[1]张 玲,常 江,吴俐勤,等.生鲜牛乳中金黄色葡萄球菌的鉴定及耐药分子特征研究[J].中国预防兽医学报,2019,(09):899-905.[doi:0.3969/j.issn.1008-0589.201901014]
 ZHANG Ling,CHANG Jiang,WU Li-qin,et al.Identification of Staphylococcus aureus from raw milk and study on the molecular characteristics of antimicrobial resistance[J].Chinese journal of preventive veterinary medicine,2019,(09):899-905.[doi:0.3969/j.issn.1008-0589.201901014]





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

张 玲12常 江12吴俐勤2裘罕琦3周沁怡4戴贤君3杨 华2夏效东1唐 标2*

 (1. 西北农林科技大学 食品科学与工程学院,陕西 杨凌 712100;
2. 浙江省农业科学院 农产品质量标准研究所,浙江 杭州 310021;3. 中国计量大学 现代科技学院,浙江 杭州 310018;
4. 南京农业大学 动物医学院,江苏 南京 210095)

ZHANG Ling12 CHANG Jiang12 WU Li-qin2 QIU Han-qi3 ZHOU Qin-yi4 DAI Xian-jun3 YANG Hua2 XIA Xiao-dong1 TANG Biao2*


(1. College of Food Science and Engineering, Northwest Agriculture and Forestry University, Yangling 712100, China;
2. Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China;
3. College of Modern Science and Technology, China Jiliang University, Hangzhou 310018, China;
4. College of Veterinary Medicine, Nanjing Agriculture University, Nanjing 210095, China)

raw milk  Staphylococcus aureus  matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)  antimicrobial resistance plasmid
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 rDNA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 HiSeq 4000平台对其进行全基因组测序,通过ResFinder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 rDNA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pNTUH_3874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.


[1]Hennekinne J A, Buyser M L D, Dragacci S. Staphylococcus aureus and its food poisoning toxins: characterization and outbreak investigation [J]. Fems Microbiol Rev, 2012, 36(4): 815-836.
[2]Bessede E, Angla-Gre M, Delagarde Y, et al. Matrix-assisted laser-desorption/ionization biotyper: experience in the routine of a university hospital [J]. Clin Microbiol Infect, 2011, 17(4): 533-538.
[3]Clark A E, Kaleta E J, Arora A, et al. Matrix-assisted laser desorption ionization-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology [J]. Clin Microbiol Rev, 2013, 26(3): 547-603.
[4]van Veen S Q, Claas E C, Kuijper E J. High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories [J]. J Clin Microbiol, 2010, 48(3): 900-907.
[5]Clark C M, Costa M S, Sanchez L M, et al. Coupling MALDI-TOF mass spectrometry protein and specialized metabolite analyses to rapidly discriminate bacterial function [J]. PNAS, 2018, 115(19): 4981-4986.
[6]Krasny L, Rohlova E, Ruzickova H, et al. Differentiation of Cronobacter spp. by tryptic digestion of the cell suspension followed by MALDI-TOF MS analysis [J]. J Microbiol Methods, 2014, 98: 105-113.
[7]Lawton S J, Weis A M, Byrne B A, et al. Comparative analysis of Campylobacter isolates from wild birds and chickens using MALDI-TOF MS, biochemical testing, and DNA sequencing [J]. J Vet Diagn Invest, 2018, 30(3): 354-361.
[8]Giebel R, Worden C, Rust S M, et al. Microbial fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) applications and challenges [J]. Adv Appl Microbiol, 2010, 71: 149-184.
[9]Malachowa N, Deleo F R. Mobile genetic elements of Staphylococcus aureus [J]. Cell Mol Life Sci, 2010, 67(18): 3057-3071.
[10]Islam T, Kubra K, Hassan Chowdhury M M. Prevalence of methicillin-resistant Staphylococcus aureus in hospitals in Chittagong, Bangladesh: A threat of nosocomial infection [J]. J Microsc Ultrastruct, 2018, 6(4): 188-191.
[11]Schulz J, Boklund A, Toft N, et al. Drivers for livestock-associated methicillin-resistant Staphylococcus aureus spread among danish pig herds - a simulation study [J]. Sci Rep, 2018, 8(1): 16962.
[12]Kostrzewa M. Application of the MALDI Biotyper to clinical microbiology: progress and potential [J]. Expert Rev Proteomics, 2018, 15(3): 193-202.
[13]PA.2015. CLSI Document M100-S25. Performance standards for antimicrobial susceptibility testing; 25th Informational Supplement [S].
[14]Rizzotti L, Simeoni D, Cocconcelli P, et al. Contribution of enterococci to the spread of antibiotic resistance in the production chain of swine meat commodities [J]. J Food Prot, 2005, 68(5): 955-965.
[15]Caldwell S J, Huang Y, Berghuis A M. Antibiotic binding drives catalytic activation of aminoglycoside kinase APH(2’’)-Ia [J]. Structure, 2016, 24(6): 935-945.
[16]Costa S S, Palma C, Kadlec K, et al. Plasmid-borne antimicrobial resistance of Staphylococcus aureus isolated in a hospital in Lisbon, Portugal [J]. Microb Drug Resist, 2016, 22(8): 617-626.
[17]Ghanbari F, Ghajavand H, Havaei R, et al. Distribution of erm genes among Staphylococcus aureus isolates with inducible resistance to clindamycin in Isfahan, Iran [J]. Adv Biomed Res, 2016, 5: 62.
[18]Chavez-Bueno S, Bozdogan B, Katz K, et al. Inducible clindamycin resistance and molecular epidemiologic trends of pediatric community-acquired methicillin-resistant Staphylococcus aureus in Dallas, Texas [J]. Antimicrob Agents Chemother, 2005, 49(6): 2283-2288.
[19]Kumar D, Pornsukarom S, Sivaraman G K, et al. Environ!敐? mental dissemination of multidrug methicillin-resistant


