江苏北部地区猪源屎肠球菌耐药性分析及其酰胺醇类耐药基因的检测
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摘要
[目的]本试验分析了江苏北部地区2016—2017年分离的135株猪源屎肠球菌的耐药表型,并对其携带的酰胺醇类耐药基因fex A、fex B、flo R、cfr、optr A进行检测,以了解猪源屎肠球菌的耐药现状及其对酰胺醇类抗生素的耐药机制,为抗菌药物的合理用药提供参考。[方法]猪肛门拭子采样,分离、纯化并鉴定屎肠球菌后,采用微量肉汤稀释法检测菌株对12种抗菌药物的耐药表型,采用PCR方法检测酰胺醇类耐药基因在屎肠球菌中的分布情况。[结果]分离的135株屎肠球菌对红霉素、四环素、氟苯尼考的耐药率较高,分别为99.26%、98.52%和82.22%,对利奈唑胺的耐药率最低,为2.96%,对其他药物的耐药率由高到低分别为氨苄西林(75.56%)、链霉素(74.07%)、庆大霉素(71.11%)、麻保沙星(69.63%)、青霉素G(45.19%)、利福平(34.07%)和磷霉素(20.00%);未发现万古霉素耐药菌株。结果还显示所有菌株都为多药耐药菌株,以7耐为主(39株),还有3株表现为10耐。111株氟苯尼考耐药的菌株中,耐药基因fex B、fex A、optr A、flo R和cfr的携带率分别为58.56%(65)、27.03%(30)、20.72%(23)、17.12%(19)和0.90%(1),部分菌株同时携带多种耐药基因。[结论]本试验分离的猪源屎肠球菌均为多药耐药菌株,并对兽医专用抗生素氟苯尼考产生较高的耐药性。该结果为猪源肠球菌耐药性流行病学调查及其对酰胺醇类耐药机制的研究奠定基础,并为江苏地区猪场抗菌药的合理使用提供依据。
[Objectives]The aim of the study is to analyze the resistant phenotype of 135 strains of Enterococcus faecium isolated from the northern of Jiangsu Province during 2016-2017 and detect the amphenicols resistance genes fex A,fex B,flo R,cfr and optr A for further knowing the prevalence of resistant E. faecium and the mechanisms involved in phenicols resistance. [Methods]E. faecium was isolated from swine anal swabs and further identified by PCR. The broth microdilution method was used to determine the minimal inhibitory concentration( MIC) of 12 antimicrobials against E. faecium. Amphenicols resistance genes fex B,fex A,optr A,flo R,and cfr were detected by PCR in florfenicol resistant strains. [Results]Total of 135 strains of E. faecium were isolated from 300 samples. The resistant rates of erythromycin,tetracycline and florfenicol were the highest with rates of 99.26%,98.52% and 82.22%,respectively.The resistance rates of other drugs from high to low were ampicillin( 75. 56%),streptomycin( 74. 07%),gentamicin( 71. 11%),marbofloxacin( 69.63%),penicillin G( 45. 19%),rifampicin( 34. 07%) and fosfomycin( 20. 00%),respectively. The strains had a lowest resistance toward linezolid and the rate was 2.96%. Vancomycin resistant strains were not found in this study. Further results showed that most of the strains presented multidrug resistance profiles. Among all the resistant strains,39 strains were resistant to 7 kinds of drugs and three strains showed resistance to 10 kinds of drugs. Among the 111 strains resistant to amphenicols,the carrying rates of the resistance genes fex B,fex A,optr A,flo R,and cfr were 58. 56%( 65),27.03%( 30),20. 72%( 23),17. 12%( 19),and0.90%( 1),respectively. Some of strains carried multiple drug resistance genes at the same time. [Conclusions]The clinical isolates of Enterococcus faecium from swine are resistant to commonly used antibacterial drugs,in particular,resistance to florfenicol is particularly serious and it should be payed attention to. The results of the study laid the foundations for clarifying the transmission mechanisms of antibiotic-resistant genes and further provided a guidance for reasonable use of antimicrobials in piggeries of Jiangsu Province.
