无乳链球菌中可接合性转移元件的检测及携带菌株的分子流行特征调查
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摘要
目的了解临床分离的无乳链球菌中可接合性转移元件(ICE)的分布,分析其耐药和分子流行病学特征。方法对临床分离的无乳链球菌进行纸片扩散法药敏试验,通过PCR检测ICESa2603家族和类ICESa2603家族ICE不同的整合酶基因及插入位点基因筛选可能携带ICE的菌株;对携带ICE的菌株进行多位点序列分型(MLST),了解其分子流行特征;对部分ICE阳性菌株进行接合试验,了解ICE及相关耐药基因的转移性并通过脉冲场凝胶电泳(PFGE)、MLST及药敏试验证实。结果 178株无乳链球菌中检出携带ICE菌株27株(15.2%),其中ICESa2603家族14株,类ICESa2603家族19株,两类家族皆为阳性的有6株。27株携带ICE的无乳链球菌MLST结果显示:最多的为ST-12型10株(37.0%),其次为ST-27型4株(14.8%)、ST-24型3株(11.1%)、ST-19型3株(11.1%)。15株红霉素耐药ICE阳性的无乳链球菌中,10株(66.7%)检测到了ICE与红霉素耐药性的接合转移。ICE阳性的无乳链球菌对红霉素、克林霉素和四环素的耐药率明显高于ICE阴性的菌株,但对左氧氟沙星的耐药率前者明显低于后者。结论 ICE在临床分离的无乳链球菌中具有较高的检出率,且可能在红霉素、四环素等耐药基因的水平传播上起着一定作用,ICE阳性菌株中的主要克隆型为ST-12型。
Objective To screen the integrative and conjugative elements(ICEs) in clinical isolates of S. agalactiae and investigate the molecular epidemiology of the ICE-carrying strains. Methods The S. agalactiae strains were subjected to antimicrobial susceptibility testing by disc diffusion method. The genes encoding ICE insertion site and various integrases were detected by PCR to identify ICESa2603 family or ICESa2603-like ICEs in clinical S. agalactiae strains. Multilocus sequence typing(MLST)was conducted to investigate the molecular epidemiological characteristics of the ICE-carrying strains. The transferability of the ICEs was investigated by conjugation experiment, and verified by pulse field gel electrophoresis typing(PFGE), antimicrobial susceptibility test and MLST. Results Among the 178 clinical S. agalactiae strains, 27(15.2%) carried ICEs, including ICESa2603 family in 14 strains, ICESa2603-like ICEs in 19 strains, and both types of ICEs in 6 strains. MLST results of the 27 ICE-carrying strains showed that the predominant sequence type was ST-12(37.0%, 10/27), followed by ST-27(14.8%, 4/27), ST-24(11.1%, 3/27), and ST-19(11.1%, 3/27). The ICE and erythromycin resistance were simultaneously transferred to the recipient strains from 10 of 15(66.7%) clinical strains.ICE-positive strains showed significantly higher resistance rate to erythromycin, clindamycin, and tetracycline than ICEnegative strains. Conclusions ICEs are highly prevalent in clinical S. agalactiae strains and play a role in the spread of antibiotic resistance at genetic level, such as erythromycin and tetracycline resistance. The main clone of ICE-carrying S. agalactiae is ST12.
Objective To screen the integrative and conjugative elements(ICEs) in clinical isolates of S. agalactiae and investigate the molecular epidemiology of the ICE-carrying strains. Methods The S. agalactiae strains were subjected to antimicrobial susceptibility testing by disc diffusion method. The genes encoding ICE insertion site and various integrases were detected by PCR to identify ICESa2603 family or ICESa2603-like ICEs in clinical S. agalactiae strains. Multilocus sequence typing(MLST)was conducted to investigate the molecular epidemiological characteristics of the ICE-carrying strains. The transferability of the ICEs was investigated by conjugation experiment, and verified by pulse field gel electrophoresis typing(PFGE), antimicrobial susceptibility test and MLST. Results Among the 178 clinical S. agalactiae strains, 27(15.2%) carried ICEs, including ICESa2603 family in 14 strains, ICESa2603-like ICEs in 19 strains, and both types of ICEs in 6 strains. MLST results of the 27 ICE-carrying strains showed that the predominant sequence type was ST-12(37.0%, 10/27), followed by ST-27(14.8%, 4/27), ST-24(11.1%, 3/27), and ST-19(11.1%, 3/27). The ICE and erythromycin resistance were simultaneously transferred to the recipient strains from 10 of 15(66.7%) clinical strains.ICE-positive strains showed significantly higher resistance rate to erythromycin, clindamycin, and tetracycline than ICEnegative strains. Conclusions ICEs are highly prevalent in clinical S. agalactiae strains and play a role in the spread of antibiotic resistance at genetic level, such as erythromycin and tetracycline resistance. The main clone of ICE-carrying S. agalactiae is ST12.
