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.

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.     

同一标本分离的2株碳青霉烯类耐药肠杆菌科细菌耐药机制相关性分析

目的 对从同一标本中分离的碳青霉烯类药物耐药的肺炎克雷伯菌和摩根摩根菌进行耐药机制相关性分析.方法 琼脂稀释法进行药物敏感试验;特异性PCR扩增和序列分析检测介导耐碳青霉烯类药物的相关基因;接合试验、质粒提取和耐药基因周围序列分析耐药的可传递性、耐药质粒的同源性及耐药基因的遗传背景;提取外膜蛋白分析菌株外膜蛋白的改变.结果 2株分离菌均产碳青霉烯酶并扩增出介导碳青霉烯类耐药的KPC-2型基因;质粒接合试验成功传递了对碳青霉烯类药物的耐药性,耐药基因被携带在2个大小不同但耐药基因周围序列完全相同的质粒上;其中摩根摩根菌耐药株缺失了相对分子质量约为38×103的外膜蛋白同时出现36×103的外膜蛋白而肺炎克雷伯菌则缺失了外膜蛋白OMPK36.结论 2株分离菌均携带KPC-2基因.该基因由2个大小不同的质粒携带,同一复合转座子介导了KPC-2基因在2株细菌的不同质粒上转移.同时外膜蛋白缺失参与了对碳青霉烯类抗生素耐药.

Abstract：

Objective To investigate the relationship of resistance mechanisms of a Klebsiella pneumoniae strain and a Morganella morganii strain resistance to carbapenems isolated from a single specimen. Methods Sensibility of antimicrobial agents was detected by agar dilution method. Specific PCR and DNA sequence analysis were performed to detect resistance genes. Plasmid feature was detected by plasmid conjugation and electrophoresis analysis. Genetic environment around blaKPC was analyzed with sequencing. The changes of outer membrane permeability were analyzed with electrophoresis of outer membrane proteins. Results blaKPC-2 was detected in 2 original isolates strains and their transconjugants. Carbapenem-resistance was successfully transfered by conjugation experiments. blaKPC-2 was located on dissimilar plasmids, but genetic environment around blaKPC-2 was the same sequence. The Morganella morganii isolate showed a loss of 38 ×103 OMPs and an additional 36 ×103 OMPs appearance, while the Klebsiella pneumoniae isolate showed a loss of OMPK36. Conclusion blaKPC-2 was detected in 2 isolates. This gene encoded by two plasmids with different sizes was located on the same composite transposon. The lack of outer membrane proteins could also play an important role causing isolates to exhibite resistance to carbapenems.     

Study of Klebsiella pneumoniae producing extendedspectrum β-lactamases against aminoglycosides

Klebsiella pneumoniae （ K. pneumoniae） is one of the main gmn-negative bacilli in clinical practice. Nosocomial infections caused by K. pneumoniae producing extended-spectrum β-lactamases （ESBLs） are very difficult to treat. This paper investigated the resistant characteristics of K. pneumoniae producing ESBLs and their aminoglycoside-modifying enzyme gene expressions including Nacetyltransferases and O-adenyltransferases. Bacteria identification and ESBLs confirmatory tests were performed by Phoenix^TM-100 system. And minimum inhibitory concentrations （MICs） of gentamicin, amikacin, kanamycin, tobranycin, netilmicin and neomycin in 53 K. pneumoniae isolates were detected by agar dilution. In addition, six aminoglycoside-modifying enzyme genes were amplified by polymerase chain reaction （PCR） and verified by DNA sequencer. It was found that imipenem and meropenem against 120 K. pneumoniae isolates produced powerful antimicrobial activities. The resistant rates of gentamicin and amikacin were 55.0% and 46.7%, respectively. Except neomycin, MIC50 and MIC90 of gentamicin, amikacin, kanamycin, tobramycin and netilmicin in 53 K. pneumoniae were all 〉 128 μg/ml, and the resistant rates were 83.0%, 52.3%, 75.5%, 81.1% and 69.8%, respectively. However, neomycin was only 39.6%. In addition, five modifying enzyme genes, including aac（3）-Ⅰ, aac（3）-Ⅱ, aac（6＇）-Ⅰb, ant（3＂）-Ⅰ, ant（2＂）-Ⅰ genes, were found in 53 isoaltes except aac （6＇）-Ⅱ, and their positive rates were 11.3%, 67.9%, 47.2%, 1.9% and 39.6%, respectively. It was also confirmed by nucleotide sequence analysis that the above resistant genes shared nearly 100% identities with GenBank published genes. The results obtained in the present study indicated that K. pneumoniae producing ESBLs strains are rapidly spreading in our hospital, and their resistance to aminoglycosides may be associated with aminoglycoside-modifying enzyme gene expressions.     