[Objectives]The aim of the study is to analyze the resistant phenotype of 135 strains of Enterococcus faecium isolated from the northern of Jiangsu Province during 2016-2017 and detect the amphenicols resistance genes fex A,fex B,flo R,cfr and optr A for further knowing the prevalence of resistant E. faecium and the mechanisms involved in phenicols resistance. [Methods]E. faecium was isolated from swine anal swabs and further identified by PCR. The broth microdilution method was used to determine the minimal inhibitory concentration( MIC) of 12 antimicrobials against E. faecium. Amphenicols resistance genes fex B,fex A,optr A,flo R,and cfr were detected by PCR in florfenicol resistant strains. [Results]Total of 135 strains of E. faecium were isolated from 300 samples. The resistant rates of erythromycin,tetracycline and florfenicol were the highest with rates of 99.26%,98.52% and 82.22%,respectively.The resistance rates of other drugs from high to low were ampicillin( 75. 56%),streptomycin( 74. 07%),gentamicin( 71. 11%),marbofloxacin( 69.63%),penicillin G( 45. 19%),rifampicin( 34. 07%) and fosfomycin( 20. 00%),respectively. The strains had a lowest resistance toward linezolid and the rate was 2.96%. Vancomycin resistant strains were not found in this study. Further results showed that most of the strains presented multidrug resistance profiles. Among all the resistant strains,39 strains were resistant to 7 kinds of drugs and three strains showed resistance to 10 kinds of drugs. Among the 111 strains resistant to amphenicols,the carrying rates of the resistance genes fex B,fex A,optr A,flo R,and cfr were 58. 56%( 65),27.03%( 30),20. 72%( 23),17. 12%( 19),and0.90%( 1),respectively. Some of strains carried multiple drug resistance genes at the same time. [Conclusions]The clinical isolates of Enterococcus faecium from swine are resistant to commonly used antibacterial drugs,in particular,resistance to florfenicol is particularly serious and it should be payed attention to. The results of the study laid the foundations for clarifying the transmission mechanisms of antibiotic-resistant genes and further provided a guidance for reasonable use of antimicrobials in piggeries of Jiangsu Province.
引文
[1] Akpaka P E,Kissoon S,Wilson C,et al. Molecular characterization of vancomycin resistant Enterococcus faecium isolates from Bermuda[J].PLoS One,2017,12(3):e0171317.
[2] Donald E L,Keller N,Barth A,et al. Clinical prevalence antimicrobial susceptibility and geographic resistance patterns of Enterococci:results from the SENTRY antimicrobial surveillance program,1997-1999[J]. Clinical Infectious Diseases,2001,32(12):133-145.
[3]瞿婷婷.多重耐药肠球菌耐药基因筛选及传播机制研究[D].杭州:浙江大学,2007.Qu T T. Study on the resistance genes and resistance transmission mechanism in multidrug-resistant Enterococcus[D]. Hangzhou:Zhejiang University,2007(in Chinese with English abstract).
[4]廖国林,刘建,李芳,等.肠球菌属医院感染分布及耐药性分析[J].中华医院感染学杂志,2009,19(13):1735-1736.Liao G L,Liu J,Li F,et al. Distribution and drug resistance of Enterococcus in hospital:analysis and prevention[J]. Chinese Journal of Nosocomiology,2009,19(13):1735-1736(in Chinese with English abstract).
[5]吴海鹏,潘国宏.肠球菌感染的临床分布及耐药性分析[J].中国医药指南,2015,13(23):77-78.Wu H P,Pan G H. Clinical distribution and drug resistance analysis of infection by Enterococcus[J]. Guide of China Medicine,2015,13(23):77-78(in Chinese).
[6]贾伟,赵梅,王文,等.肠球菌属耐药性与耐药基因的比较研究[J].中华医院感染学杂志,2015,25(24):5551-5554.Jia W,Zhao M,Wang W,et al. Comparative study on drug resistances and drug-resistant genes of Enterococcus species in clinic[J]. Chinese Journal of Nosocomiology,2015,25(24):5551-5554(in Chinese with English abstract).
