猪链球菌对大环内酯类抗生素的耐药性研究
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摘要
为测定猪链球菌对大环内酯类抗生素的耐药性和相关耐药基因分布特点,分析分离株对大环内酯类抗生素耐药的分子机制。从河南等7省份不同猪场采集来的疑似猪链球菌患病猪的脑脊液、关节液、肝等部位分离、纯化疑似菌落,采用镜检、16S rRNA基因方法鉴定猪链球菌。采用肉汤微量稀释法检测分离猪链球菌对大环内酯类抗生素红霉素、泰乐菌素、替米考星的耐药情况,采用PCR方法检测相关耐药基因(包括编码核糖体甲基化酶的erm基因ermA、ermB、ermC、ermTR和介导外排泵机制的mefA、mefE及msrD基因)分布情况。最终从573份病料分离鉴定出猪链球菌165株,分离率为28.8%。药敏试验结果显示:猪链球菌对大环内酯类抗生素耐药率高达98.1%,对红霉素、泰乐菌素、替米考星耐药率分别达96.9%、95.1%、94.5%,有153株菌株对3种抗生素同时耐药。耐药基因检测发现大环内酯类耐药基因ermA、ermB、ermC、ermTR、mefA、mefE、msrD检出率分别为5.4%、81.8%、13.3%、6.6%、11.5%、7.2%、5.4%,其中ermB检出率最高。除发现有93株菌株仅携带1个耐药基因外,分别有30、17、2、1个菌株分别携带2、3、4、6个大环内酯类耐药基因。在检出耐药基因的菌株中有82.5%(118/143)的菌株仅携带erm基因,有13.9%(20/143)的菌株同时携带erm以及mef、msr基因,另有3%(5/143)的菌株仅携mef、msr基因。猪链球菌对大环内酯类药物广泛耐药,大环内酯类耐药基因在猪链球菌中广泛分布,细菌的耐药性和菌株耐药基因携带情况基本呈正相关。以ermB介导的核糖体23S rRNA甲基化耐药机制占主导地位,由mefA、mefE、msrD介导的外排泵机制亦存在于分离菌株中。
To determine the resistance of Streptococcus suis to macrolides and the distribution characteristics of related resistance genes, and to analyze the molecular mechanism of resistance of the isolates to macrolide antibiotics, suspected colonies were isolated and purified from the cerebrospinal fluid, joint fluid and liver of pigs infected with Streptococcus suis from different piggery of seven provinces like Henan. Streptococcus suis was identified by microscopic examination and 16 S rRNA. The resistance of Streptococcus suis to macrolides including erythromycin, tylosin and timicosin was detected by broth microdilution method. The PCR method was used to detect the distribution of the related genes(including the erm genes encoding the ribosome methylase, ermA, ermB, ermC, and ermTR, and the mefA, mefE and msrD genes that mediate the efflux pump mechanism). 165 Streptococcus suis isolates were obtained from 573 samples and the isolation rate was 28.8%. The susceptibility test showed that the resistance rate of Streptococcus suis isolates to macrolides was 98.1%(162/165), and the resistance rate to erythromycin, tylosin and tymmicosin was 96.9%, 95.1% and 94.9%, respectively. 153 of 165 isolated strains were resistant to all 3 antibiotics. Resistance gene detection found that the detection rate of macrolide resistance gene ermA, ermB, ermC, ermTR, mefA, mefE and msrD was 5.4%, 81.8%, 13.3%, 6.6%, 11.5%, 7.2% and 5.4%, respectively, among which the detection rate ermB gene was the highest. In addition to finding 93 strains carrying only 1 resistance genes, 30, 17, 2 and 1 strains carried 2, 3, 4 and 6 macrolide resistant genes, respectively. Among the resistant gene-positive strains, 82.5%(118/143) strains only carried single erm gene, and 13.9%(20/143) strains carried erm, mef and msr genes simultaneously, and 3%(5/143) strains only carried mef and msr genes. Streptococcus suis is widely resistant to macrolides, and macrolide resistance genes are widely distributed in Streptococcus suis, and basically bacterial resistance and drug resistance gene carrying are positively correlated. The mechanism of ermB mediated ribosome 23 S rRNA methylation is dominant, and the efflux pump mechanism mediated by mefA, mefE and msrD also exists among the tested isolates.
