摘要
【目的】调查产生生物被膜的金黄色葡萄球菌流行病学特征,探究生物被膜形成能力与分子型之间的相关性,为治疗动物金黄色葡萄球菌感染提供理论依据。【方法】以广东省兽药研制与安全评价重点实验室保存的98株金黄色葡萄球菌为研究对象,用结晶紫半定量法测定所有菌株生物被膜形成能力,将产膜菌株进行22种常见抗菌药的最小抑菌浓度测定,通过金黄色葡萄球菌常见3种(spa、MLST和PFGE)分型方式进行分子分型,并分析产膜能力与分子型之间的相关性,最后通过全基因组测序技术分析生物被膜产生菌株中耐药基因和毒力基因携带状况。【结果】结晶紫半定量结果显示,98株金黄色葡萄球菌中能产生生物被膜的菌株共计23株,占23.47%。包括牛乳源22株(占比25.29%,22/87),其中14株(占比60.87%,14/22)来自浙江奶牛养殖场,8株(占比39.13%,8/22)来自福建奶牛养殖场,以及猪源1株(占比9.10%,1/11),来自广东屠宰场,说明牛乳源金黄色葡萄球菌产膜潜力高于猪源;将产膜能力划分为强、中、弱3个等级,23株产膜菌株中,其中强产膜菌株2株(占比8.70%,2/23),中产膜菌株9株(占比39.13%,9/23),以及弱产膜菌株12株(占比52.17%,12/23)。药敏试验显示,牛乳源产膜菌株对所有受试抗菌药物敏感,而猪源产膜菌株对青霉素、阿莫西林、头孢噻呋、头孢西丁、恩诺沙星、环丙沙星、克林霉素、多西环素、红霉素、氟苯尼考、复方新诺明、泰妙菌素、替米考星13种抗菌药均表现出耐药;spa分型结果显示,98株金黄色葡萄球菌共获得8种spa型,产膜的23株金黄色葡萄球菌占其中3种:1株t2922型来自广东猪源,14株t2119来自浙江牛乳源,8株t189来自福建牛乳源;MLST分型结果显示,98株菌共分为9种ST型,其中6种ST型不具有产生生物被膜的能力,分别为ST398、ST522、ST705、ST1651、ST479和ST151,仅3种ST型的菌株具有生物被膜产生能力,分别为ST9、ST7和ST188。结果表明,强产膜菌株分子型主要为ST7-t2119,中产膜菌株主要为ST7-t2119和ST188-t189,弱产膜菌株为ST9-t2922、ST7-t2119和ST188-t189;同时结合PCA分析不同ST型组间差异性发现,弱产膜菌株的ST型能与中强产膜菌株很好的区分开来,只有特定ST型的金黄色葡萄球菌才具有产生生物被膜的能力。23株产膜菌株PFGE分型全部成功,结果显示,广东、福建、浙江三省产膜菌株共分为3种PFGE型,且PFGE型存在地域分布特征,同一地区的分离株可能存在克隆传播,但克隆株之间生物被膜产生能力有明显差异;全基因组测序结果显示,产膜菌株中耐药基因和毒力基因根据分子型不同携带情况呈现多样性。【结论】不同来源的金黄色葡萄球菌有不同程度产生生物被膜的潜力,且均携带多种不同的产膜基因,牛乳源金黄色葡萄球菌产膜潜力远远高于猪源;菌株能否产膜与ST型存在强相关性,特定的ST型如ST9、ST7和ST188更容易产生生物被膜。
【Objective】The aim of this study was to investigate the epidemiological characteristics of Staphylococcus aureus(S.aureus)in the biofilm producing strains and to explore the correlation between biofilm forming ability and molecular typing,so as to provide the theoretical basis for the treatment of S.aureus infection.【Method】The biofilm producing ability of all strains of S.aureus was determined by crystal violet semi-quantitative method.The minimum inhibitory concentrations of 22 common antibiotics were determined by the membrane producing strains.Molecular typing was conducted by three common typing methods of S.aureus,including spa typing,MLST typing and PFGE typing,and the correlation between membrane production capacity and molecular typing was analyzed.Finally,whole genome sequencing technology was used to analyze the antibiotics resistance gene and virulence genes in the biofilm producing strains.【Result】The semi-quantitative results of crystalline violet showed that a total of 23 strains(23.47%)of 98 S.aureus strains were able to produce biofilm,including 22 strains(25.29%,22/87)from cow milk source,14 strains(60.87%,14/22)from Zhejiang dairy farms,8 strains(39.13%,8/22)from Fujian dairy farms,and 1 strain from pig source(9.10%,1/11)from Guangdong slaughterhouse,indicating that the film-producing potential of S.aureus from cow's milk source was higher than that of pig source,22 strains(95.65%)of which were from cow's milk source and 1 strain(4.35%)was from pig source.The film-producing ability was classified into strong,medium and weak,and among the 23 film-producing strains,2 strains(8.70%,2/23)were strong film-producing strains,9 strains(39.13%,9/23)were medium,and 12 strains(52.17%,12/23)were weak.The results of the drug sensitivity test showed that the bovine milk-derived membrane-producing strains were sensitive to all the