纹带棒状杆菌临床分离株分子分型和耐药性分析
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摘要
目的揭示我国临床分离的纹带棒状杆菌分子分型及耐药性,为制定纹带棒状杆菌感染防治措施提供依据。方法采用多中心横断面研究方法,收集广东、唐山、北京地区各一家三级甲等医院在2017年10月—2018年1月间从临床标本中分离的197株纹带棒状杆菌。用微量肉汤法检测最低抑菌浓度(minimal inhibitory concentration, MIC);聚合酶链式反应检测菌株的氨基糖苷修饰酶基因[aph(3'')-Ib、aph(6)-Id、aac(6')-Ib、aad A、aph(3')-IIIa以及AAC(6')-Ie-APH(2'')-Ia]、核糖体甲基化酶基因(erm X)、核糖体保护蛋白基因(tetW)、外输泵蛋白基因(tetL)以及碳青霉烯酶基因(bla_(IMP)、bla_(VIM)、bla_(OXA-48)、bla_(NDM)和bla_(KPC)),目的产物经测序和BLAST比对并确定其基因型;采用脉冲场凝胶电泳(pulsed field gel electrophoresis, PFGE)对菌株进行分子分型,用Bio Numerics软件对所有实验菌株的PFGE图谱进行聚类分析。结果 99.5%的被测菌株至少对一种抗生素表现耐药;庆大霉素耐药菌株中耐药基因aph(3'')-Ib、aph(6)-Id、aac(6')-Ib和aad A检出率分别为89.3%(67/75)、84%(63/75)、57.3%(43/75)和4%(3/75);红霉素以及克林霉素耐药菌株中耐药基因ermX的检出率均为88.3%(153/173);四环素和多西环素耐药菌株中耐药基因tetW和tetL的检出率分别95.5%(105/110)、97.2%(70/72)和100%(110/110)、100%(72/72)。(英文摘要也要改)197株临床分离纹带棒状杆菌共分为82个型别,每个型别分别包含1～27株菌,其中11个型别包含的菌株数大于5株,广东的菌株优势型别为CSS01.CN0058;唐山的菌株优势型别为CSS01.CN0035、CSS01.CN0051、CSS01.CN0052和CSS01.CN0048;北京的菌株优势型别为CSS01.CN0058、CSS01.CN0070、CSS01.CN0108和CSS01.CN0015。结论我国纹带棒状杆菌临床分离株耐药情况严峻,而且存在多重耐药菌株的院内传播,需要开展更大范围的系统监测为制定控制措施提供实验依据。
Objective To reveal the molecular typing and drug resistance of clinical isolates of Corynebacterium striatum in China, and provide evidence for the prevention and treatment of Corynebacterium striatum infections. Methods A multi-center cross-sectional study method was used to collect 197 isolates of Corynebacterium striatumfrom clinical specimens between October 2017 and January 2018 in a tertiary hospital from Guangdong, Tangshan, and Beijing, respectively. We used the microdilution broth method to measure the minimal inhibitory concentration. Aminoglycoside modifying enzyme(AME) genes [aph(3'')-Ib, aph(6)-Id, aac(6')-Ib, aadA, aph(3')-IIIa and AAC(6')-Ie-APH(2'')-Ia], ribosomal RNA methylases genes(ermX) ribosomal protection proteins genes(tetW), efflux pump proteins genes(tet L), and carbapenemase genes(bla_(IMP), bla_(VIM), bla_(OXA-48), bla_(NDM), bla_(KPC)) were detected by PCR using primers specific for each gene and by sequencing of the PCR products. The desired target products were sequenced, and then compared with BLAST. Pulsed field gel electrophoresis(PFGE) was performed to typing all isolates. BioNumerics was used to develop the molecular type database based on PFGE patterns. Results The antibiotic susceptibility of 12 different antibiotics was tested based on CLSI M45 document 2016, and 99.5% of all the isolates showed resistant to at least one group of antibiotics. The detection rates of aph(3')-Ib, aph(6)-I, aac(6')-Ib and aadA genes within gentamycin resistant isolates were 89.3%(67/75), 84%(63/75), 57.3%(43/75) and 4%(3/75), respectively. the detection rates of tet W and tet Lgenes within tetracycline and doxycycline were 95.5%(105/110) and 97.2%(70/72), and 100%(110/110) and 100%(72/72), respectively. Eighty-two PFGE patterns were identified among the 197 Corynebacterium striatum isolates, among which eleven types were detected in more than five strains. PFGE dominant patterns of Guangdong isolates was CSS01.CN0058; PFGE dominant patterns of Tangshan isolates were CSS01.CN0035, CSS01.CN0051, CSS01.CN0052, and CSS01.CN0048; PFGE dominant patterns of Beijing isolates were CSS01.CN0058, CSS01.CN0070, CSS01.CN0108, and CSS01.CN0015. Conclusions Clinical isolates of Corynebacterium striatum in China are severely resistant against multiple antibiotics, and there exists transmission of multi-drug resistant isolates within a hospital. More extensive systematic monitoring is needed to provide experimental basis for developing control measures.