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 ZHAO Yan,ZHANG Ai-rong,HAO Yong-qing.Cloning and expression of the D fragment of fibronectin-bindingprotein A gene from Staphylococcus aureus[J].Chinese journal of preventive veterinary medicine,2009,(09):361.
 ZHANG Bao-jun,CUI Jing-chun*,ZHANG Xian,et al.Development and evaluation of Staphylococcusinduced -induced mastitis model[J].Chinese journal of preventive veterinary medicine,2009,(09):365.
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 LIU Hong-gui,WU Rui*.Rapid detection of Staphylococcus aureus strains by immunochromatographic assay[J].Chinese journal of preventive veterinary medicine,2009,(09):283.
[4]张宝君,崔京春*,张 献,等.无毒突变肠毒素免疫对金黄色葡萄球菌乳腺感染的免疫效果研究 [J].中国预防兽医学报,2009,(07):557.
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[5]崔京春,张宝君,钱爱东,等.葡萄球菌肠毒素C基因的克隆表达及其生物学活性鉴定 [J].中国预防兽医学报,2009,(08):600.
 CUI Jing-chun,ZHANG Bao-jun,QIAN Ai-dong,et al.Cloning and expression of staphylococcal enterotoxin C[J].Chinese journal of preventive veterinary medicine,2009,(09):600.
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 LI Jian-ping,ZHOU Hai-jian,YUAN Lin,et al.Genotyping of Staphylococcus aureus isolated from bovine mastitis using pulsed-field gel electrophoresis [J].Chinese journal of preventive veterinary medicine,2009,(09):684.
 CHI Jia-qi,LI Run-ting,ZHU Zhan-bo,et al.PCR-based detection of genes encoding virulence determinants and capsular polysaccharides in Staphylococcus aureus from bovine subclinical mastitis cases [J].Chinese journal of preventive veterinary medicine,2009,(09):860.
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 DING Zhao-feng,TANG Wei,TONG Chun-yu,et al.Analysis of the tetracycline resistance of Staphylococcus aureus from cow mastitis[J].Chinese journal of preventive veterinary medicine,2010,(09):892.[doi:10.3969/j.issn.1008-0589.2010.10.15]
[10]任宪刚,朱战波*,崔 莉,等.不同凝固酶基因型牛源金黄色葡萄球菌主要致病因子的检测及分析[J].中国预防兽医学报,2008,(08):608.
 REN Xian-gang,ZHU Zhan-bo*,CUI Li,et al.Virulence factors of Staphylococcus aureus of different coagulase genotype[J].Chinese journal of preventive veterinary medicine,2008,(09):608.
 ZHANG Zhao-xing,LI Pei-gou*,LI Yun-yu,et al.Analyzing phylogenetic groups, drug resistance phenotypes, and drug resistance genotypes of Escherichia coli isolates causing salpingoperitonitis in layer chicken[J].Chinese journal of preventive veterinary medicine,2019,(09):885.[doi:10.3969/j.issn.1008-0589.201812012]

更新日期/Last Update: 2019-10-28