tested antibacterial drugs,while the pig-derived membrane-producing strains showed resistance to 13 antibacterial drugs,including penicillin,amoxicillin,ceftiofur,cefoxitin,enrofloxacin,ciprofloxacin,clindamycin,doxycycline,erythromycin,flupenthixol,cotrimoxazole,tiamulin,and tilmicosin.The spa typing results showed that 98 strains of S.aureus obtained 8 spa types,and 23 strains of film-producing S.aureus accounted for 3 of them:1 strain t2922 from porcine origin in Guangdong,14 strains t2119 from Zhejiang cow milk source,and 8 strains t189 from Fujian cow milk source.MLST typing results showed that 98 strains were classified into 9 ST types,of which 6 ST types did not have the ability to produce biofilm,namely ST398,ST522,ST705,ST1651,ST479 and ST151,and only 3 strains of ST type had biofilm production ability,namely ST9,ST7 and ST188.It was found that the molecular types of strong film-producing strains were mainly ST7-t2119,the medium film-producing strains were mainly ST7-t2119 and ST188-t189,and the weak film-producing strains were ST9-t2922,ST7-t2119 and ST188-t189.The ST type of weak film-producing strains could be well distinguished from the medium and strong film-producing strains,and only the specific ST type of S.aureus had the ability to produce biofilm.23 film-producing strains PFGE typing all successful PFGE typing results showed that the results show that each strain of film-producing bacteria in Guangdong,Fujian and Zhejiang provinces were divided into 3 PFGE types,and there were geographical distribution characteristics of PFGE types;the strains isolating from the same region had clonal transmission,and the strains in the province were clonal to each other,but there were significant differences in biofilm production ability between clones.;the whole genome sequencing results showed that the drug resistance genes and virulence genes in the film-producing strains were diverse according to the molecular type.【Conclusion】S.aureus from different sources had different potential to produce biofilm and all carried different film-producing genes.The film-producing potential of S.aureus from bovine milk source was much higher than that of porcine source,and all carrid different film-producing genes.Whether strains could produce film or not may be strongly correlated with ST type,and the specific ST types,such as ST9,ST7 and ST188,were more likely to produce biofilm;however,at the same time,the strains with the same molecular type had different abilities to produce biofilm.
作者
唐子云
胡健欣
陈进
陆毅兴
孔伶俐
刁露
张发福
熊文广
曾振灵
TANG ZiYun;HU JianXin;CHEN Jin;LU YiXing;KONG LingLi;DIAO Lu;ZHANG FaFu;XIONG WenGuang;ZENG ZhenLing(College of Veterinary Medicine,South China Agricultural University/Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation/Guangdong Laboratory for Lingnan Modern Agriculture/National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria,Guangzhou 510642)
出处
《中国农业科学》
CAS
CSCD
北大核心
2022年第3期602-612,共11页
Scientia Agricultura Sinica
基金
“十三五”国家重点研发计划(2018YFD0500300)。
关键词
金黄色葡萄球菌
生物被膜
耐药性
分子分型
毒力基因
Staphylococcus aureus
biofilm
antibiotics resistance gene
molecular typing
virulence genes