[7] Mutschler M,Trojan S,Defosse J M,et al. Severe sepsis caused by a linezolid-resistant Enterococcus faecium in a 10-year-old girl after multiple trauma[J]. International Journal of Infectious Diseases,2013,17:466-467.
[8] Alatorre-Fernandez P,Mayoral-Teran C,Velazquez-Acosta C,et al. A polyclonal outbreak of bloodstream infections by Enterococcus faecium in patients with hematologic malignancies[J]. American Journal of Infection Control,2017,45:260-266.
[9]胡付品,朱德妹,汪复,等. 2015年CHINET细菌耐药性监测[J].中国感染与化疗杂志,2016,16(6):685-694.Hu F P,Zhu D M,Wang F,et al. Report of CHINET antimicrobial resistance surveillance program in 2015[J]. Chinese Journal of Infection and Chemotherapy,2016,16(6):685-694(in Chinese with English abstract).
[10]杨青,俞云松,林洁,等. 2005—2014年CHINET肠球菌属细菌耐药性监测[J].中国感染与化疗杂志,2016,16(2):146-152.Yang Q,Yu Y S,Lin J,et al. Resistance profile of Enterococcus in hospitals across China:results of CHINET antimicrobial resistance surveillance program,2005-2014[J]. Chinese Journal of Infection and Chemotherapy,2016,16(2):146-152(in Chinese with English abstract).
[11]胡付品,郭燕,朱德妹,等. 2016年中国CHINET细菌耐药性监测[J].中国感染与化疗杂志,2017,17(5):481-491.Hu F P,Guo Y,Zhu D M,et al. CHINET surveillance of bacterial resistance across China:report of the results in 2016[J]. Chinese Journal of Infection and Chemotherapy,2017,17(5):481-491(in Chinese with English abstract).
[12]刘永安,李学瑞,周建华,等.一株羊源致病性屎肠球菌的分离鉴定及耐药性分析[J].中国畜牧兽医,2017,44(2):530-537.Liu Y A,Li X R,Zhou J H,et al. Isolation,identification and drug resistance analysis of pathogenic Enterococcus faecium of sheep[J]. China Animal Husbandry&Veterinary Medicine,2017,44(2):530-537(in Chinese with English abstract).
[13]卢彩景,张秀平,陈陆,等.猪源屎肠球菌致病力与耐药性及生物被膜形成能力的相关性分析[J].中国预防兽医学报,2014,36(5):350-353.Lu C J,Zhang X P,Chen L,et al. Correlation analysis among pathogenicity and antibiotic resistance and capability of biofilm formation of Enterococcus faecium isolated from swine[J]. Chinese Journal of Preventive Veterinary Medicine,2014,36(5):350-353(in Chinese with English abstract).
[14] Tamang M D,Moon D C,Kim S R,et al. Detection of novel oxazolidinone and phenicol resistance gene optr A in enterococcal isolates from food animals and animal carcasses[J]. Veterinary Microbiology,2017,201:252-256.
[15]刘佳,陈霞,赵爱兰,等.河南省某地区健康人源及动物源肠球菌种属分布及耐药性差异研究[J].中国畜牧兽医,2014,41(12):172-177.Liu J,Chen X,Zhao A L,et al. Study on the differences of prevalence and drug resistance of Enterococcus species of human and animal origins in Henan Province,China[J]. China Animal Husbandry&Veterinary Medicine,2014,41(12):172-177(in Chinese with English abstract).
[16]刘凌.不同来源肠球菌的耐药性调查及对氟苯尼考耐药机制的研究[D].郑州:河南农业大学,2015.Liu L. Investigation on the resistance situation in enterococci strains from various resources and its resistance mechanism to florfenicol[D].Zhengzhou:Henan Agricultural University,2015(in Chinese with English abstract).
