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.     

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.     

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.     

Characterization of Clinical Multidrug-Resistant Escherichia coli and Klebsiella pneumoniae Isolates, 2007-2009, China

Various resistance mechanisms facilitate the emergence and spread of multidrug-resistance (MDR) phenotypes of Escherichia coli and Klebsiella pneumoniae. To elucidate the MDR mechanisms of E. coli and K. pneumoniae in China, we analyzed the antimicrobial susceptibilities of strains isolated from clinical samples in a large tertiary care hospital in Beijing, China, during 2007-2009 and characterized the isolates with a cefotaxime-ciprofloxacin-amikacin (CTX-CIP-AK) resistance pattern. In total, 98 and 52 clinical isolates of E. coli and K. pneumoniae, respectively, with a CTX-CIP-AK resistance pattern were subjected to antimicrobial susceptibility testing and screening of common β-lactamase genes, plasmid-mediated quinolone resistance (PMQR) genes, quinolone resistance-determining region (QRDR) substitutions, and 16S rRNA methylase genes by polymerase chain reaction amplification and DNA sequencing. Pulsed-field gel electrophoresis (PFGE) was used to determine the genetic relatedness of the isolates. Approximately 6.86% and 8.05% of the clinical E. coli and K. pneumoniae isolates, respectively, exhibited MDR phenotypes. The MDR K. pneumoniae isolates exhibited significantly higher ceftazidime resistance than the MDR E. coli isolates (90.4% vs. 76.5%, p=0.0339); a similar result was noted for piperacillin-tazobactam resistance (28.8% vs. 2%, p=0.0001). The common resistance determinants among the MDR E. coli and K. pneumoniae isolates were as follows: CTX-M (88.8% vs. 82.7%), PMQR genes (70.4% vs. 90.4%), gyrA mutations (100% vs. 90.4%), and 16S rRNA methylase genes (93.9% vs. 94.2%). Half (50%) of the MDR E. coli isolates belonged to phylogenetic group D, followed by group A (39.8%). For the E. coli isolates, 94 PFGE patterns and 23 clusters were identified, whereas 51 PFGE patterns and 11 clusters were identified for the K. pneumoniae isolates. Clinical E. coli and K. pneumoniae isolates seem to have a low prevalence of MDR phenotypes in China. The great genetic variation indicates a considerable transmission of common resistance determinants, including a high prevalence of QRDR substitutions in E. coli and K. pneumoniae.     

Genetic relatedness among extended-spectrum beta-lactamase-producing Klebsiella pneumoniae outbreak isolates associated with colonization and invasive disease in a neonatal intensive care unit

Pulsed-field gel electrophoresis typing of 60 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBLKp) isolates obtained in a neonatal intensive care unit during an outbreak indicated the dissemination of two major bacterial genotypes associated with colonization and invasive disease: one composed by aminoglycoside-resistant isolates and the other by aminoglycoside-susceptible isolates. A urease-negative phenotype was observed among aminoglycoside-resistant ESBLKp. Six pairs of isolates from gastrointestinal (GI) colonization and isolates from invasive disease that occurred 3-23 days later were shown to belong to the same genotype, reinforcing a direct association between colonization and subsequent disease. These data indicate that screening for ESBLKp GI colonization in an outbreak setting may be useful to detect neonates at a higher risk of invasive disease.     

Prevalence and characterisation of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates in healthy volunteers in Tunisia

