16S rRNA甲基化酶基因由两条不同的质粒编码,不存在相关性.临床医院应加强监控,以防止交叉感染.     

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.     

Occurrence of extended spectrum beta-lactamases among isolates of Enterobacteriaceae from urinary tract infections in southern Italy

The present investigation was undertaken to assess the prevalence of extended-spectrum beta-lactamases (ESbetaLs) among urinary tract infection (UTI) isolates. During 4 months in 2004, a total of 650 Enterobacteriaceae strains from UTIs was collected by five clinical microbiology laboratories located in southern Italy and the beta-lactamase production was investigated. A total of 50 of the 650 isolates were double-disk positive and suspected of producing an ESbetaL; Escherichia coli (36.0%) and Klebsiella pneumoniae (32.0%) were the most common species among all ESbetaL producers. Characterization of ESbetaL determinants was carried out by the colony blot hybridization method, and polymerase chain reaction (PCR) and DNA sequencing in order to identify the presence of bla (TEM), bla (SHV), bla (PER), and bla (CTX-M) determinants. The ESbetaL variants found in this study were the following: TEM-15, TEM-24, TEM-52, TEM-134, SHV-12, CTX-M-1, CTX-M-3, CTX-M-15, and PER-1. As expected, the majority of the isolates were found to be susceptible to imipenem (94%), cefepime (54%) and piperacillin-tazobactam (54%). The results of this survey show the prevalence of ESbetaL enzymes among enterobacterial pathogens causing UTIs in southern Italy.     

Effects of inoculum and beta-lactamase activity in AmpC- and extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae clinical isolates tested by using NCCLS ESBL methodology

Escherichia coli and Klebsiella pneumoniae isolates with extended-spectrum beta-lactamases (ESBLs) or AmpC cephalosporinases generally respond as predicted to NCCLS tests for ESBL production. However, inoculum size may affect MICs. The effect of inoculum level in clinical isolates expressing beta-lactamases were studied at inocula within 0.5 log unit of the standard inoculum, using broth microdilution methodology with ceftazidime, cefotaxime, cefepime, cefpodoxime, and aztreonam. Strains with TEM-1 or no beta-lactamases gave consistent MIC results with inocula of 10(5) and 10(6) CFU/ml. When the bacteria were screened for ESBL production and the lower inoculum was used, several strains with ESBLs, including CTX-M-10, TEM-3, TEM-10, TEM-12, TEM-6, SHV-18, and K1, gave false-negative results for one or more antimicrobial agents (MICs below the NCCLS screening concentration for detecting suspected ESBLs). When the higher inoculum was used, MICs of at least one antimicrobial agent increased at least fourfold in strains producing TEM-3, TEM-10, TEM-28, TEM-43, SHV-5, SHV-18, and K1. All antimicrobial agents showed an inoculum effect with at least one ESBL producer. Confirmatory clavulanate effects were seen for both inocula for all ESBL-producing strains with all antimicrobial agents tested, except for the CTX-M-10-producing E. coli with ceftazidime and the SHV-18-producing K. pneumoniae with cefotaxime. In kinetic studies, cefpodoxime and cefepime were hydrolyzed by ESBLs in a manner similar to that of cefotaxime. When total beta-lactamase activity and hydrolysis parameters were evaluated, however, no single factor was predictive of inoculum effects. These results indicate that the NCCLS screening and confirmation tests are generally predictive of ESBL production, but false-negative results can arise when a lower inoculum is used in testing.     

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.     

Occurrence of extended-spectrum beta-lactamases in members of the genus Shigella in the Republic of Korea

A nationwide survey was carried out in Korea to assess the prevalence of Shigella strains producing extended-spectrum beta-lactamases (ESBLs). From 1991 to 2002, 5,911 clinical strains were isolated and screened for resistance to extended-spectrum cephalosporins. Twenty of the Shigella isolates were ESBL positive, based on the synergistic effects between clavulanate and selected beta-lactams (ceftazidime and cefotaxime). Nucleotide sequence analysis of these isolates revealed that they harbored bla(TEM-19) (eight isolates), bla(TEM-15) (five isolates), bla(TEM-52) (six isolates), bla(TEM-17) (one isolate), bla(TEM-20) (one isolate), and bla(CTX-M-14) (three isolates). All the ESBL-encoding genes in this study were carried in conjugable plasmids. Thus, TEM-19, TEM-15, TEM-52, and CTX-M-14 beta-lactamases can be considered common Korean ESBL types in Shigella sonnei and are probably transmitted through interspecies spread between medical facilities and the community in Korea. This is the first report of the presence of TEM-17, TEM-19, and TEM-20 in Korea and in S. sonnei.     