[17] Macovei L,Zurek L. Ecology of antibiotic resistance genes:characterization of Enterococci from houseflies collected in food settings[J]. Applied and Environmental Microbiology,2006,72(6):4028-4035.
[18] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk and diffusion susceptibility tests for bacteria isolated from animals:second informational supplement VET01-S2[R]. Wayne:CLSI,2013.
[19] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing:twenty-third informational supplement M100-S23[R]. Wayne:CLSI,2013.
[20] Kehrenberg C,Schwarz S. Florfenicol-chloramphenicol exporter gene fex A is part of the novel transposon Tn558[J]. Antimicrobial Agents and Chemotherapy,2005,49(2):813-815.
[21] Faldynova M,Pravcova M,Sisak F,et al. Evolution of antibiotic resistance in Salmonella enterica serovar typhimurium strains isolated in the Czech Republic between 1984 and 2002[J]. Antimicrobial Agents and Chemotherapy,2003,47(6):2002-2005.
[22] Kehrenberg C,Schwarz S. Distribution of florfenicol resistance genes fex A and cfr among chloramphenicol-resistant Staphylococcus isolates[J].Antimicrobial Agents and Chemotherapy,2006,50(4):1156-1163.
[23] Wang Y,LüY,Cai J C,et al. A novel gene,optr A,that confers transferable resistance to oxazolidinones and phenicols and its presence in Enterococcus faecalis and Enterococcus faecium of human and animal origin[J]. Journal of Antimicrobial Chemotherapy,2015,70:2182-2190.
[24]廖云凤,罗鹏,孙珊. 2015年重庆市粪肠球菌和屎肠球菌耐药性监测结果分析[J].国际检验医学杂志,2017,38(12):1585-1588.Liao Y F,Luo P,Sun S. Surveillance of antibiotic resistance of Enterococcus faecalis and Enterococcus faecium in Chongqing in 2015[J].International Journal of Laboratory Medicine,2017,38(12):1585-1588(in Chinese with English abstract).
[25]杜凡姝,吕茜,段锻,等.江苏苏北地区猪链球菌耐药性调查及万古霉素耐药基因检测[J].畜牧与兽医,2018,50(12):32-36.Du F S,LüX,Duan D,et al. Antimicrobial susceptibility and emergence of vancomycin resistance genes in Streptococcus suis[J]. Animal Husbandry&Veterinary Medicine,2018,50(12):32-36(in Chinese with English abstract).
[26]陈霞,刘玉庆,李文革,等.山东省猪源肠球菌酰胺醇类药物耐药表型及耐药机制研究[J].中国人兽共患病学报,2013,29(10):959-964.Chen X,Liu Y Q,Li W G,et al. Characterization phenotypes and mechanisms of phenicols resistance among Enterococcus species of swine origin from Shandong Province[J]. Chinese Journal of Zoonoses,2013,29(10):959-964(in Chinese with English abstract).
[27]赵丽青.广东某猪场噁唑烷酮类耐药基因optr A的流行特点[D].广州:华南农业大学,2016.Zhao L Q. The epidemic characteristics of oxazolidinone-resistant gene optr A in a pig farm in Guangdong[D]. Guangzhou:South China Agricultural University,2016(in Chinese with English abstract).
[2] Donald E L,Keller N,Barth A,et al. Clinical prevalence antimicrobial susceptibility and geographic resistance patterns of Enterococci:results from the SENTRY antimicrobial surveillance program,1997-1999[J]. Clinical Infectious Diseases,2001,32(12):133-145.
[3]瞿婷婷.多重耐药肠球菌耐药基因筛选及传播机制研究[D].杭州:浙江大学,2007.Qu T T. Study on the resistance genes and resistance transmission mechanism in multidrug-resistant Enterococcus[D]. Hangzhou:Zhejiang University,2007(in Chinese with English abstract).