Objective To reveal the molecular typing and drug resistance of clinical isolates of Corynebacterium striatum in China, and provide evidence for the prevention and treatment of Corynebacterium striatum infections. Methods A multi-center cross-sectional study method was used to collect 197 isolates of Corynebacterium striatumfrom clinical specimens between October 2017 and January 2018 in a tertiary hospital from Guangdong, Tangshan, and Beijing, respectively. We used the microdilution broth method to measure the minimal inhibitory concentration. Aminoglycoside modifying enzyme(AME) genes [aph(3'')-Ib, aph(6)-Id, aac(6')-Ib, aadA, aph(3')-IIIa and AAC(6')-Ie-APH(2'')-Ia], ribosomal RNA methylases genes(ermX) ribosomal protection proteins genes(tetW), efflux pump proteins genes(tet L), and carbapenemase genes(bla_(IMP), bla_(VIM), bla_(OXA-48), bla_(NDM), bla_(KPC)) were detected by PCR using primers specific for each gene and by sequencing of the PCR products. The desired target products were sequenced, and then compared with BLAST. Pulsed field gel electrophoresis(PFGE) was performed to typing all isolates. BioNumerics was used to develop the molecular type database based on PFGE patterns. Results The antibiotic susceptibility of 12 different antibiotics was tested based on CLSI M45 document 2016, and 99.5% of all the isolates showed resistant to at least one group of antibiotics. The detection rates of aph(3')-Ib, aph(6)-I, aac(6')-Ib and aadA genes within gentamycin resistant isolates were 89.3%(67/75), 84%(63/75), 57.3%(43/75) and 4%(3/75), respectively. the detection rates of tet W and tet Lgenes within tetracycline and doxycycline were 95.5%(105/110) and 97.2%(70/72), and 100%(110/110) and 100%(72/72), respectively. Eighty-two PFGE patterns were identified among the 197 Corynebacterium striatum isolates, among which eleven types were detected in more than five strains. PFGE dominant patterns of Guangdong isolates was CSS01.CN0058; PFGE dominant patterns of Tangshan isolates were CSS01.CN0035, CSS01.CN0051, CSS01.CN0052, and CSS01.CN0048; PFGE dominant patterns of Beijing isolates were CSS01.CN0058, CSS01.CN0070, CSS01.CN0108, and CSS01.CN0015. Conclusions Clinical isolates of Corynebacterium striatum in China are severely resistant against multiple antibiotics, and there exists transmission of multi-drug resistant isolates within a hospital. More extensive systematic monitoring is needed to provide experimental basis for developing control measures.
引文
[1]Alatoom A A,Cazanave C J,Cunningham S A,et al.Identification of non-diphtheriae Corynebacterium by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry:Table1[J].J Clin Microbiol,2011,50(1):160-163.
[2]Renom F,Garau M,Rubi M,et al.Nosocomial outbreak of Corynebacterium striatum infection in patients with chronic obstructive pulmonary disease[J].J Clin Microbiol,2007,45(6):2064-2067.