Toxin（s）, Other Than Cholera Toxin, Produced by Environmental Non O1 Non O139 Vibrio cholerae

A total of 39 Vibrio cholerae non O1 non O139 strains were isolated from surface waters of different parts of Dhaka City, Bangladesh. All these strains showed lack of ctx or zot gene, as demonstrated by the PCR analysis. Eighteen representative strains were tested for enterotoxin production using a rabbit ileal loop model, of which live cells of 8 strains and culture filtrates of 6 strains produced fluid accumulation in ileal loops. However, none of them produced heat stable toxin （ST）, as detected by suckling mouse assay. On the other hand, 15% of isolates produced cytotoxin as detected by the Chinese Hamster Ovary （CHO） cell assay. Fifty times concentrated culture filtrates of the representative strains did not give any precipitin band against the anti-cholera toxin, suggesting the strains produced an enterotoxin, which is antigenically different from known cholera toxin （CT）. Eighty percent of the total isolates were found to be positive for heat labile haemolysin detected by tube method, whereas, 39% were found positive by the Christie-Atkins-Munch-Petersen （CAMP） method. However, 87% of the isolates were positive for haemagglutinin/protease and all of the strains were positive for mannose-sensitive-haemagglutinin assay.     

Molecular mechanism of the qnrA genemediated quionlone resistance in Gram-negative bacteria

To explore the prevalence of the plasmid-mediated quinolone resistance gene qnrA in Gramnegative bacteria and to investigate its molecular genetic background and resistance profile in isolates harboring this gene, a total of 629 nalidixic acid-resistant isolates of non-repetitive Gram-negative bacteria were collected from clinical specimens between April 2004 and April 2006 and these isolates were screened for qnrA gene by PCR using specific primers combined with DNA sequencing. The extended spectrum β-1actamase （ESBL） or AmpC-producing isolates were distinguished by the phenotypic confirmatory test combined with DNA sequencing, and the antibiotics susceptibility test for qnrA-positive isolates was carried out by Kirby-Bauer and E-test method. To detect the location of the qnrA gene, plasmid conjugation and Southern hybridization were performed and the integron structure containing the qnrA gene was cloned by PCR strategy and sequenced by primer walking. It was demonstrated that the incidence of the qnrA-positive strains in nalidixic acid-resistant bacteria was 1.9% （12/629）, in which the detection rates for Klebiesiella pneumoniae. Enterobacter cloacae, Enterobacter aerogenes, Citrobacterfreundii and Salmonella choeraesuis were 2.2% （3/138）, 17. 1% （6/35）, 9. 1% （1/11）, 12.5% （1/8）, and 14.3% （1/7）, respectively. The qnrA gene was found to be embedded in the complex sull-type integron located on plasmids with varied size （80-180 kb）. Among them, 4 qnrA-positive isolates carried integron In37 and 8 isolates carried a novel integron, temporarily desig- nated as InX. All the qnrA-positive isolates were ESBL-producing and transferable for the multi-drug resistance. It is concluded that the plasmid-mediated drug-resistance mechanism exists in the quinolone resistant strains of isolates from hospitals in Guangdong area, but the incidence was rather low. Nevertheless, it is still possible that the horizontal transfer of the resistant qnrA gene might lead to the spreading of drug-resista     

