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.     

The characteristics of extended-spectrum beta-lactamases in Korean isolates of Enterobacteriaceae

Extended-spectrum beta-lactamases (ESBLs) in gram-negative organisms have been implicated as the enzymes responsible for resistance to oxyimino-cephalosporins. The incidence of ESBL-producers in Korean isolates of Escherichia coli and Klebsiella pneumoniae were in the range of 4.8-7.5% and 22.5-22.8%, respectively. The ESBL-producing isolates revealed variable levels of resistance to cefotaxime, ceftazidime and aztreonam. They also showed the elevated MIC values of non-beta-lactam antibiotics. SHV-12 and SHV-2a were the enzymes most frequently found in K. pneumoniae strains, but TEM-52 was the most prevalent in E. coli isolates. About 15% of ESBL-producing isolates of Enterobacteriaceae produced CMY-1 enzyme, which conferred resistance to cephamycins such as cefoxitin as well as oxyimino-cephalosporins. Thus, the most common types of ESBLs in Korea are TEM-52, SHV-12 SHV-2a, and CMY-1.     

Molecular typing and virulence analysis of serotype K1 Klebsiella pneumoniae strains isolated from liver abscess patients and stool samples from noninfectious subjects in Hong Kong, Singapore, and Taiwan

Serotype K1 Klebsiella pneumoniae with multilocus sequence type 23 (ST23) has been strongly associated with liver abscess in Taiwan. Few data regarding the strain types and virulence of this serotype from other Asian countries are available. Serotype K1 K. pneumoniae strains isolated from liver abscess and stool samples from subjects hospitalized in Hong Kong, Singapore, and Taiwan hospitals were examined. Forty-seven serotype K1 isolates were identified: 26 from liver abscess samples and 21 from stool samples. MLST revealed 7 sequence types: 85.1% (40 of 47 isolates) belonged to ST23, 1 isolate belonged to ST163 (a single-locus variant of ST23), and 2 isolates were ST249 (a 3-locus variant of ST23). New STs, namely, ST367, ST425, and ST426, were allocated to 3 of 4 isolates from stool samples. The virulence of these strains was determined by neutrophil phagocytosis and mouse infection models. Except for two ST23 isolates, all Klebsiella pneumoniae isolates were resistant to phagocytosis. Resistance to serum killing varied in isolates of ST23, while all non-ST23 strains were susceptible to serum killing except one with ST249 from a liver abscess. All hypervirulent isolates with a 50% lethal dose of <10(2) CFU were from ST23, were resistant to phagocytosis and serum killing, and also carried both virulence-associated genes, rmpA and aerobactin. Multilocus sequence typing genotype 23 was the most prevalent sequence type among serotype K1 K. pneumoniae isolates from both liver abscess and stool samples in the Asia Pacific region. Serotype K1 K. pneumoniae isolates with capsule expression leading to phagocytic resistance and with the aerobactin gene were associated with hypervirulence.     

Multilocus sequence typing versus pulsed-field gel electrophoresis for characterization of extended-spectrum beta-lactamase-producing Escherichia coli isolates

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strains are emerging pathogens. Molecular typing of ESBL-producing E. coli is useful for surveillance purposes, to monitor outbreaks and track nosocomial spread. Although pulsed-field gel electrophoresis (PFGE) is the current "gold standard" for bacterial molecular typing, multilocus sequence typing (MLST) may offer advantages. Forty ESBL-producing E. coli isolates were selected at random from a cohort of intensive care unit patients who had active surveillance perirectal cultures done. PFGE identified 19 unique PFGE types (PT) among the 40 isolates; MLST identified 22 unique sequence types. MLST had greater discriminatory ability than PFGE for ESBL-producing E. coli. Simpson's indices of diversity for PFGE and MLST were 0.895 and 0.956, respectively. There were five clonal complexes (CCs) (isolates with differences of no more than two loci) that each contained multiple PT, but each PT was found in only one CC, indicating genetic consistency within a CC. MLST has clear utility in studies of ESBL-producing E. coli, based on a greater discriminatory ability and reproducibility than PFGE and the ability to a priori define genetically related bacterial strains.     

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.     

