1株马乳源PVL+ST22型金黄色葡萄球菌致病性分析

doi:10.11843/j.issn.0366-6964.2024.02.029

摘要/Abstract

摘要： 国内鲜有马乳源PVL⁺ST22型金黄色葡萄球菌(Staphylococcus aureus)相关报道,本研究对1株新疆马乳源PVL⁺ST22型金黄色葡萄球菌的致病性进行评估,以期为马乳源金黄色葡萄球菌的相关研究提供基础数据。使用选择性培养基从新疆健康马乳中分离金黄色葡萄球菌,然后通过生化鉴定和分子鉴定方法对金黄色葡萄球菌进行鉴定。对所获得的金黄色葡萄球菌进行多位点序列分型(multilocus sequence typing,MLST)、spa分型、药物敏感性检测、生物被膜形成能力、溶血活性、毒力基因检测、小鼠皮肤脓肿和致病性试验。再对分离株进行小鼠细胞的黏附、侵袭和胞内增殖试验,以评估其致病性。结果显示,从马乳中分离得到1株PVL⁺ST22型金黄色葡萄球菌(1/65,1.5%),spa分型为t304。仅对青霉素耐药,无生物被膜形成能力,但携带9种毒力基因(sec、seg、seh、tsst-1、hla、hlb、clfA、clfB和cna),并且具有溶血活性。小鼠皮肤脓肿和致病性试验表明,其可导致小鼠形成皮肤脓肿,并且在高剂量感染时可导致小鼠死亡。ST22型金黄色葡萄球菌可在小鼠巨噬细胞中黏附(4.26%)、侵袭(6.45%)和胞内增殖,并可造成细胞凋亡。综上,马乳源PVL⁺ST22型金黄色葡萄球菌具有较强的致病力,可能在宿主致病和公共卫生安全等方面存在隐患。

关键词: 马乳, PVL⁺, ST22型金黄色葡萄球菌, 致病性

Abstract: There are few reports on PVL⁺ ST22 Staphylococcus aureus (S. aureus) isolated from equine milk in China. This study was conducted to evaluate the pathogenicity of an S. aureus (PVL⁺ ST22) isolated from equine milk in Xinjiang, hoping to provide basic data for the study of S. aureus from equine milk in China. S. aureus were isolated from apparently healthy equine milk in Xinjiang using the selective medium and then identified by biochemical and molecular identification methods. The obtained S. aureus were subjected to multilocus sequence typing (MLST), spa typing, antimicrobial susceptibility testing, and biofilm-forming ability, hemolytic activity, virulence gene detection, along with mouse skin abscess and pathogenicity assay. The isolated strain was then subjected to mouse macrophage adhesion, invasion, and intracellular proliferation assays to assess their pathogenicity. The results indicated that a PVL⁺ ST22 S. aureus (1/65, 1.5%) strain was isolated from equine milk, with spa typing t304. It was resistant to penicillin only, had no biofilm-forming ability, but carried 9 virulence genes (sec, seg, seh, tsst-1, hla, hlb, clfA, clfB, and cna ) and has hemolytic activity. Mouse skin abscess and pathogenicity tests also showed that it caused skin abscess formation and death in mice at high doses of infection. ST22 S. aureus has adhered (4.26%), invaded (6.45%), and proliferated intracellularly in mouse macrophages and caused apoptosis. In conclusion, the PVL⁺ ST22 S. aureus isolated from equine milk is highly pathogenic and may pose potential risks in terms of host pathogenicity and public health safety.

Key words: equine milk, PVL⁺, ST22 Staphylococcus aureus, pathogenicity

中图分类号:

吴自豪, 蔡依龙, 陀海欣, 陈伟. 1株马乳源PVL⁺ST22型金黄色葡萄球菌致病性分析[J]. 畜牧兽医学报, 2024, 55(2): 718-726.

WU Zihao, CAI Yilong, TUO Haixin, CHEN Wei. Pathogenicity Analysis of a PVL⁺ ST22 Staphylococcus aureus Isolated from Equine Raw Milk[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(2): 718-726.