引文
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[12]JIANG H,CHEN M,LI T,et al.Molecular characterization of Streptococcus agalactiae causing community-and hospitalacquired infections in Shanghai,China[J].Front Microbiol,2016,7:1308.
[13]WANG H,ZHAO C,HE W,et al.High prevalence of fluoroquinolone-resistant group B Streptococci among clinical isolates in China and predominance of sequence type 19 with serotype III[J].Antimicrob Agents Chemother,2013,57(3):1538-1541.
[2]LU B,CHEN X,WANG J,et al.Molecular characteristics and antimicrobial resistance in invasive and noninvasive Group B Streptococcus between 2008 and 2015 in China[J].Diagn Microbiol Infect Dis,2016,86(4):351-357.
[3]DELAVAT F,MIYAZAKI R,CARRARO N,et al.The hidden life of integrative and conjugative elements[J].FEMS Microbiol Rev,2017,41(4):512-537.
[4]ZHOU K,XIE L,HAN L,et al.ICESag37,a novel integrative and conjugative element carrying antimicrobial resistance genes and potential virulence factors in Streptococcus agalactiae[J].Front Microbiol,2017,8:1921.
[5]JONES N,BOHNSACK J F,TAKAHASHI S,et al.Multilocus sequence typing system for group B Streptococcus[J].J Clin Microbiol,2003,41(6):2530-2536.
[6]ELLIOTT J A,FARMER K D,FACKLAM R R.Sudden increase in isolation of group B streptococci,serotype V,is not due to emergence of a new pulsed-field gel electrophoresis type[J].J Clin Microbiol,1998,36(7):2115-2116.
[7]HUANG J,LIANG Y,GUO D,et al.Comparative genomic analysis of the ICESa2603 family ICEs and spread of erm(B)-and tet(O)-carrying transferable 89K-subtype ICEs in swine and bovine isolates in China[J].Front Microbiol,2016,7:55.
[8]PALMIERI C,MAGI G,CRETI R,et al.Interspecies mobilization of an ermT-carrying plasmid of Streptococcus dysgalactiae subsp.equisimilis by a coresident ICE of the ICESa2603 family[J].J Antimicrob Chemother,2013,68(1):23-26.
[9]TETTELIN H,MASIGNANI V,CIESLEWICZ M J,et al.Complete genome sequence and comparative genomic analysis of an emerging human pathogen,serotype V Streptococcus agalactiae[J].Proc Natl Acad Sci U S A,2002,99(19):12391-12396.
[10]WANG P,TONG J J,MA X H,et al.Serotypes,antibiotic susceptibilities,and multi-locus sequence type profiles of Streptococcus agalactiae isolates circulating in Beijing,China[J].PLoS One,2015,10(3):e0120035.
[11]WANG X,CAO X,LI S,et al.Phenotypic and molecular characterization of Streptococcus agalactiae colonized in Chinese pregnant women:predominance of ST19/III and ST17/III[J].Res Microbiol,2018,169(2):101-107.
[12]JIANG H,CHEN M,LI T,et al.Molecular characterization of Streptococcus agalactiae causing community-and hospitalacquired infections in Shanghai,China[J].Front Microbiol,2016,7:1308.
[13]WANG H,ZHAO C,HE W,et al.High prevalence of fluoroquinolone-resistant group B Streptococci among clinical isolates in China and predominance of sequence type 19 with serotype III[J].Antimicrob Agents Chemother,2013,57(3):1538-1541.