泛耐药肺炎克雷伯菌株的医院内流行特性的研究

目的 了解华山医院泛耐药肺炎克雷伯菌株及其流行的特点.方法 收集2006年8月-2009年12月对CLSI推荐常规检测药物均耐药的肺炎克雷们菌临床分离株,共57株.所有菌株都进行药物敏感试验、超广谱β-内酰胺酶(ESBLs)初筛及表型确证试验、改良Hodge试验、等电聚焦电泳,聚合酶链反应及其产物测序、接合试验、肠杆菌基因间重复共有序列PCR(ERIC-PCR)和多位点序列分型(MLST).结果 所有菌株都携带blaKPC-2、blaCTX-M-14、blaSHV12和blaTEM-1及qnrB和aac(6')-I b-cr基因.57株细菌中ST423型5株,MIST ST11型52株.ST423型散发,而ST11型呈医院内流行.57株细菌都对替加环素耐药,对多黏菌素、米诺环素和多西环素部分敏感.结论 本次泛耐药肺炎克雷们流行主要为ST11型菌株;不同的肺炎克雷伯菌株,播散能力不同;检出泛耐药肺炎克雷伯菌时应增加检测药物的种类.     

泛耐药肺炎克雷伯菌株的医院内流行特性的研究

目的 了解华山医院泛耐药肺炎克雷伯菌株及其流行的特点.方法 收集2006年8月-2009年12月对CLSI推荐常规检测药物均耐药的肺炎克雷们菌临床分离株,共57株.所有菌株都进行药物敏感试验、超广谱β-内酰胺酶(ESBLs)初筛及表型确证试验、改良Hodge试验、等电聚焦电泳,聚合酶链反应及其产物测序、接合试验、肠杆菌基因间重复共有序列PCR(ERIC-PCR)和多位点序列分型(MLST).结果 所有菌株都携带blaKPC-2、blaCTX-M-14、blaSHV12和blaTEM-1及qnrB和aac(6')-I b-cr基因.57株细菌中ST423型5株,MIST ST11型52株.ST423型散发,而ST11型呈医院内流行.57株细菌都对替加环素耐药,对多黏菌素、米诺环素和多西环素部分敏感.结论 本次泛耐药肺炎克雷们流行主要为ST11型菌株;不同的肺炎克雷伯菌株,播散能力不同;检出泛耐药肺炎克雷伯菌时应增加检测药物的种类.

Abstract：

Objective To understand the epidemic characteristics of an outbreak of panresistance Klebsiella pneumoniae occurred between 2006 and 2009 in a university hospital of Shanghai, China. Methods A total of 57 panresistance K. pneumoniae isolates were collected from August 2006 to December 2009.Antibiotic susceptibility of the isolates were determined by Kirby-Bauer disc diffusion method and microbroth dilution (MBD). ESBLs-producing initial screen test and phenotypic confirmatory test and carbapenemase-producing modified Hodge test ( MHT) were performed to detect the resistance phenotype of the isolates. Be-ta-lactamases were studied by IEF, PCR and the product sequencing. While conjugation assay were conducted to understand the transferability of these genes. The genetic relationship between isolates was established by ERIC-PCR and multilocus sequence typing (MLST). Except for the antibiotics recommended by CLSI guideline in the routine test, the other antibiotics were added to find out the effective drugs to treat the infection. Results All 57 isolates were highly resistant to all examined antibiotics. All isolates produced ESBLs and carbapenemase. IEF revealed that each isolate produced four beta-lactamases. All isolates carried blaKPC-2,blaCTX-M-14,blaSHV12,blaTEM-1,qnrB and aac(6') - I b-cr. Forty-four of the 57 (77.2% ) isolates were successful to transfer their resistance genes to E. coli recipient J53 by conjugation assay. By RAPD, all 57 isolates were grouped into two genotypes that were further identified as members of MUST types 423 and 11.Sequence types 423(ST423) only occurred before May 2008 and ST11 occurred (52 isolates) after May 2008. Most of isolates of the outbreak were ST11 (91. 2% ). A part of isolates were susceptive to added antibiotics. Conclusion The outbreak of panresistance K.pneumoniae was caused by those isolates which carried multiple resistant genes. There is a different ability of dissemination between different ST types K. pneumoniae isolate. It was necessary to add the antibiotics to find out the effective drugs to treat the infection.     