Analysis on drug-resistance and molecular epidemiology of Acinetobacter baumannii isolated from the clinical samples in two Chinese hospitals

In the present study, the drug-resistance genes encodingβ-lactamases, aminoglycoside modifying enzymes, DNA topoisomerases and integron as well as their molecular epidemiology were investigated by means of analyzing the drug-resistance and molecular epidemiology of Acinebacter bau mannii isolated from the clinical samples in two hospitals in Qiangzhou and Huzhou city of Jiangsu and Zhejiang province from July 2000 to March 2005. The minimal inhibitory concentrations (MICs) of these 307 isolates were detected by automatic microbiological system, and 35 strains against 5-fluoro-quinolones were performed by agar dilution assay. Meanwhile, the resistant genes in 80 isolates were amplified by PCR with identification by DNA sequencer. It was found that most of the 307 isolates of A . baumannii were resistant to multiple antibiotics tested, in which the resistance rates of the isolates against piperacillin, piperacillin/tazobactam, amoxacillin/clavulanic acid, cefotaxime, ceftazidime, cefepime, gentamicin, amikacin, ciprofloxacin, chloramphenicol and sulfamethoxazole/trimethoprim were all above 35% , but those of imipenem and meropenem were quite low, ranged only 2.6% and 3.3%. In addition, it was also demonstrated that the positive rates of TEM and SHVβ-lactamase genes accounted for 93.8% and 22.5% respectively, and those of the aminoglycoside-modifying enzyme genes including aacC1, aacC2, aacC3, aacC4, aacC4A, aphA6, ant(2")-I and ant(3") I were 58.8%, 8.8%, 7.5%, 28.8%, 45.0%, 2.5%, 28.8% and 65.0% respectively. The mutations in the quinolone-resistant determining region (QRDR) of gyrA and parC genes indicated that substitution in Ser-83 residue of GyrA protein was most frequently occurred among strains with MIC for ciprofloxacin of more than 4μg/ml, whereas a double mutation at Ser-83 residue of gyrA and Ser-80 of parC was found in strains with MIC of ciprofloxacin of more than 8μg/ml. As to the positive rates of class 1 integron (Int I -1) and qacE△1-sul-1, it was found to be 60.0% and 77.5% respectively, and the rates of resistant genes of strains isolated in these two hospitals varied considerably. The results obtained in the present study indicate the presence of the multiple resistant genes in strains of A. baumannii , and great measures should be taken to control the spread of the resistant strains carrying the resistant genes.     

肠道病毒71型VP1及2A基因特征研究

目的 检测临床分离的肠道病毒71型(EV71)轻型、重型毒株VP1基因、2A基因的核苷酸序列,进行遗传进化途径分析.方法 将50份临床手足口病患者粪便标本处理后分离病毒,用EV71特异性引物进行RT-PCR扩增,获得EV71毒株,针对不同临床背景的标本,分别选取轻型、重型的毒株,用特异性引物分别对VP1基因及2A基因进行RT-PCR扩增,对其产物进行测序及遗传进化分析,并探讨它们之间的差别.结果 50份临床手足口病标本中有30份检测是EV71,其中轻型(手足口)13份,重型(手足口并发脑炎或心肌炎)17份.轻型和重型中各自选取5株对VP1基因和2A基因进行测序和遗传学分析,通过同源性比较和构建系统发生树发现,此10株EV71病毒和中国大陆已发表的5株EV71病毒(fuyangEU703814.1、xi_anHM003207.1、shandongEU753418.1、shenzhen-FJ607337.1、henanGU366191.1)全部属于C基因型,且核苷酸同源性较高,VP1基因和2A基因的同源性分别在94.7%～99.4%和93.6%～99.3%范围内.本次分离的10株EV71病毒与A、B基因型代表株比较,核苷酸同源性分别为81.0%～84.6%和78.4%～82.2%,差异较大.与已知的C1、C2、C3亚型代表株比较,核苷酸同源性在87.8%～90.2%,差异≥10%,与已知的C4亚型代表株比较,核苷酸同源性在96.8%～99.6%,因此认为可将这10株病毒划分为C4亚型.在系统发生树上,这10株病毒形成一个较独立的分支.结论 EV71 C4亚型病毒在中国大陆有较广泛的传播,且毒株之间VP1基因的遗传关系紧密;2A基因在轻型和重型病例毒株之间遗传差异不大.