参考文献

[1] SMITH T C,PEARSON N. The emergence of Staphylococcus aureus ST398[J].Vector Borne Zoonotic Dis,2011,11(4):327-339.
[2] JIAN Y,ZHAO L,ZHAO N,et al. Increasing prevalence of hypervirulent ST5 methicillin susceptible Staphylococcus aureus subtype poses a serious clinical threat[J].Emerg Microbes Infect,2021,10(1):109-122.
[3] ZHOU W X,JIN Y,LIU X,et al. Comparison of genetic features and evolution of global and Chinese strains of community-associated methicillin-resistant Staphylococcus aureus ST22[J].Microbiol Spectr,2022,10(1):e02037-21.
[4] BÖCHER S,SKOV R L,KNUDSEN M A,et al. The search and destroy strategy prevents spread and long-term carriage of methicillin-resistant Staphylococcus aureus:results from the follow-up screening of a large ST22 (E-MRSA 15) outbreak in Denmark[J].Clin Microbiol Infect,2010,16(9):1427-1434.
[5] LIM K T,HANIFAH Y A,YUSOF M Y M,et al. Comparison of methicillin-resistant Staphylococcus aureus strains isolated in 2003 and 2008 with an emergence of multidrug resistant ST22:SCCmec IV clone in a tertiary hospital,Malaysia[J].J Microbiol Immunol Infect,2013,46(3):224-233.
[6] BUKOWSKI M,WLADYKA B,DUBIN G.Exfoliative toxins of Staphylococcus aureus[J].Toxins (Basel),2010,2(5):1148-1165.
[7] RÁJOVÁ J,PANTŮ AČG EK R,PETRÁŠ P,et al.Necrotizing pneumonia due to clonally diverse Staphylococcus aureus strains producing Panton-Valentine leukocidin:the Czech experience[J].Epidemiol Infect,2016,144(3):507-515.
[8] XIAO N,YANG J H,DUAN N,et al.Community-associated Staphylococcus aureus PVL⁺ ST22 predominates in skin and soft tissue infections in Beijing,China[J].Infect Drug Resist,2019,12:2495-2503.
[9] PINTO A N,SETH R,ZHOU F,et al.Emergence and control of an outbreak of infections due to Panton-Valentine leukocidin positive,ST22 methicillin-resistant Staphylococcus aureus in a neonatal intensive care unit[J].Clin Microbiol Infect,2013,19(7):620-627.
[10] PETINAKI E,SPILIOPOULOU I.Methicillin-resistant Staphylococcus aureus colonization and infection risks from companion animals:Current perspectives[J].Vet Med Res Rep,2015,6:373-382.
[11] CUNY C,ABDELBARY M M H,KÖCK R,et al.Methicillin-resistant Staphylococcus aureus from infections in horses in Germany are frequent colonizers of veterinarians but rare among MRSA from infections in humans[J].One Health,2016,2:11-17.
[12] GROVES M D,CROUCH B,COOMBS G W,et al.Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolated from Australian veterinarians[J].PLoS One,2016,11(1):e0146034.
[13] WALTHER B,MONECKE S,RUSCHER C,et al.Comparative molecular analysis substantiates zoonotic potential of equine methicillin-resistant Staphylococcus aureus[J].J Clin Microbiol,2009,47(3):704-710.
[14] WORTHING K A,ABRAHAM S,PANG S,et al.Molecular characterization of methicillin-resistant Staphylococcus aureus isolated from Australian animals and veterinarians[J].Microb Drug Resist,2018,24(2):203-212.
[15] 中华人民共和国农业部.NY/T 2962-2016 奶牛乳房炎乳汁中金黄色葡萄球菌、凝固酶阴性葡萄球菌、无乳链球菌分离鉴定方法[S].北京:中国农业出版社,2016.