The objective of this investigation was to analyse the carriage rate of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in faecal samples of healthy humans in Tunisia and to characterise the recovered isolates. One hundred and fifty samples were inoculated on MacConkey agar plates supplemented with cefotaxime (2 μg/ml) for ESBL-positive E. coli recovery. The characterisation of ESBL genes and their genetic environments, detection of associated resistance genes, multilocus sequence typing (MLST) and phylogroup typing were performed by polymerase chain reaction (PCR) and sequencing. The presence and characterisation of integrons and virulence factors were studied by PCR and sequencing. ESBL-positive E. coli isolates were detected in 11 of 150 faecal samples (7.3%) and one isolate/sample was further characterised. These isolates contained the blaCTX-M-1 (ten isolates) and blaTEM-52c genes (one isolate). The ISEcp1 (truncated by IS10 in four strains) and orf477 sequences were found upstream and downstream, respectively, of all bla (CTX-M-1) genes. Seven different sequence types (STs) and three phylogroups were identified among CTX-M-1-producing isolates [ST/phylogroup (number of isolates)]: ST58/B1 (3), ST57/D (2), ST165/A (1), ST155/B1 (1), ST10/A (1), ST398/A (1) and ST48/B1 (1). The TEM-52-producing isolate was typed as ST219 and phylogroup B2. Six ESBL isolates contained class 1 integrons with the gene cassettes dfrA17-aadA5 (five isolates) and dfrA1-aadA1 (one). Healthy humans in the studied country could be a reservoir of CTX-M-1-producing E. coli.     

Multiclonal epidemic of Klebsiella pneumoniae isolates producing DHA-1 in a Spanish hospital

Between June 2007 and January 2008, 26 Klebsiella pneumoniae isolates carrying bla_DHA-1 on an IncL/M plasmid were obtained from clinical samples at Granollers Hospital, Barcelona, Spain. Three of the isolates also carried a bla_CTX-M-15 gene. The 26 isolates showed 11 pulsed-field gel electrophoresis (PFGE) patterns. Multilocus sequence typing showed that PFGE patterns A, B and C belonged to sequence type (ST)17, D to ST13, E to ST427, F and G to ST416, H to ST37, I to ST440, J to ST326, and K to ST428. Results demonstrated the effectiveness of the infection control programme in place at the centre. This study reports the first characterization of STs for bla_DHA-1-producing K. pneumoniae isolates.     

Comparison of pulsed-field gel electrophoresis and randomly amplified DNA polymorphism analysis for typing extended-spectrum-beta-lactamase-producing Klebsiella pneumoniae.

The incidence and transmission patterns of extended-spectrum-beta-lactamase (ESBL)-producing Klebsiella pneumoniae in patients admitted to the intensive care unit (ICU) of a university hospital were investigated over a 3-year period. K. pneumoniae isolates were characterized by antibiotic susceptibility, capsular serotyping, plasmid profiles, and pulsed-field gel electrophoresis (PFGE) of genome macrorestriction patterns with XbaI, and the results were compared with those obtained by typing with the randomly amplified polymorphic DNA (RAPD) patterns. The discriminatory power of RAPD typing was evaluated for three primers. The incidence of isolation of ESBL-producing K. pneumoniae was 2.5 cases per 1,000 admissions to the ICU versus 0.35 cases per 1,000 admissions to other units (relative risk, 7.03; 95% confidence interval, 3.89 to 12.69). Infection developed in 53% of evaluable patients. Thirty-six percent of the cases were possibly acquired in other institutions. Isolates from ICU patients were subdivided into six capsular serotypes and into four clonal groups based on antibiotype, plasmid content, and PFGE and RAPD patterns. Two clones were associated with clusters of cross-infection, involving 5 and 12 patients, respectively. Following implementation of contact isolation precautions, the incidence of nosocomial acquisition of ESBL-producing K. pneumoniae decreased from 0.55 to 0.26 cases per 1,000 admissions (P = 0.03). PFGE and RAPD analysis showed concordant results and comparable discrimination for differentiation between groups of epidemiologically related strains of ESBL-producing K. pneumoniae. More subclonal variants were determined among epidemic clones by PFGE analysis than by RAPD analysis. Both methods are useful for typing K. pneumoniae strains in epidemiological investigations, although RAPD analysis is more efficient.     