Abstract：

Objective To detect VP1 and 2A genes of Enterovirus type 71 (EV71) isolated from clinical specimens of patients with light or heavy symptoms and analyze the homogeneity and phylogenetic tree. Methods Fifty clinical specimens of children with hand-foot-and-mouth disease ( HFMD) were dealed with, which were tested by RT-PCR assay with specific primer pairs for EV71. EV71 isolates from patients with light or heavy clinical symptoms were tested by RT-PCR assay with two specific primer pairs for VP1 and 2A genes of EV71 respectively. All of the PCR products were sequenced and compared with that of previously isolated EV71 isolates available from GenBank by homogeneity and phylogenetic tree analyses. Results The RT-PCR results indicated that 30 isolates were EV71, 13 of 30 isolates were from clinical specimens of patients with light symptoms of hand-foot and mouth, the other were from clinical specimens of patients with heavy symptoms of complications. VP1 genes and 2A genes of 10 EV71 isolated strains including 5 light strains and 5 heavy strains were sequenced and compared with that of previously isolated 5 EV71 Chinese isolates available from GenBank (fuyangEU703814.1, xi_anHM003207. 1, shandongEU753418.1, shenzhenFJ607337.1, henanGU366191. 1) by homogeneity and phylogenetic tree analyses. The homogeneity of VP1 and 2A genes of the 10 EV71 isolated strains and 5 previously isolated strains were between 94.7% -99.4% and 93.6% -99.3% respectively, with the representative isolates of A and B genotypes was between 81.0%-84. 6% and 78. 4%-82. 2% respectively. The data suggested that all of the 10 Chinese isolates belong to EV71 genotype C. There were only 87.8% -90.2% homology among these 10 strains and the representative strains of C1, C2, C3 sub-genotypes of EV71 but 96. 8% -99.6% homology among these 10 strains and the representative strains of C4 sub-genotypes of EV71, this suggested that these 10 Chinese isolates composed the C4 sub-genotype, of the C genotype, that formed a single branch in the phylogenetic tree. Conclusion EV71 of sub-genotype C4 distributed in Mainland China, and VP1 genes have close genetic relationship between isolated strains. There is no obvious difference in 2A genes between clinical specimens of patients with light or heavy symptoms by homogeneity and phylogenetic tree analyses.     

Characterization of integrons and novel cassette arrays in bacteria from clinical isloates in China,2000-2014

Rapid dissemination of antibiotic resistance genes among bacterial isolates is an increasing problem in China.Integron,a conserved DNA sequence,which is carried on episomal genetic structures,plays a very important role in development of antibiotic resistance.This systematic analysis was based on MEDLINE and EMBASE databases.We summarized the distribution and proportion of different types of gene cassette arrays of integrons(including class 1,2,3 and atypical class 1 integron) from clinical bacteria isolates in China.Fifty-six literatures were included in this study.Most of the strains were Gram-negative bacteria(94.1%,7,364/7,822) while only 5.9% strains were Grampositive bacteria.Class 1 integrons were detected in 54.2%(3956/7295) Gram-negative strains.aadA2 was the most popular gene cassette array detected from 60 Gram-positive bacteria while dfrA17-aadA5 were detected in 426 Gramnegative bacteria.This study identified 12 novel gene cassette arrays which have not been previously found in any species.All the novel gene cassette arrays were detected from Gram-negative bacteria.A regional characteristic of distribution of integrons was presented in this study.The results highlight a need for continuous surveillance of integrons and provide a guide for future research on integron-mediated bacteria resistance.     

