生鲜牛乳中金黄色葡萄球菌的鉴定及耐药分子特征研究

Identification of Staphylococcus aureus from raw milk and study on the molecular characteristics of antimicrobial resistance

为了解生鲜牛乳中金黄色葡萄球菌(S.aureus)的污染及耐药情况,研究其耐药的分子特征,本研究利用国标法分离了来自浙江杭州市某奶牛场99份生鲜乳中的S.aureus,通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行鉴定,对其进行质谱聚类分析和16S r DNA系统发育分析,采用微量肉汤稀释法测定其最小抑菌浓度(MIC),利用Illlumina Hi Seq 4000平台对其进行全基因组测序,通过Res Finder数据库分析其耐药基因,利用EasyFig和BRIG软件进行耐药质粒比对分析。结果显示在99份生鲜牛乳中共分离到5株S.aureus (5.1%),其质谱聚类和16S r DNA系统发育分析树形具有较高一致性。发现两株耐甲氧西林S.aureus (MRSA) SABHZ053和SABHZ079,且其多位点序列分型(MLST)同为ST965,其中菌株SABHZ079对青霉素、红霉素、克林霉素、头孢西丁、氧氟沙星、替米考星和庆大霉素7种抗生素耐药。全基因组测序分析后显示菌株SABHZ053和SABHZ079的染色体中均含有aph(3’)-III、ant(6)-Ia和mecA耐药基因。菌株SABHZ053存在2个耐药质粒,pSA053-1 (4 439 bp)介导tet(K)对四环素类耐药,pSA053-2 (34 609 bp)介导blaZ对青霉素耐药。菌株SABHZ079有3个耐药质粒,其中pSABHZ079-1 (39 227 bp)与pSA053-2同源,但相对插入了Tn551-erm(B)-aph(2’’)-Ia转座单元,可以介导其对大环内脂类和庆大霉素的耐药,质粒pSABHZ079-2 (2 473 bp)介导erm(C)对大环内脂类耐药,而质粒pSABHZ079-3(4 439 bp)与pSA053-1完全相同。另外,本研究显示耐药质粒pSABHZ079-1可分为骨架I和II两部分,可能分别由来自人源的pNTUH3874与pN315或其相似质粒杂合而来。本研究揭示了该奶牛场生鲜乳中S.aureus耐药性的分子特征,表明耐药质粒和转座元件是其耐药性产生的重要因子,为奶牛场合理用药和控制S.aureus的抗药性提供重要参考。

To investigate the distribution and antimicrobial resistance of Staphylococcus aureus in raw milk of a dairy farm in Hangzhou,and the molecular characteristics of antimicrobial resistance,S.aureus was isolated based on National Standard of the People's Republic of China and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF MS).Then,cluster analysis of MALDI-TOF MS and phylogenetic analysis of 16S rDNA were carried out.Additionally,minimum inhibitory concentration(MIC)of S.aureus was determined using the broth micro dilution methods.The whole genome sequences were performed using the Illumina HiSeq 4000 sequencing platform.Antimicrobial resistant genes were analyzed by ResFinder while comparative analysis of plasmids was conducted via EasyFig and BRIG softwares.The results showed that five S.aureus isolates(5.1%)were isolated from 99 raw milk samples.The results of MALDI-TOF MS and 16S rDNA were in good agreement.The cluster analysis and phylogenetic analysis showed high consistency.Two methicillin-resistant Staphylococcus aureus(MRSA)strains(SABHZ053 and SABHZ079)with the same ST965 type were identified.Strain SABHZ079 was resistant to seven antibiotics including penicillin,erythromycin,clindamycin,cefoxitin,ofloxacin,gentamycin and tilmicosin.The aph(3')-III,ant(6)-Ia and mecA genes were found in the chromosomes of both strains SABHZ053 and SABHZ079 according to the whole genome sequencing.The SABHZ053 strain contained tet(K)-carrying plasmid pSA053-1(4,439bp)and blaZ-carrying plamid pSA053-2(34,609bp).The SABHZ079 stain harbored three plasmids including pSABHZ079-1(39,227bp),pSABHZ079-2(2,473bp)and pSABHZ079-3(4,439bp).Comparative analysis showed that pSABHZ079-1 was homologous to pSABHZ053-2 except for the insertion of Tn551-erm(B)-aph(2'')-Ia encoding macrolides and gentamicin,but pSABHZ079-2 carried another macrolides gene erm(C),and the third plasmid pSABHZ079-3 was identical to pSA053-1 completely.Further analysis showed that pSABHZ079-1 could be divided into two parts,backbone I and II,which might be derived from the hybrid of pNTUH_3874 and pN315 or their similar plasmids from human sources,respectively.This study revealed the molecular characteristics of antimicrobial resistance of S.aureus in raw milk of the dairy farm indicating that drug-resistant plasmids and transposable elements were important factors for antimicrobial resistance.It would provide important references for rationale drugs use and the control of antimicrobial resistance in dairy cows.