[3]Martin M,Melon O,Celada M,et al.Septicaemia due to Corynebacterium striatum:Molecular confirmation of entry via the skin[J].J Med Microbiol,2003,52(7):599-602.
[4]Fernández-Ayala M,Nan D,Fari?as M.Vertebral osteomyelitis due to Corynebacterium striatum[J].Am JMed,2001.doi:10.1016/s0002-9343(01)00739-2.
[5]Oliva A,Belvisi V,Iannetta M,et al.Pacemaker lead endocarditis due to multidrug-resistant Corynebacterium striatum detected with sonication of the device[J].J Clin Microbiol,2010,48(12):4669-4671.
[6]Otsuka Y,Ohkusu K,Kawamura Y,et al.Emergence of multidrug-resistant Corynebacterium striatum as a nosocomial pathogen in long-term hospitalized patients with underlying diseases[J].Diagn Microbiol Infect Dis,2006,54(2):109-114.
[7]Leonard R B,Nowowiejski D J,Warren J J,et al.Molecular evidence of person-to-person transmission of a pigmented strain of Corynebacterium striatum in intensive care units[J].J Clin Microbiol,1994,32(1):164-169.
[8]Brandenburg A H,van Belkum A,van Pelt C,et al.Patientto-patient spread of a single strain of Corynebacterium striatum causing infections in a surgical intensive care unit[J].J Clin Microbiol,1996,34(9):2089-2094.
[9]Baio P V,Mota H F,Freitas A D,et al.Clonal multidrugresistant Corynebacterium striatum within a nosocomial environment,Rio de Janeiro,Brazil[J].Mem Inst Oswaldo Cruz,2013,108(1):23-29.
[10]CLSI.2016.Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria,3rd ed.CLSI guidelineM45[S].Clinical and Laboratory Standards Institute,Wayne,PA.
[11]Navas J,Fernández-Martínez M,Salas C,et al.Susceptibility to aminoglycosides and distribution of aph and aac(3)-XI genes among Corynebacterium striatum clinical isolates[J].PLoS One,2016,11(12):e167856.doi:10.1371/journal.pone.0167856.
[12]Nordmann P,Naas T,Poirel L.Global Spread of Carbapenemase-producing Enterobacteriaceae[J].Emerg Infect Dis,2011,17(10):1791-1798.
[13]Gomila M,Renom F,Gallegos M D C,et al.Identification and diversity of multi-resistant Corynebacterium striatum clinical isolates by MALDI-TOF mass spectrometry and by a multigene sequencing approach[J].BMC Microbiol,2012,12(1):e52.doi:10.1186/1471-2180-12-52.
[14]Alibi S,Ferjani A,Boukadida J,et al.Occurrence of Corynebacterium striatum as an emerging antibioticresistant nosocomial pathogen in a Tunisian hospital[J].Scientif Rep,2017,7(1):9704-9712.
[15]Wang J,Wang Y,Du X,et al.Rapid transmission of multidrug-resistant Corynebacterium striatum among susceptible patients in a tertiary hospital in China[J].J Infect Develop Count,2016,10(12):1299-1305.
[16]Hahn W O,Werth B J,Butler-Wu S M,et al.Multidrugresistant Corynebacterium striatum associated with increased use of parenteral antimicrobial drugs[J].Emerg Infect Dis,2016,22(11):1908-1914.
[17]Campanile F,Carretto E,Barbarini D,et al.Clonal multidrug-resistant Corynebacterium striatum strains,Italy[J].Emerg Infect Dis,2009,15(1):75-78.
[18]陈东科,许宏涛,胡付品.纹带棒杆菌医院内感染状况分析[J].中华流行病学杂志,2013,34(9):947-948.
[19]陈东科,周海健.耐碳青霉烯类抗生素肺炎克雷伯菌种群结构和耐药机制研究[J].中国抗生素杂志,2018,43(5):507-511.