Ministry of Agriculture of the PRC.NY/T 2962-2016 Methods for isolation and identification of Staphylococcus aureus,coagulase-negative Staphylococcus,and Streptococcus agalactis in milk from dairy cow with mastitis[S].Beijing:China Agriculture Press,2016.(in Chinese)
[16] LIAO G H,WU Z H,LV J F,et al.Investigation of clonal diversity,virulence genes,and antibiotic resistance of Staphylococcus aureus recovered from raw cow milk in southern Xinjiang,China[J].Folia Microbiol,2022,67(2):245-252.
[17] 吴自豪,吕俊凡,廖光华,等.南疆巴州地区奶牛隐性乳腺炎性金黄色葡萄球菌的MLST分型及其药物敏感性分析[J].塔里木大学学报,2020,32(2):10-16.
WU Z H,LV J F,LIAO G H,et al.Antimicrobial resistance features and MLST typing of Staphylococcus aureus isolates from subclinical bovine mastitis in Bayingol Mongolian Autonomous Prefecture of southern Xinjiang,China[J].Journal of Tarim University,2020,32(2):10-16.(in Chinese)
[18] 廖光华,代佳君,邓钰蓱,等.基于nuc基因的奶牛乳房炎性金黄色葡萄球菌PCR检测方法的建立[J].塔里木大学学报,2019,31(4):7-11.
LIAO G H,DAI J J,DENG Y P,et al.Identification of PCR amplification based on nuc gene for Staphylococcus aureus from raw milk[J].Journal of Tarim University,2019,31(4):7-11.(in Chinese)
[19] REN Q,LIAO G H,WU Z H,et al.Prevalence and[LL] characterization of Staphylococcus aureus isolates from subclinical bovine mastitis in southern Xinjiang,China[J].J Dairy Sci,2020,103(4):3368-3380.
[20] FANG B,LIU B H,SUN B L.Transcriptional regulation of virulence factors Hla and phenol-soluble modulins α by AraC-type regulator Rbf in Staphylococcus aureus[J].Int J Med Microbiol,2020,310(5):151436.
[21] 李华明,项维,卢文兵,等.1株猪源ST-35型肺炎克雷伯菌的致病性和药物敏感性分析[J].畜牧兽医学报,2022,53(12):4356-4366.
LI H M,XIANG W,LU W B,et al.Pathogenicity and drug sensitivity analysis of a porcine Klebsiella pneumoniae type ST-35[J].Acta Veterinaria et Zootechnica Sinica,2022,53(12):4356-4366.(in Chinese)
[22] 张立梅.奶牛乳房炎性金葡菌毒力基因和耐药性及其与SCVs致病力差异[D].北京:中国农业大学,2018.
ZHANG L M.Virulence and resistance characteristics of Staphylococcus aureus isolated from bovine mastitis and its pathogenic differences with SCVs[D].Beijing:China Agricultural University,2018.(in Chinese)
[23] TURNER N A,SHARMA-KUINKEL B K,MASKARINEC S A,et al.Methicillin-resistant Staphylococcus aureus:an overview of basic and clinical research[J].Nat Rev Microbiol,2019,17(4):203-218.
[24] ARGUDÍN M Á,MENDOZA M C,RODICIO M R.Food poisoning and Staphylococcus aureus enterotoxins[J].Toxins,2010,2(7):1751-1773.
[25] KOOSHA R Z,HOSSEINI H M,AGHDAM E M,et al.Distribution of tsst-1 and mecA genes in Staphylococcus aureus isolated from clinical specimens[J].Jundishapur J Microbiol,2016,9(3):e29057.
[26] CHEN Y,LIU Z X,DUO L B,et al.Characterization of Staphylococcus aureus from distinct geographic locations in China:an increasing prevalence of spa-t030 and SCCmec type III[J].PLoS One,2014,9(4):e96255.
[27] RUDKIN J K,EDWARDS A M,BOWDEN M G,et al.Methicillin resistance reduces the virulence of healthcare-associated methicillin-resistant Staphylococcus aureus by interfering with the agr quorum sensing system[J].J Infect Dis,2012,205(5):798-806.
[28] BALDAN R,TESTA F,LORÈ N I,et al.Factors contributing to epidemic MRSA clones replacement in a hospital setting[J].PLoS One,2012,7(8):e43153.