To determine the resistance of Streptococcus suis to macrolides and the distribution characteristics of related resistance genes, and to analyze the molecular mechanism of resistance of the isolates to macrolide antibiotics, suspected colonies were isolated and purified from the cerebrospinal fluid, joint fluid and liver of pigs infected with Streptococcus suis from different piggery of seven provinces like Henan. Streptococcus suis was identified by microscopic examination and 16 S rRNA. The resistance of Streptococcus suis to macrolides including erythromycin, tylosin and timicosin was detected by broth microdilution method. The PCR method was used to detect the distribution of the related genes(including the erm genes encoding the ribosome methylase, ermA, ermB, ermC, and ermTR, and the mefA, mefE and msrD genes that mediate the efflux pump mechanism). 165 Streptococcus suis isolates were obtained from 573 samples and the isolation rate was 28.8%. The susceptibility test showed that the resistance rate of Streptococcus suis isolates to macrolides was 98.1%(162/165), and the resistance rate to erythromycin, tylosin and tymmicosin was 96.9%, 95.1% and 94.9%, respectively. 153 of 165 isolated strains were resistant to all 3 antibiotics. Resistance gene detection found that the detection rate of macrolide resistance gene ermA, ermB, ermC, ermTR, mefA, mefE and msrD was 5.4%, 81.8%, 13.3%, 6.6%, 11.5%, 7.2% and 5.4%, respectively, among which the detection rate ermB gene was the highest. In addition to finding 93 strains carrying only 1 resistance genes, 30, 17, 2 and 1 strains carried 2, 3, 4 and 6 macrolide resistant genes, respectively. Among the resistant gene-positive strains, 82.5%(118/143) strains only carried single erm gene, and 13.9%(20/143) strains carried erm, mef and msr genes simultaneously, and 3%(5/143) strains only carried mef and msr genes. Streptococcus suis is widely resistant to macrolides, and macrolide resistance genes are widely distributed in Streptococcus suis, and basically bacterial resistance and drug resistance gene carrying are positively correlated. The mechanism of ermB mediated ribosome 23 S rRNA methylation is dominant, and the efflux pump mechanism mediated by mefA, mefE and msrD also exists among the tested isolates.
引文
[1] KATAOKA Y, YOSHIDA T, SAWADA T. A 10-year survey of antimicrobial susceptibility of Streptococcus suis isolates from swine in Japan[J]. Journal of Veterinary Medical Science, 2000, 62(10): 1053-1057.
[2] 杨建江, 韩文瑜, 雷连成, 等. 猪链球菌耐药性的检测[J]. 中国兽医科技, 2004, 34(7): 48-50.
[3] AARESTRUP F M, RASMUSSEN S R, ARTURSSON K, et al. Trends in the resistance to antimicrobial agents of Streptococcus suis isolates from Denmark and Sweden[J]. Veterinary Microbiology, 1998, 63(1): 71-80.
[4] VELA A I, MORENO M A, CEBOLLA J A, et al. Antimicrobial susceptibility of clinical strains of Streptococcus suis isolated from pigs in Spain[J]. Veterinary Microbiology, 2005, 105(2): 143-147.
[5] 杨建江,韩文瑜,雷连成,等.长春地区猪链球菌对大环内酯和林克酰胺类耐药的分子机制研究[J].中国人兽共患病杂志,2004,20(8):698-701.
[6] 李晓云, 苑青艳, 闫明, 等. 猪链球菌对大环内酯类药物耐药性研究进展[J]. 动物医学进展, 2008, 29(6): 80-83.
[7] MARTEL A, DEVRIESE L A, DECOSTERE A, et al. Presence of macrolide resistance genes in streptococci and enterococci isolated from pigs and pork carcasses.[J]. International Journal of Food Microbiology, 2003, 84(1):27-32.
[8] BRENCIANI A, BACCIAGLIA A, VECCHI M, et al. Genetic elements carrying erm(B) in Streptococcus pyogenes and association with tet(M) tetracycline resistance gene[J]. Antimicrob Agents Chemother, 2007, 51(4):1209-1216.
[9] PALMIERI C, PRINCIVALLI M S, BRENCIANI A, et al. Different genetic elements carrying the tet(W) gene in two human clinical isolates of Streptococcus suis[J]. Antimicrobial Agents & Chemotherapy, 2011, 55(2):631-636.