Antimicrobial resistance and genetic diversity of Shigella sonnei isolates from western Ireland,an area of low incidence of infection

Shigella sonnei is a significant cause of gastroenteritis in both developing and industrialized countries. Definition of the diversity and antimicrobial susceptibility of S. sonnei isolates may be helpful in the management of individual cases and outbreaks. Antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) were performed with 67 isolates of S. sonnei predominantly (n = 59) from three counties in the west of Ireland. Phage typing (n = 17), plasmid profiling (n = 28), and integron analysis (n = 24) were performed with subsets of strains. PFGE typing permitted recognition of two major clusters: PFGE type A (n = 53) and PFGE type B (n = 14). PFGE type A was associated with resistance to ampicillin, streptomycin, and sulfonamides (51 of 53 isolates), and those that were phage typed (n = 6) were phage type 3. PFGE type B was associated with resistance to streptomycin, sulfonamides, tetracycline, and trimethoprim (11 of 14 isolates) and phage type 6 (9 of 11 isolates). Fifteen different plasmid profiles were identified among the 28 isolates analyzed. A class 2 integron was present in all 14 PFGE type B isolates. One of these isolates also contained a class 1 integron and showed a unique variant of the PFGE type B pattern. Sequence analysis of the gene cassette structures contained within these integrons identified distinct open reading frames that encoded determinants of resistance to trimethoprim, streptomycin, and streptothricin. Our data demonstrate two predominant PFGE types among S. sonnei isolates circulating in this region. The limited diversity of the S. sonnei isolates in this region means that detection of isolates indistinguishable by PFGE and according to their antibiograms in two or more patients is not persuasive evidence of a common-source food- or waterborne outbreak. Indistinguishable plasmid profiles in addition to indistinguishable PFGE and antibiogram types may be more suggestive of an epidemiologically relevant link between cases.     

A nosocomial outbreak of KPC-2-producing Klebsiella pneumoniae in a Chinese hospital: dissemination of ST11 and emergence of ST37, ST392 and ST395

In China, Klebsiella pneumoniae carbapenemase (KPC) -producing K. pneumoniae isolates have been identified. However, little is known about the spread and outbreak of KPC-producing enterobacterial pathogens. In this study, 48 non-duplicated KPC-producing isolates were analysed for genetic relatedness by pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility by E-test, and sequence type (ST) by multilocus sequence typing. S1-PFGE and Southern blot were used for plasmid profiling, and PCR and subsequent sequencing were performed to determine the effects of genetic background on the blaKPC gene. From December 2011 to June 2012, an outbreak of the KPC-2-producing K. pneumoniae was observed. The 48 isolates of K. pneumoniae are categorized into eight PFGE types (A1, A2, A3, A4, B, C, D and E). The predominant pathogens of the outbreak were strains with PFGE types A1, A2 and A3, which all belong to ST11. Furthermore, ST37, ST392 and ST395 KPC-2-producing K. pneumoniae isolates have also been sporadically identified. The blaKPC-2-carrying plasmids vary in size from 30 to 220 kb. The genetic environments of the blaKPC-2 gene for most strains were consistent with the genetic structure of blaKPC-2 on the plasmid pKP048. In conclusion, the dissemination and outbreak of KPC-2-producing K. pneumoniae isolates in this study appeared to be clonal, and ST11 K. pneumoniae was the predominant clone attributed to the outbreak. This is the first study to report the emergence and spread of KPC-producing K. pneumoniae ST392 and ST395 worldwide. Our findings suggest that horizontal transfer of Tn3-based transposons might mediate the spread of blaKPC-2 gene between different K. pneumoniae clones in China.     

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.     

Laboratory investigation of a multistate food-borne outbreak of Escherichia coli O157:H7 by using pulsed-field gel electrophoresis and phage typing.