Molecular characterization of extended-spectrum beta-lactamases produced by clinical isolates of Klebsiella pneumoniae and Escherichia coli from a Korean nationwide survey

To determine the prevalence and genotypes of extended-spectrum beta-lactamases (ESBLs) among clinical isolates of Klebsiella pneumoniae and Escherichia coli, we performed antibiotic susceptibility testing, pI determination, induction testing, transconjugation, and DNA sequencing analysis. Among the 509 isolates collected from 13 university hospitals in Korea, 39.2% produced ESBLs. ESBL-producing isolates were detected in every region in Korea. A total of 44.6% of the isolates produced both TEM- and SHV-type ESBLs, and 52% of ESBL-producing isolates transferred resistance to ceftazidime by transconjugation. The ESBLs were TEM-19, TEM-20, TEM-52, SHV-2a, SHV-12, and one new variant identified for the first time in Korea, namely, TEM-116. TEM-1 and SHV-12 were by far the most common variants. TEM-1, TEM-116, and SHV-12 showed a high prevalence in K. pneumoniae. Two isolates (E. coli SH16 and K. pneumoniae SV3) produced CMY-1-like beta-lactamases, which play a decisive role in resistance to cefoxitin and cefotetan, as well as TEM-type enzymes (TEM-20 and TEM-52, respectively). Using MIC patterns and DNA sequencing analysis, we postulated a possible evolution scheme among TEM-type beta-lactamases in Korea: from TEM-1 to TEM-19, from TEM-19 to TEM-20, and from TEM-20 to TEM-52.     

Occurrence of qnrA-positive clinical isolates in French teaching hospitals during 2002–2005

Bacteria harbouring the novel qnrA plasmid-mediated mechanism of quinolone resistance have been described in different countries, but the frequency of their occurrence has not been investigated. In total, 1,468 clinical isolates of Enterobacteriaceae with quinolone resistance or extended-spectrum beta-lactamase (ESBL) phenotypes were collected from eight teaching hospitals in France during 2002-2005 and screened for qnrA. Overall, 28 isolates (22 Enterobacter cloacae, three Klebsiella pneumoniae, one Citrobacter freundii, one Klebsiella oxytoca and one Proteus mirabilis) were positive for qnrA, representing 1.9% of all isolates, 3.3% of ESBL-producing isolates (22% of the E. cloacae isolates) and 0% of non-ESBL-producing isolates. The prevalence of qnrA among consecutive ESBL-producing isolates in 2004 from the eight hospitals was 2.8% (18/639). Of the qnrA-positive isolates, 100% were intermediately-resistant or resistant to nalidixic acid, and 75% to ciprofloxacin. Twenty-one of the 22 qnrA-positive E. cloacae isolates were obtained from two hospitals in the Paris area, and molecular typing and plasmid content analysis showed clonal relationships for five, three and two isolates, respectively. The qnrA genetic environment was similar to that of the In36 integron. The remaining two isolates had qnrA variants (30 and 29 nucleotide differences, respectively, compared with the original sequence) and an unknown genetic environment. The ESBL gene associated with qnrA was bla(SHV-12) in most of the isolates, but bla(PER-1) and bla(SHV-2a) were found in two isolates. In France, it appears that qnrA-positive isolates are predominantly E. cloacae isolates producing SHV-12, and may be associated with the dissemination of an In36-like integron.     