[10] BRENCIANI A, AVIGNAROLI B. Phim 46.1, the main streptococcus pyogenes element carrying mef(A) and tet(O) genes.[J]. Antimicrobial Agents & Chemotherapy, 2010, 54(1):221-229.
[11] OKWUMABUA O, O’CONNOR M, SHULL E. A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase[J]. FEMS microbiology letters, 2003, 218(1): 79-84.
[12] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: Twenty-fourth informational supplement, M100-S24[J]. Clinical and Laboratory Standards Institute , 2014, 34(1):120-129.
[13] GURUNG M, TAMANG M D, MOON D C, et al. Molecular basis of resistance to selected antimicrobial agents in the emerging zoonotic pathogen Streptococcus suis[J]. Journal of Clinical Microbiology, 2015, 53(7): 2332-2336.
[14] SUTCLIFFE J, GREBE T, TAIT-KAMRADT A, et al. Detection of erythromycin-resistant determinants by PCR[J]. Antimicrobial Agents and Chemotherapy, 1996, 40(11): 2562-2566.
[15] 陈惠玲, 邓家德, 叶惠芬, 等. 侵入性感染 B 族链球菌对红霉素及四环素的耐药基因检测[J]. 中华医院感染学杂志, 2010,20 (10): 1354-1357.
[16] BOJARSKA A, MOLSKA E, JANAS K, et al. Streptococcus suis in invasive human infections in Poland: clonality and determinants of virulence and antimicrobial resistance[J]. European Journal of Clinical Microbiology & Infectious Diseases, 2016, 35(6): 917-925.
[17] 刘丽英. 大环内酯类抗生素的临床研究进展[J]. 兽药与饲料添加剂, 2001, 6(6):13-15.
[18] ZHANG C, NING Y, ZHANG Z, et al. In vitro antimicrobial susceptibility of Streptococcus suis strains isolated from clinically healthy sows in China[J]. Veterinary Microbiology, 2008, 131(3/4): 386-392.
[19] WEI Z, LI R, ZHANG A, et al. Characterization of Streptococcus suis isolates from the diseased pigs in China between 2003 and 2007[J]. Veterinary Microbiology, 2009, 137(1/2): 196-201.
[20] 应薇芳, 赵焕灿, 钱娅, 等. 浙江部分地区屠宰场待宰猪链球菌的检测及药敏试验[J]. 动物医学进展, 2007, 28(6): 24-27.
[21] 张卓然, 夏梦岩, 倪语星. 微生物耐药的基础与临床[J].北京: 人民卫生出版社, 2007.
[22] LóPEZ A C, ORT. Tetracycline and oxytetracycline resistance determinants detected in Bacillus cereus strains isolated from honey samples[J]. Rev Arg Microbiol, 2009, 40(4): 231-237.
[23] 汪华, 胡晓抒,朱凤才,等. 人—猪链球菌感染性综合征的流行病学调查[J]. 现代预防医学, 2000, 27(3): 312-314.
[24] 张桂枝, 罗永煌. 大环内酯类抗生素研究进展[J]. 动物医学进展, 2004, 25(3):33-36.
[25] 刘耀川. 细菌对大环内酯类药物耐药机制简介[J]. 现代畜牧兽医, 2012(10):52-53.
[2] 杨建江, 韩文瑜, 雷连成, 等. 猪链球菌耐药性的检测[J]. 中国兽医科技, 2004, 34(7): 48-50.
[3] AARESTRUP F M, RASMUSSEN S R, ARTURSSON K, et al. Trends in the resistance to antimicrobial agents of Streptococcus suis isolates from Denmark and Sweden[J]. Veterinary Microbiology, 1998, 63(1): 71-80.
[4] VELA A I, MORENO M A, CEBOLLA J A, et al. Antimicrobial susceptibility of clinical strains of Streptococcus suis isolated from pigs in Spain[J]. Veterinary Microbiology, 2005, 105(2): 143-147.
[5] 杨建江,韩文瑜,雷连成,等.长春地区猪链球菌对大环内酯和林克酰胺类耐药的分子机制研究[J].中国人兽共患病杂志,2004,20(8):698-701.