[4]廖国林,刘建,李芳,等.肠球菌属医院感染分布及耐药性分析[J].中华医院感染学杂志,2009,19(13):1735-1736.Liao G L,Liu J,Li F,et al. Distribution and drug resistance of Enterococcus in hospital:analysis and prevention[J]. Chinese Journal of Nosocomiology,2009,19(13):1735-1736(in Chinese with English abstract).
[5]吴海鹏,潘国宏.肠球菌感染的临床分布及耐药性分析[J].中国医药指南,2015,13(23):77-78.Wu H P,Pan G H. Clinical distribution and drug resistance analysis of infection by Enterococcus[J]. Guide of China Medicine,2015,13(23):77-78(in Chinese).
[6]贾伟,赵梅,王文,等.肠球菌属耐药性与耐药基因的比较研究[J].中华医院感染学杂志,2015,25(24):5551-5554.Jia W,Zhao M,Wang W,et al. Comparative study on drug resistances and drug-resistant genes of Enterococcus species in clinic[J]. Chinese Journal of Nosocomiology,2015,25(24):5551-5554(in Chinese with English abstract).
[7] Mutschler M,Trojan S,Defosse J M,et al. Severe sepsis caused by a linezolid-resistant Enterococcus faecium in a 10-year-old girl after multiple trauma[J]. International Journal of Infectious Diseases,2013,17:466-467.
[8] Alatorre-Fernandez P,Mayoral-Teran C,Velazquez-Acosta C,et al. A polyclonal outbreak of bloodstream infections by Enterococcus faecium in patients with hematologic malignancies[J]. American Journal of Infection Control,2017,45:260-266.
[9]胡付品,朱德妹,汪复,等. 2015年CHINET细菌耐药性监测[J].中国感染与化疗杂志,2016,16(6):685-694.Hu F P,Zhu D M,Wang F,et al. Report of CHINET antimicrobial resistance surveillance program in 2015[J]. Chinese Journal of Infection and Chemotherapy,2016,16(6):685-694(in Chinese with English abstract).
[10]杨青,俞云松,林洁,等. 2005—2014年CHINET肠球菌属细菌耐药性监测[J].中国感染与化疗杂志,2016,16(2):146-152.Yang Q,Yu Y S,Lin J,et al. Resistance profile of Enterococcus in hospitals across China:results of CHINET antimicrobial resistance surveillance program,2005-2014[J]. Chinese Journal of Infection and Chemotherapy,2016,16(2):146-152(in Chinese with English abstract).
[11]胡付品,郭燕,朱德妹,等. 2016年中国CHINET细菌耐药性监测[J].中国感染与化疗杂志,2017,17(5):481-491.Hu F P,Guo Y,Zhu D M,et al. CHINET surveillance of bacterial resistance across China:report of the results in 2016[J]. Chinese Journal of Infection and Chemotherapy,2017,17(5):481-491(in Chinese with English abstract).
[12]刘永安,李学瑞,周建华,等.一株羊源致病性屎肠球菌的分离鉴定及耐药性分析[J].中国畜牧兽医,2017,44(2):530-537.Liu Y A,Li X R,Zhou J H,et al. Isolation,identification and drug resistance analysis of pathogenic Enterococcus faecium of sheep[J]. China Animal Husbandry&Veterinary Medicine,2017,44(2):530-537(in Chinese with English abstract).
[13]卢彩景,张秀平,陈陆,等.猪源屎肠球菌致病力与耐药性及生物被膜形成能力的相关性分析[J].中国预防兽医学报,2014,36(5):350-353.Lu C J,Zhang X P,Chen L,et al. Correlation analysis among pathogenicity and antibiotic resistance and capability of biofilm formation of Enterococcus faecium isolated from swine[J]. Chinese Journal of Preventive Veterinary Medicine,2014,36(5):350-353(in Chinese with English abstract).
[14] Tamang M D,Moon D C,Kim S R,et al. Detection of novel oxazolidinone and phenicol resistance gene optr A in enterococcal isolates from food animals and animal carcasses[J]. Veterinary Microbiology,2017,201:252-256.