泛耐药肺炎克雷伯菌株的医院内流行特性的研究

目的 了解华山医院泛耐药肺炎克雷伯菌株及其流行的特点.方法 收集2006年8月-2009年12月对CLSI推荐常规检测药物均耐药的肺炎克雷们菌临床分离株,共57株.所有菌株都进行药物敏感试验、超广谱β-内酰胺酶(ESBLs)初筛及表型确证试验、改良Hodge试验、等电聚焦电泳,聚合酶链反应及其产物测序、接合试验、肠杆菌基因间重复共有序列PCR(ERIC-PCR)和多位点序列分型(MLST).结果 所有菌株都携带blaKPC-2、blaCTX-M-14、blaSHV12和blaTEM-1及qnrB和aac(6')-I b-cr基因.57株细菌中ST423型5株,MIST ST11型52株.ST423型散发,而ST11型呈医院内流行.57株细菌都对替加环素耐药,对多黏菌素、米诺环素和多西环素部分敏感.结论 本次泛耐药肺炎克雷们流行主要为ST11型菌株;不同的肺炎克雷伯菌株,播散能力不同;检出泛耐药肺炎克雷伯菌时应增加检测药物的种类.     

bla KPC and rmtB on a single plasmid in Enterobacter amnigenus and Klebsiella pneumoniae isolates from the same patient

Enterobacter amnigenus (EA76) and Klebsiella pneumoniae (KP76) isolates with multidrug-resistant (MDR) patterns were identified from the same patient in the neurosurgery department of our hospital. An outbreak of MDR K. pneumoniae had also occurred in this department. To characterize the resistance mechanism and molecular epidemiology of these isolates, sequential experiments including antimicrobial susceptibility testing, polymerase chain reaction (PCR), plasmid analysis, pulsed field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were performed. EA76 and KP76 were resistant to all of the antibiotics tested, except colistin and tigecycline. blaKPC-2, blaTEM-1, blaSHV-12, blaCTX-M-3, blaCTX-M-14, and rmtB genes were identified in both isolates, with blaKPC-2, blaTEM-1, blaCTX-M-14, and rmtB being co-carried on one plasmid in each isolate. Further analysis showed different restriction patterns between the two KPC-carrying plasmids. Of the 11 carbapenem-resistant isolates found in the outbreak, all were resistant to all of the β-lactams tested, with 63.64% (7/11) also exhibiting resistance to aminoglycosides and 72.73% (8/11) exhibiting resistance to quinolones. PCR analysis and molecular typing of the 11 K. pneumoniae strains revealed that the seven aminoglycoside-resistant isolates shared the same antibiotic-resistant gene pattern and identical or one-band-difference PFGE profiles relative to KP76. In addition, all of the eight aminoglycoside-resistant isolates, including KP76, belonged to the national epidemic clone ST11. The overall results indicate the emergence of E. amnigenus and outbreak of ST11 K. pneumoniae, with both co-harboring blaKPC and rmtB genes on a single plasmid in our neurosurgery wards.     

SHV-5 extended-spectrum beta-lactamases in clinical isolates of Escherichia coli in Malaysia

Escherichia coli isolates resistant to ceftazidime isolated in the University Malaya Medical Center (UMMC) Kuala Lumpur, Malaysia, between the years 1998 and 2000 were studied for extended-spectrum beta-lactamase (ESBL) production. All strains were analysed phenotypically and genotypically and found to be ESBL-producing organisms harbouring SHV-5 beta-lactamase. This was confirmed by PCR-SSCP and nucleotide sequencing of the blaSHV amplified gene. As there was no evidence of ESBL activity in E. coli prior to this, coupled with the fact that there was a predominance of SHV-5 beta-lactamases in Klebsiella pneumoniae isolates in UMMC, we postulate that the E. coli obtained the SHV-5 beta-lactamase genes by plasmid transfer from the ESBL-producing K. pneumoniae.     

Concurrent emergence of multidrug resistance and heat resistance by CTX-M-15-encoding conjugative plasmids in Klebsiella pneumoniae

A plasmid-encoded ClpK protein was recently identified as a predictor of a heat-resistant phenotype in the opportunistic pathogen Klebsiella pneumoniae. This study was undertaken to evaluate the presence of the clpK gene in extended-spectrum β-lactamase (ESBL)-producing K. pneumoniae and to assess the probable co-transfer of multi-resistance with the heat resistance phenotype. A Danish collection of 80 ESBL-producing K. pneumoniae bloodstream infection isolates was screened for clpK by colony hybridization. Nineteen isolates (24%) were positive for clpK; some of them representing major clones identified in Denmark. Among these, nine isolates belonged to a single K. pneumoniae CTX-M-15 clone with sequence type (ST)16 exhibiting a heat-resistant phenotype. This clone has a multi-hospital occurrence and has also been detected outside Denmark. Horizontal co-transfer of multiple antibiotic resistances, including the CTX-M-15 resistance determinant, and the heat resistance phenotype was observed. Thus, the clpK gene is harbored by different ESBL-producing K. pneumoniae isolates including a clone of ST16 internationally spread. The co-localization of clpK on transferable ESBL-encoding plasmids allowing co-dissemination of multiple drug resistance with bacterial heat resistance is a highly interesting phenomenon that may further complicate the prevention of spreading of certain successful clones of multi-resistant K. pneumoniae.     