[6] 李晓云, 苑青艳, 闫明, 等. 猪链球菌对大环内酯类药物耐药性研究进展[J]. 动物医学进展, 2008, 29(6): 80-83.
[7] MARTEL A, DEVRIESE L A, DECOSTERE A, et al. Presence of macrolide resistance genes in streptococci and enterococci isolated from pigs and pork carcasses.[J]. International Journal of Food Microbiology, 2003, 84(1):27-32.
[8] BRENCIANI A, BACCIAGLIA A, VECCHI M, et al. Genetic elements carrying erm(B) in Streptococcus pyogenes and association with tet(M) tetracycline resistance gene[J]. Antimicrob Agents Chemother, 2007, 51(4):1209-1216.
[9] PALMIERI C, PRINCIVALLI M S, BRENCIANI A, et al. Different genetic elements carrying the tet(W) gene in two human clinical isolates of Streptococcus suis[J]. Antimicrobial Agents & Chemotherapy, 2011, 55(2):631-636.
[10] BRENCIANI A, AVIGNAROLI B. Phim 46.1, the main streptococcus pyogenes element carrying mef(A) and tet(O) genes.[J]. Antimicrobial Agents & Chemotherapy, 2010, 54(1):221-229.
[11] OKWUMABUA O, O’CONNOR M, SHULL E. A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase[J]. FEMS microbiology letters, 2003, 218(1): 79-84.
[12] Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: Twenty-fourth informational supplement, M100-S24[J]. Clinical and Laboratory Standards Institute , 2014, 34(1):120-129.
[13] GURUNG M, TAMANG M D, MOON D C, et al. Molecular basis of resistance to selected antimicrobial agents in the emerging zoonotic pathogen Streptococcus suis[J]. Journal of Clinical Microbiology, 2015, 53(7): 2332-2336.
[14] SUTCLIFFE J, GREBE T, TAIT-KAMRADT A, et al. Detection of erythromycin-resistant determinants by PCR[J]. Antimicrobial Agents and Chemotherapy, 1996, 40(11): 2562-2566.
[15] 陈惠玲, 邓家德, 叶惠芬, 等. 侵入性感染 B 族链球菌对红霉素及四环素的耐药基因检测[J]. 中华医院感染学杂志, 2010,20 (10): 1354-1357.
[16] BOJARSKA A, MOLSKA E, JANAS K, et al. Streptococcus suis in invasive human infections in Poland: clonality and determinants of virulence and antimicrobial resistance[J]. European Journal of Clinical Microbiology & Infectious Diseases, 2016, 35(6): 917-925.
[17] 刘丽英. 大环内酯类抗生素的临床研究进展[J]. 兽药与饲料添加剂, 2001, 6(6):13-15.
[18] ZHANG C, NING Y, ZHANG Z, et al. In vitro antimicrobial susceptibility of Streptococcus suis strains isolated from clinically healthy sows in China[J]. Veterinary Microbiology, 2008, 131(3/4): 386-392.
[19] WEI Z, LI R, ZHANG A, et al. Characterization of Streptococcus suis isolates from the diseased pigs in China between 2003 and 2007[J]. Veterinary Microbiology, 2009, 137(1/2): 196-201.
[20] 应薇芳, 赵焕灿, 钱娅, 等. 浙江部分地区屠宰场待宰猪链球菌的检测及药敏试验[J]. 动物医学进展, 2007, 28(6): 24-27.
[21] 张卓然, 夏梦岩, 倪语星. 微生物耐药的基础与临床[J].北京: 人民卫生出版社, 2007.
[22] LóPEZ A C, ORT. Tetracycline and oxytetracycline resistance determinants detected in Bacillus cereus strains isolated from honey samples[J]. Rev Arg Microbiol, 2009, 40(4): 231-237.
[23] 汪华, 胡晓抒,朱凤才,等. 人—猪链球菌感染性综合征的流行病学调查[J]. 现代预防医学, 2000, 27(3): 312-314.
[24] 张桂枝, 罗永煌. 大环内酯类抗生素研究进展[J]. 动物医学进展, 2004, 25(3):33-36.
[25] 刘耀川. 细菌对大环内酯类药物耐药机制简介[J]. 现代畜牧兽医, 2012(10):52-53.