[2]Renom F,Garau M,Rubi M,et al.Nosocomial outbreak of Corynebacterium striatum infection in patients with chronic obstructive pulmonary disease[J].J Clin Microbiol,2007,45(6):2064-2067.
[3]Martin M,Melon O,Celada M,et al.Septicaemia due to Corynebacterium striatum:Molecular confirmation of entry via the skin[J].J Med Microbiol,2003,52(7):599-602.
[4]Fernández-Ayala M,Nan D,Fari?as M.Vertebral osteomyelitis due to Corynebacterium striatum[J].Am JMed,2001.doi:10.1016/s0002-9343(01)00739-2.
[5]Oliva A,Belvisi V,Iannetta M,et al.Pacemaker lead endocarditis due to multidrug-resistant Corynebacterium striatum detected with sonication of the device[J].J Clin Microbiol,2010,48(12):4669-4671.
[6]Otsuka Y,Ohkusu K,Kawamura Y,et al.Emergence of multidrug-resistant Corynebacterium striatum as a nosocomial pathogen in long-term hospitalized patients with underlying diseases[J].Diagn Microbiol Infect Dis,2006,54(2):109-114.
[7]Leonard R B,Nowowiejski D J,Warren J J,et al.Molecular evidence of person-to-person transmission of a pigmented strain of Corynebacterium striatum in intensive care units[J].J Clin Microbiol,1994,32(1):164-169.
[8]Brandenburg A H,van Belkum A,van Pelt C,et al.Patientto-patient spread of a single strain of Corynebacterium striatum causing infections in a surgical intensive care unit[J].J Clin Microbiol,1996,34(9):2089-2094.
[9]Baio P V,Mota H F,Freitas A D,et al.Clonal multidrugresistant Corynebacterium striatum within a nosocomial environment,Rio de Janeiro,Brazil[J].Mem Inst Oswaldo Cruz,2013,108(1):23-29.
[10]CLSI.2016.Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria,3rd ed.CLSI guidelineM45[S].Clinical and Laboratory Standards Institute,Wayne,PA.
[11]Navas J,Fernández-Martínez M,Salas C,et al.Susceptibility to aminoglycosides and distribution of aph and aac(3)-XI genes among Corynebacterium striatum clinical isolates[J].PLoS One,2016,11(12):e167856.doi:10.1371/journal.pone.0167856.
[12]Nordmann P,Naas T,Poirel L.Global Spread of Carbapenemase-producing Enterobacteriaceae[J].Emerg Infect Dis,2011,17(10):1791-1798.
[13]Gomila M,Renom F,Gallegos M D C,et al.Identification and diversity of multi-resistant Corynebacterium striatum clinical isolates by MALDI-TOF mass spectrometry and by a multigene sequencing approach[J].BMC Microbiol,2012,12(1):e52.doi:10.1186/1471-2180-12-52.
[14]Alibi S,Ferjani A,Boukadida J,et al.Occurrence of Corynebacterium striatum as an emerging antibioticresistant nosocomial pathogen in a Tunisian hospital[J].Scientif Rep,2017,7(1):9704-9712.
[15]Wang J,Wang Y,Du X,et al.Rapid transmission of multidrug-resistant Corynebacterium striatum among susceptible patients in a tertiary hospital in China[J].J Infect Develop Count,2016,10(12):1299-1305.
[16]Hahn W O,Werth B J,Butler-Wu S M,et al.Multidrugresistant Corynebacterium striatum associated with increased use of parenteral antimicrobial drugs[J].Emerg Infect Dis,2016,22(11):1908-1914.
[17]Campanile F,Carretto E,Barbarini D,et al.Clonal multidrug-resistant Corynebacterium striatum strains,Italy[J].Emerg Infect Dis,2009,15(1):75-78.
[18]陈东科,许宏涛,胡付品.纹带棒杆菌医院内感染状况分析[J].中华流行病学杂志,2013,34(9):947-948.
[19]陈东科,周海健.耐碳青霉烯类抗生素肺炎克雷伯菌种群结构和耐药机制研究[J].中国抗生素杂志,2018,43(5):507-511.
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