Solexa测序法研究肺炎克雷伯菌质粒基因组的耐药基因

目的 通过Solexa高通量测序法研究肺炎克雷伯菌的质粒与耐药性的关系.方法 菌株为7年临床分离的206株肺炎克雷伯菌.提取所有菌的全部质粒DNA,Solexa高通量测序获得大规模的短序列.SOAP软件对质粒基因进行分析拼接,分析结果与相关数据库进行比对.MAQ软件分析质粒基因组包含的超广谱β-内酰胺酶(ESBL)多样性及单核苷酸多态性(SNP)情况.结果 肺炎克雷伯菌质粒基因组中已知的直接与耐药相关的基因就有13种.质粒基因组中存在多个ABC主动外排转运系统.发现4种编码β-内酰胺酶的ORF,其中SHV型ESBLs分布最广.系统分析了206株肺炎克雷伯菌质粒基因组中SHV型ESBLs的SNPs位点,发现存在着大量的非同义替换SNPs位点.结论 发现质粒中SHV型ESBLs基因可能受到选择压力,存在着大量的非同义替换SNPs位点.肺炎克雷伯菌质粒存在外排药物耐药方式,从而形成低水平的非特异多重耐药.     

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.     

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.     

Clonality of Streptococcus pneumoniae serotype 1 isolates from pediatric patients in the United States

We compared Streptococcus pneumoniae serotype 1 isolates causing disease among children in six geographic regions of the United States to determine genetic relatedness. Genomic fingerprints were determined by repetitive element polymorphism PCR (Rep-PCR). Multilocus sequence type characterization was performed on selected isolates. Four different genomic banding patterns were identified by Rep-PCR. One profile (clone 1) was predominant and matched sequence type 227.     

Fecal colonization of Enterobacteriaceae carrying plasmid-mediated quinolone resistance determinants in Korea

The aims of the current study were to investigate the prevalence and molecular characteristics of plasmid-mediated quinolone resistance (PMQR) genes from colonizing fecal organisms and to compare the incidence and subtype of these genes according to bacterial species and hospital at five tertiary-care hospitals in Korea. A total of 500 nonduplicated clinical isolates of Enterobacteriaceae were obtained from fecal specimens at five tertiary-care hospitals between March and May 2008. The PMQR genes (qnrA, qnrB, qnrS, aac(6')-Ib-cr, and qepA) were amplified by PCR and confirmed by direct sequencing of the PCR products. A total of 83 (16.6%) qnr-positive isolates were detected. The prevalence rates of qnrA, qnrB, and qnrS were 1.4%, 13.6%, and 1.6%, respectively. The species distributions of qnrB-positive isolates were Klebsiella pneumoniae (37/109; 33.9%), Citrobacter freundii (10/34; 29.4%), Citrobacter braakii (8/13; 61.5%), and Escherichia coli (8/275; 2.9%). Sixteen subtypes of qnrB were detected, including seven novel variants. The prevalences of aac(6')-Ib-cr and qepA were 15.6% (n=78) and 0.6% (n=3), respectively. The aac(6')-Ib-cr gene was detected in 39 (47.0%) of 83 qnr-positive isolates and 39 (9.4%) of 417 qnr-negative isolates There was one qepA variant containing a novel mutation (Ala231Val). The prevalence of PMQR genes was high in Enterobacteriaceae from stool specimens in Korea, and there was a close relation between qnr and aac(6')-Ib-cr.     

High rates of plasmid-mediated quinolone resistance QnrB variants among ciprofloxacin-resistant Escherichia coli and Klebsiella pneumoniae from urinary tract infections in Korea