Two hundred thirty-three isolates of Escherichia coli O157:H7 were analyzed by both pulsed-field gel electrophoresis (PFGE) and bacteriophage typing. All 26 isolates from persons whose illness was associated with a recent multistate outbreak of E. coli O157:H7 infections linked to the consumption of undercooked hamburgers and all 27 isolates from incriminated lots of hamburger meat had the same phage type and the same PFGE pattern. Twenty-five of 74 E. coli O157:H7 isolates from Washington State and 10 of 27 isolates from other states obtained during the 6 months before the outbreak had the same phage type as the outbreak strain, but only 1 isolate had the same PFGE pattern. PFGE thus appeared to be a more sensitive method than bacteriophage typing for distinguishing outbreak and non-outbreak-related strains. The PFGE patterns of seven preoutbreak sporadic isolates and five sporadic isolates from the outbreak period differed from that of the outbreak strain by a single band, making it difficult to identify these isolates as outbreak or non-outbreak related. Phage typing and PFGE with additional enzymes were helpful in resolving this problem. While not as sensitive as PFGE, phage typing was helpful in interpreting PFGE data and could have been used as a simple, rapid screen to eliminate the need for performing PFGE on unrelated isolates.     

First outbreak of multidrug-resistant Klebsiella pneumoniae producing both SHV-12-type extended-spectrum beta-lactamase and DHA-1-type AmpC beta-lactamase at a Korean hospital

PURPOSE: Coexistence of different classes of beta-lactamases in a single bacterial isolate may pose diagnostic and therapeutic challenges. We investigated a spread of Klebsiella pneumoniae isolates co-producing an AmpC beta-lactamase and an extended-spectrum beta-lactamase (ESBL) in a university hospital. MATERIALS AND METHODS: Over a three-month period, a total of 11 K. pneumoniae isolates, which exhibited resistance to cefotaxime, aztreonam, and cefoxitin, were isolated. These isolates showed positive to ESBLs by double disk tests. Minimal inhibitory concentrations (MICs) were determined by broth microdilution testing. All isolates were examined by isoelectric focusing, PCR and sequence analysis to identify bla(SHV) and bla(DHA), and molecular typing by pulsed-field gel electrophoresis (PFGE). RESULTS: All 11 isolates were highly resistant (MIC, > or = 128microg/ml) to ceftazidime, aztreonam, and cefoxitin, while they were susceptible (MIC, < or = 2microg/ml) to imipenem. The bla(SHV-12) and bla(DHA-1) genes were detected by PCR and sequence analysis. PFGE revealed a similar pattern in 10 of the 11 strains tested. CONCLUSION: This is the first outbreak report of K. pneumoniae in Korea which co-produced SHV-12 and DHA-1 beta-lactamase, and we suggest a clonal spread of multidrug-resistant K. pneumoniae at a hospital.     

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.     

Identification of multidrug-resistant Streptococcus pneumoniae strains isolated in Poland by multilocus sequence typing

Multilocus sequence typing (MLST) of 35 isolates of multidrug-resistant Streptococcus pneumoniae recovered in Poland during 1995-1996 distinguished 10 different sequence types (ST). The majority of the isolates were assigned to two Polish clones of serotypes 6B and 23F, although the international clones, Spain23F-1 and Spain9V-3, were also identified. Similar results were obtained using pulsed-field gel electrophoresis (PFGE), providing a direct comparison of these two typing methods.     

Performance of phenotypic and genotypic methods to determine the clinical relevance of serial blood isolates of staphylococcus epidermidis in patients with septicemia

Five typing methods, including biotyping (API ID32; BioMérieux, Marcy l'Etoile, France), quantitative antibiogram typing based on actual zone sizes, plasmid typing, randomly amplified polymorphic DNA (RAPD) analysis (with primer M13 and primer set ERIC-2-1026), and pulsed-field gel electrophoresis (PFGE), were compared with a previously performed method of DNA fingerprinting by AFLP (amplified fragment length polymorphism analysis) for their performance in the typing of blood isolates of Staphylococcus epidermidis. Sixteen epidemiologically unrelated strains and 11 sets of four blood culture isolates from 11 patients with septicemia were used. The stabilities and reproducibilities of the patterns, the discriminatory capacities of the methods, and the ability to apply the methods to blood culture isolates were used as performance criteria. All strains tested were typeable by each method, and the patterns were stable and reproducible. The numbers of different types within the collection of 16 epidemiologically different isolates were 5 by biotyping, 14 by antibiogram typing, 4 by plasmid typing, 9 by the RAPD assay (combination of results with primer M13 and primer set ERIC-2-1026), and 16 by PFGE. Within the 11 sets of four blood culture isolates the types found by quantitative antibiogram typing, plasmid typing, and PFGE were unique for each set, whereas by biotyping and RAPD analysis some types were observed in more than one set. The results of biotyping did not correspond with the results of the other methods or the results of AFLP. For 6 of the 11 sets, the results of all methods except those of biotyping corresponded completely. Quantitative antibiogram typing, PFGE, and AFLP proved to be the most accurate of the six typing methods tested.     