Emergence of CTX-M-15 extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolates in Bosnia and Herzegovina

Fifty-seven nosocomial Klebsiella pneumoniae isolates producing extended-spectrum β-lactamases (ESBLs) were collected between February 2007 and November 2007 in different wards of the Sarajevo (Bosnia-Herzegovina) reference hospital. These isolates comprise two major epidemic pulsed-field electrophoresis-defined clones plus two minor clones. In addition to the ESBL-mediated resistance, all strains uniformly showed resistance to ciprofloxacin, gentamicin and tobramycin. The β-lactamases involved in this resistance phenotype were TEM-1, SHV-1, and CTX-M-15, as demonstrated by isoelectric focusing, PCR amplification, and sequencing. TEM-1 and CTX-M-15 β-lactamases, as well as the aminoglycoside resistance determinants, were encoded in plasmids that could be transferred to Escherichia coli by conjugation. In three of the infected patients with the predominant clone, cefoxitin resistance development (MICs >128 mg/L) was documented. The analysis of the outer membrane proteins of the cefoxitin-susceptible and cefoxitin-resistant isolates revealed that the former expressed only one of the two major porins, OmpK36, whereas in the latter, the expression of Ompk36 was altered or abolished. This is the first report of CTX-M-15-producing K. pneumoniae in Bosnia-Herzegovina. Furthermore, we document and characterize for the first time cefoxitin resistance development in CTX-M-15-producing K. pneumoniae.     

Expansion of ESBL-producing Klebsiella pneumoniae in hospitalized patients: A successful story of international clones (ST15, ST147, ST336) and epidemic plasmids (IncR,IncFIIK)

The aim of this study was to characterize by a multi-level approach extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae isolates other than E. coli from Portuguese hospitals. Eighty-eight ESBL-producing clinical isolates (69 Klebsiella pneumoniae, 13 Enterobacter cloacae complex, 3 Klebsiella oxytoca, 1 Enterobacter asburiae, 1 Proteus mirabilis and 1 Serratia marcescens) recovered from hospitals located in the North (A) or Centre (B, C) regions during two time periods (2006–7 and 2010) were analyzed. Standard methods were used for bacterial identification, antibiotic susceptibility testing, ESBL characterization, clonal (PFGE, MLST) and plasmid (S1-PFGE, I-CeuI-PFGE, replicon typing, hybridization) analysis. Isolates produced mostly CTX-M-15 (47%) or SHV-12 (30%), and less frequently other SHV- (15%; SHV-2, -5, -28, -55, -106) or TEM- (9%; TEM-10, -24, -199)-types, with marked local and temporal variations. The increase of CTX-M-15 and diverse SHV ESBL-types observed in Hospital A was associated with the amplification of multidrug-resistant (MDR) K. pneumoniae epidemic clones (ST15, ST147, ST336). SHV-12 and TEM-type ESBLs were mostly identified in diverse isolates of different Enterobacteriaceae species in Hospitals B and C in 2006–7. Particular plasmid types were linked to bla_CTX-M-15 (IncR or non-typeable plasmids), bla_SHV-12 (IncR or IncHI2), bla_{SHV-28/-55/-106} (IncFII_K1 or IncFII_K5), bla_TEM-10 (IncL/M) or bla_TEM-24 (IncA/C), mostly in epidemic clones. In our country, the amplification of CTX-M-15 and diverse SHV-type ESBL among non-E. coli Enterobacteriaceae is linked to international MDR K. pneumoniae clones (ST15, ST147, ST336) and plasmid types (IncR, IncFII_K). Furthermore, we highlight the potential of IncFII_K plasmids (here firstly associated with bla_{SHV-2/-28/-55/-106}) to disseminate as antibiotic resistance plasmids.     

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.