Identification of CTX-M15-, SHV-28-producing Klebsiella pneumoniae ST15 as an epidemic clone in the Copenhagen area using a semi-automated Rep-PCR typing assay

Rapid molecular typing methods can be a valuable aid in the investigation of suspected outbreaks. We used a semi-automated repetitive sequence-based polymerase chain reaction (Rep-PCR) typing assay and pulsed field gel electrophoresis (PFGE) to investigate the relationship between local Klebsiella pneumoniae (K. pneumoniae) producing extended spectrum β-lactamases (ESBLs) and their relation to recognized Danish outbreak strains. PFGE and Rep-PCR produced similar clustering among isolates. Individual isolates from each cluster were further characterized by PCR amplification and sequencing of bla _TEM, bla _SHV, and bla _CTX-M, and multilocus sequence typing (MLST). Thirty-five out of 52 ESBL-producing K. pneumoniae isolates were ST15 and bla _CTX-M15, bla _SHV-28, and bla _TEM-1 positive by PCR. Ten out of 52 were ST16 and tested positive for bla _CTX-M15, bla _SHV-1, and bla _TEM-1. Isolates from previously recognized hospital outbreaks were also ST15 and PCR positive for bla _CTX-M15, bla _SHV-28, and bla _TEM-1, and typed within the main cluster by both Rep-PCR and PFGE. In conclusion, K. pneumoniae ST15 containing bla _CTX-M15 and bla _SHV-28 constitutes an epidemic clone in the Copenhagen area and this clone can be rapidly recognized by semi-automated Rep-PCR.     

Emergence of a colistin-resistant KPC-2-producing Klebsiella pneumoniae ST258 clone in Hungary

Nine Klebsiella pneumoniae isolates showing non-susceptibility to carbapenems were collected from three centres in the north-eastern region of Hungary. The minimum inhibitory concentrations (MICs) of antibiotics were determined by Etest. The putative production of a carbapenemase was tested by the modified Hodge test. The presence of bla _KPC genes was verified by polymerase chain reaction (PCR) and sequencing. Furthermore, molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All isolates showed extensively drug-resistant (XDR) phenotype, and of these, eight isolates were highly resistant to colistin. The isolates carried bla _KPC-2, bla _SHV-12, bla _TEM-1 and bla _SHV-11. PFGE analysis of the nine KPC-2-producing Hungarian ST258 K. pneumoniae isolates, two KPC-2-producing Norwegian ST258 isolates and 33 CTX-M-15-producing ST11 isolates revealed the existence of one genetic cluster at an 88% similarity level. The overall results of the PFGE clustering, MLST and the presence of SHV-11 in both ST11 and ST258 suggest that this is the first hyperepidemic clonal complex of multidrug-resistant K. pneumoniae, probably CC258/CC340, possibly undergoing worldwide spread.     

Detection of diverse SCCmec variants in methicillin-resistant Staphylococcus aureus and comparison of SCCmec typing methods

Non-duplicate methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 436), collected from four hospitals located in three Korean cities between 2001 and 2005, were investigated by SCCmec typing and multilocus sequence typing (MLST). Variations within SCCmec, especially type II, were detected in 165 (37.8%) isolates, and these variants were characterised using four different SCCmec typing methods. The predominant SCCmec type was a type II variant that differed from type II by the absence of a pUB110 insertion. MLST analysis showed that most of the isolates carrying SCCmec variants belonged to ST5.     