[15]刘佳,陈霞,赵爱兰,等.河南省某地区健康人源及动物源肠球菌种属分布及耐药性差异研究[J].中国畜牧兽医,2014,41(12):172-177.Liu J,Chen X,Zhao A L,et al. Study on the differences of prevalence and drug resistance of Enterococcus species of human and animal origins in Henan Province,China[J]. China Animal Husbandry&Veterinary Medicine,2014,41(12):172-177(in Chinese with English abstract).
[16]刘凌.不同来源肠球菌的耐药性调查及对氟苯尼考耐药机制的研究[D].郑州:河南农业大学,2015.Liu L. Investigation on the resistance situation in enterococci strains from various resources and its resistance mechanism to florfenicol[D].Zhengzhou:Henan Agricultural University,2015(in Chinese with English abstract).
[17] Macovei L,Zurek L. Ecology of antibiotic resistance genes:characterization of Enterococci from houseflies collected in food settings[J]. Applied and Environmental Microbiology,2006,72(6):4028-4035.
[18] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial disk and diffusion susceptibility tests for bacteria isolated from animals:second informational supplement VET01-S2[R]. Wayne:CLSI,2013.
[19] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing:twenty-third informational supplement M100-S23[R]. Wayne:CLSI,2013.
[20] Kehrenberg C,Schwarz S. Florfenicol-chloramphenicol exporter gene fex A is part of the novel transposon Tn558[J]. Antimicrobial Agents and Chemotherapy,2005,49(2):813-815.
[21] Faldynova M,Pravcova M,Sisak F,et al. Evolution of antibiotic resistance in Salmonella enterica serovar typhimurium strains isolated in the Czech Republic between 1984 and 2002[J]. Antimicrobial Agents and Chemotherapy,2003,47(6):2002-2005.
[22] Kehrenberg C,Schwarz S. Distribution of florfenicol resistance genes fex A and cfr among chloramphenicol-resistant Staphylococcus isolates[J].Antimicrobial Agents and Chemotherapy,2006,50(4):1156-1163.
[23] Wang Y,LüY,Cai J C,et al. A novel gene,optr A,that confers transferable resistance to oxazolidinones and phenicols and its presence in Enterococcus faecalis and Enterococcus faecium of human and animal origin[J]. Journal of Antimicrobial Chemotherapy,2015,70:2182-2190.
[24]廖云凤,罗鹏,孙珊. 2015年重庆市粪肠球菌和屎肠球菌耐药性监测结果分析[J].国际检验医学杂志,2017,38(12):1585-1588.Liao Y F,Luo P,Sun S. Surveillance of antibiotic resistance of Enterococcus faecalis and Enterococcus faecium in Chongqing in 2015[J].International Journal of Laboratory Medicine,2017,38(12):1585-1588(in Chinese with English abstract).
[25]杜凡姝,吕茜,段锻,等.江苏苏北地区猪链球菌耐药性调查及万古霉素耐药基因检测[J].畜牧与兽医,2018,50(12):32-36.Du F S,LüX,Duan D,et al. Antimicrobial susceptibility and emergence of vancomycin resistance genes in Streptococcus suis[J]. Animal Husbandry&Veterinary Medicine,2018,50(12):32-36(in Chinese with English abstract).
[26]陈霞,刘玉庆,李文革,等.山东省猪源肠球菌酰胺醇类药物耐药表型及耐药机制研究[J].中国人兽共患病学报,2013,29(10):959-964.Chen X,Liu Y Q,Li W G,et al. Characterization phenotypes and mechanisms of phenicols resistance among Enterococcus species of swine origin from Shandong Province[J]. Chinese Journal of Zoonoses,2013,29(10):959-964(in Chinese with English abstract).
[27]赵丽青.广东某猪场噁唑烷酮类耐药基因optr A的流行特点[D].广州:华南农业大学,2016.Zhao L Q. The epidemic characteristics of oxazolidinone-resistant gene optr A in a pig farm in Guangdong[D]. Guangzhou:South China Agricultural University,2016(in Chinese with English abstract).