Molecular epidemiology of a Shigella flexneri outbreak in a mountainous township in Taiwan, Republic of China

An outbreak of shigellosis occurred in a township of Nantou Conuty in central Taiwan from August to October in 1996. The infections extended to two neighboring townships and continued to the end of 1996. Forty cases were confirmed during the period, in contrast to only one confirmed case in Nantou County in 1996 before the outbreak. All of these 41 cases in 1996 were identified as infections with Shigella flexneri serotype 2a. In order to trace the source of the infections, the 41 isolates recovered were analyzed by plasmid profile and pulsed-field gel electrophoresis (PFGE). There was no correlation between the plasmid profile results and the PFGE results, and the latter were used for subtyping of the 41 isolates. Twenty-two isolates (53%) had the same NotI and XbaI PFGE patterns, and 4 isolates (10%) had an additional unstable plasmid band in their NotI patterns but otherwise had the same NotI and XbaI patterns as the 22 isolates. These 26 isolates were designated the outbreak strain, and of these, 24 appeared in eight villages in one township and 2 appeared in a neighboring township. Fourteen of the remaining 15 isolates, including the isolate recovered 7 months before the outbreak, had both NotI and XbaI PFGE patterns closely related to those of the outbreak strain, indicating that Shigella infections were endemic in the area. By tracing the first isolation dates of the outbreak strain in individual villages and the neighboring township, it was found that the strain spread along the major arterial road and its branch road as time passed. Our molecular typing results and epidemiological data demonstrated the endemic nature of the outbreak strain as well as a person-to-person mode of transmission for the widespread infections the strain caused.     

Comparison of restriction endonuclease analysis, ribotyping, and pulsed-field gel electrophoresis for molecular differentiation of Clostridium difficile strains.

A combined clinical and molecular epidemiologic analysis of 46 strains of Clostridium difficile, including 16 nosocomial isolates from one ward (outbreak ward) plus 17 other nosocomial isolates and 13 community-acquired isolates, was performed. HindIII digests of total cellular DNA were analyzed by restriction enzyme analysis (REA) and ribotyping; SmaI digests were analyzed by pulsed-field gel electrophoresis (PFGE). Isolates were assigned to typing groups on the basis of the profiles detected; isolates with closely related profiles were assigned to subgroups. The 16 isolates from the outbreak ward were resolved by both REA and PFGE into five distinct groups; 13 isolates represented two REA groups and three PFGE groups and two isolates were resolved as distinct groups by both techniques. DNA obtained from one isolate was persistently partially degraded, precluding analysis by PFGE. Seventeen sporadic nosocomial isolates were resolved by REA and PFGE into comparable numbers of groups (i.e., nine groups) and subgroups (i.e., 15 and 14 subgroups, respectively), with two isolates not evaluable by PFGE. The 13 epidemiologically unrelated community-acquired isolates were assigned to 11 groups by REA and to 12 groups by PFGE. Overall, ribotyping identified only nine groups among the 46 isolates. We conclude that REA and PFGE have comparable discriminatory powers for epidemiologic typing of C. difficile isolates and that ribotyping is appreciably less discriminatory. For a few isolates, partial DNA degradation prevented analysis by PFGE but not by REA or ribotyping; the cause of the degradation is unknown.     

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.