Extended-spectrum beta-lactamases in ceftazidime-resistant Escherichia coli and Klebsiella pneumoniae isolates in Turkish hospitals

PURPOSE: To study the prevalence of TEM-, SHV- and GES-type beta -lactamases among Escherichia coli and Klebsiella pneumoniae strains having ceftazidime MICs higher than 2 mg/L. METHODS: A total of 63 E. coli and 41 K. pneumoniae isolated from five different university hospitals were studied for the existence of TEM-, SHV- and GES-type beta -lactamases. Susceptibility tests were carried out according to the criteria of National Committee for Clinical Laboratory Standards. MICs were obtained by agar dilution method. Existence of extended-spectrum beta -lactamases (ESBLs) were assessed by double-disc synergy test (DDST). Existence of the above-mentioned beta -lactamase genes were studied both by PCR with specific oligonucleotide primers and isoelectric focusing methods. RESULTS: None of the isolates were carbapenem-resistant. DDSTs were positive in 50 (79.3%) and 33 (80.5%) of E. coli and K. pneumoniae , respectively. TEM gene was detected in 41 (65.1%) and 19 (46.3%), whereas SHV gene in 18 (28.6%) and 20 (48.8%) of E. coli and K. pneumoniae strains, respectively. GES genes were not detected. CONCLUSIONS: TEM and SHV genes are highly prevalent among ESBL-producing E. coli and K. pneumoniae , whereas GES-type ESBLs are absent and found not to be responsible of ceftazidime resistance in Turkish hospitals.     

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.     

Molecular epidemiologic analysis and antimicrobial resistance of Helicobacter cinaedi isolated from seven hospitals in Japan

Helicobacter cinaedi colonizes the colons of human and animals and can cause colitis, cellulitis, and sepsis in humans, with infections in immunocompromised patients being increasingly recognized. However, methods for analyzing the molecular epidemiology of H. cinaedi are not yet established. A genotyping method involving multilocus sequence typing (MLST) was developed and used to analyze 50 H. cinaedi isolates from Japanese hospitals in addition to 6 reference strains. Pulsed-field gel electrophoresis (PFGE) results were also compared with the MLST results. Based on the genomic information from strain CCUG18818, 21 housekeeping genes were selected as candidates for MLST and were observed to have high homology (96.5 to 100%) between isolates. Following a comparison of the 21 housekeeping genes from 8 H. cinaedi isolates, 7 genes were chosen for MLST, revealing 14 sequence types (STs). The isolates from 3 hospitals belonged to the same STs, but the isolates from the other 4 hospitals belonged to different STs. Isolates belonging to ST6 were analyzed by PFGE and showed similar, but not identical, patterns between isolates. Isolates belonging to ST9, ST10, and ST11, which belonged to the same clonal complex, had the same pattern. All isolates were found to contain mutations in GyrA and the 23S rRNA gene that confer ciprofloxacin and clarithromycin resistance, respectively, in H. cinaedi. These results raise concerns about the increase in H. cinaedi isolates resistant to clarithromycin and ciprofloxacin in Japan.     

Molecular characterization of extended-spectrum beta-lactamases produced by nosocomial isolates of Enterobacteriaceae from an Italian nationwide survey

Extended-spectrum beta-lactamases (ESBLs) are widespread in hospital settings worldwide. The present investigation was undertaken to assess the distribution and prevalence of ESBLs belonging to the TEM and SHV families in 448 ESBL-producing clinical isolates of Enterobacteriaceae collected from 10 different Italian hospitals. The natures of TEM and SHV determinants were identified by direct sequencing of PCR-amplified genes. TEM-52 and SHV-12 were the most common variants, and they were found in most hospitals and in several different species. Other less frequent variants included TEM-5, TEM-12, TEM-15, TEM-19, TEM-20, TEM-24, TEM-26, TEM-43, TEM-60, TEM-72, TEM-87, SHV-2a, SHV-5, and SHV-11. Proteus mirabilis was the most common producer of TEM-type ESBLs, while Klebsiella pneumoniae was the most common producer of SHV-type ESBLs. The distribution of TEM- and SHV-type ESBL variants in Enterobacteriaceae from Italian hospitals exhibited notable differences from those from other geographical settings.