The aims of this study were to investigate the prevalence of qnrA, qnrB, and qnrS determinants and their molecular characteristics in ciprofloxacin-resistant isolates of Escherichia coli and Klebsiella pneumoniae from urinary tract infections (UTI) in Korea. A total of 202 nonduplicated clinical isolates of ciprofloxacin-resistant E. coli (n = 143) and K. pneumoniae (n = 59) were collected between July 2005 and August 2006. The qnr determinant screening was carried out by PCR amplification of qnrA, qnrB, and qnrS, and all positive results were confirmed by direct sequencing of the PCR products. For qnr-positive strains and their conjugants, antimicrobial susceptibility tests and pulsed-field gel electrophoresis (PFGE) were performed. The qnrB gene was detected in 41 of the 202 isolates. Among 33 of 59 (55.9%) K. pneumoniae isolates showing qnrB, 29 isolates contained the qnrB4 gene, 3 isolates had the qnrB2 gene, and 1 isolate had the qnrB6 gene. All 8 (5.6%) of the qnrB-positive isolates among the 143 E. coli strains possessed the qnrB4 gene. The minimum inhibitory concentrations (MICs) of ciprofloxacin for the transconjugants were 0.03-2 mug/ml, representing an increase of 4- to 256-fold relative to the recipient, E. coli J53Az(r). Resistances to various other antimicrobial agents also were transferred with the plasmid. The PFGE analysis revealed indistinguishable or closely related patterns in several strains and highly diverse patterns in general. QnrB variants, especially the qnrB4 subtype, are highly prevalent in ciprofloxacin-resistant E. coli and K. pneumoniae from UTI in Korea. The emergence of plasmid-mediated quinolone resistance may contribute by several means to the rapid increase in bacterial resistance to these drugs.     

Genotyping of Mycoplasma pneumoniae clinical isolates reveals eight P1 subtypes within two genomic groups

Three methods for genotyping of Mycoplasma pneumoniae clinical isolates were applied to 2 reference strains and 21 clinical isolates. By a modified restriction fragment length polymorphism (RFLP) analysis of PCR products of the M. pneumoniae cytadhesin P1 gene, 5 subtypes were discriminated among 13 P1 type 1 strains and 3 subtypes were discriminated among 8 P1 type 2 strains. Sequence analysis of the 16S-23S rRNA gene spacer region and part of the 23S rRNA gene revealed one nucleotide difference in the intergenic spacer region in 3 of the 21 isolates. In the 23S rRNA gene sequence of the 8 P1 type 2 strains an extra adenosine was present, but it was absent from the 13 P1 type 1 strains. On the basis of M. pneumoniae genome sequence data, primers were designed to amplify large interrepeat fragments by long PCR, and these fragments were subsequently analyzed by RFLP analysis. Only two types, long PCR types 1 and 2, could be discriminated among the M. pneumoniae isolates. All P1 type 1 strains were assigned to long PCR type 1, and all P1 type 2 strains were assigned to long PCR type 2. These data obtained by three independent typing methods thus confirm the existence of two distinct M. pneumoniae genomic groups but expand the possibility of strain typing on the basis of variations within their P1 genes.     

Nosocomial outbreak of septicaemia in neonatal intensive care unit due to extended spectrum β-lactamase producing Klebsiella pneumoniae showing multiple mechanisms of drug resistance

A total of 14 phenotypically similar clinical isolates of Klebsiella pneumoniae, resistant to multiple drugs including cefotaxime and ceftazidime, were isolated from blood of neonates admitted to neonatal intensive care unit (NICU) within a short span of 10 days. Alarmed at the possibility of occurrence of outbreak, a thorough investigation was done. Microbiological sampling of the NICU and labour room (LR) environment yielded 12 K. pneumoniae isolates. The presence of extended spectrum β-lactamase (ESBL) in the clinical and environmental strains was detected by double-disk synergy test (DDST), CLSI phenotypic confirmatory disk diffusion test (PCDDT) and E-test ESBL strips. Amp-C screen (disk) test was done to determine Amp-C β-lactamase production. 100% clinical strains, 57% NICU strains and 80% LR strains were ESBL positive. 57% clinical, 43% NICU and 20% LR strains were Amp-C screen positive. Polymerase chain reaction (PCR) of representative ESBL positive (10 clinical and 5 environmental) strains showed CTX gene and TEM and/or SHV gene in all. K. pneumoniae showing multiple mechanisms of drug resistance was responsible for the outbreak.     

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.     

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.     

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.