基于不同ST型肺炎克雷伯菌感染的小鼠乳腺炎模型的差异分析

doi:10.16431/j.cnki.1671-7236.2025.01.038

摘要/Abstract

摘要： 【目的】肺炎克雷伯菌感染是奶牛乳腺炎的常见病因,其诱导的临床型乳腺炎发病急、临床症状重,牛奶产量和质量降低,给奶牛场造成巨大的经济损失。本研究旨在探究奶牛乳腺炎源性不同序列型(sequence type,ST)肺炎克雷伯菌的毒力作用,筛选肺炎克雷伯菌发挥感染作用的潜在关键毒力基因。【方法】采用多位点序列分型(multi-locus sequence typing,MLST)分析山东、湖北2个大型奶牛场的129株临床乳腺炎源性肺炎克雷伯菌的ST型分布,并选择3种检出率较高的ST型肺炎克雷伯菌感染小鼠建立乳腺炎模型,探究小鼠乳腺的形态学损伤及炎性因子表达变化。通过分析肺炎克雷伯菌的感染特性与毒力基因分布,筛选与肺炎克雷伯菌感染相关的关键毒力基因。【结果】 129株肺炎克雷伯菌共分为79种ST型,其中ST107占比最高,为19.37%;其次为ST2324(7.75%)和ST13(3.87%)。与ST107和ST13型相比,感染后24 h,ST2324型肺炎克雷伯菌诱导小鼠临床评分和乳腺载菌量均显著升高(P＜0.05);乳腺内腺泡轮廓消失和大量炎性细胞浸润,乳腺中白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)和细胞色素C(Cyt C)) mRNA表达量均显著升高(P＜0.05)。毒力基因检测结果显示,3种不同ST型分离株共检测到74个毒力基因,其中ST2324型分离株中检出了ST13和ST107型分离株没有检出的毒力基因manB、pla和pilW。【结论】本研究从129株肺炎克雷伯菌中共检出79种ST型,其中ST2324、ST107和ST13检出率较高,ST2324型肺炎克雷伯菌对小鼠致病性最强,且携带的毒力基因manB、pla和pilW与肺炎克雷伯菌的高致病性密切相关。本研究结果为进一步解析肺炎克雷伯菌感染奶牛乳腺炎的机制提供了理论参考。

关键词: 奶牛乳腺炎; 肺炎克雷伯菌; 序列型(ST); 毒力基因

Abstract: 【Objective】 Klebsiella pneumoniae infection is a common cause of mastitis in dairy cows,which induces acute clinical mastitis with severe clinical symptoms,resulting in reduced milk production and quality,thereby causing significant economic losses to dairy farms.The purpose of this study was to explore the virulence effects of different sequence types (ST) of Klebsiella pneumoniae originating from cow mastitis,and screen for potential key virulence genes that contribute to the infectious role of Klebsiella pneumoniae. 【Method】 The multi-locus sequence typing (MLST) method was employed to analyze the distribution of ST among 129 strains of Klebsiella pneumoniae isolated from clinical mastitis cases in two large dairy farms located in Shandong and Hubei provinces.Subsequently,3 of the most prevalent ST of Klebsiella pneumoniae were selected to establish mastitis model in mice,and the morphological damage and expression changes of inflammatory factors in mammary glands of mice were investigated.The key virulence genes associated with Klebsiella pneumoniae infection were screened by analyzing the infection characteristics and virulence gene distribution of Klebsiella pneumoniae. 【Result】 129 strains of Klebsiella pneumoniae were categorized into 79 ST types,of which ST107 accounted for the highest proportion of 19.37%,followed by ST2324 (7.75%) and ST13 (3.87%).Compared with ST107 and ST13,ST2324 induced Klebsiella pneumoniae clinical score and mammary gland bacteria load were significantly increased 24 h after infection (P ＜ 0.05).The acinar contour disappeared and a large number of inflammatory cells infiltrated,and the mRNA expressions of interleukin-1β (IL-1β),tumor necrosis factor-α (TNF-α),and cytochrome C (Cyt C) were significantly increased (P ＜ 0.05).Virulence gene detection results showed that 74 virulence genes were detected in 3 different ST isolates,among which the virulence genes manB, pla and pilW were detected in ST2324 but not in ST13 and ST107. 【Conclusion】 In this study,a total of 79 ST types were detected from 129 strains of Klebsiella pneumoniae,among which ST2324,ST107 and ST13 were detected with high rates,and ST2324 had the strongest pathogenicity to mice,and the virulence genes manB,pla and pilW were closely related to the high pathogenicity of Klebsiella pneumoniae.The results of this study provided a theoretical reference for further analysis of the mechanism of Klebsiella pneumoniae infection of dairy cow mastitis.

Key words: dairy cow mastitis; Klebsiella pneumoniae; sequence type (ST); virulence gene

中图分类号:

S858.292

任美奕, 梁洪秀, 李璨, 冯鸣鹊, 谢琴娜, 宋德源, 刘明超, 高健, 程佳. 基于不同ST型肺炎克雷伯菌感染的小鼠乳腺炎模型的差异分析[J]. 中国畜牧兽医, 2025, 52(1): 422-431.

REN Meiyi, LIANG Hongxiu, LI Can, FENG Mingque, XIE Qinna, SONG Deyuan, LIU Mingchao, GAO Jian, CHENG Jia. Differential Analysis of Mastitis Model in Mice Infected with Klebsiella pneumoniae of Different ST Types[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(1): 422-431.

参考文献

[1] 张辉殷,王丽娟.奶牛乳腺炎的病因与治疗[J].中国畜牧业,2023,22:115-116. ZHANG H Y,WANG L J.Etiology and treatment of mastitis in dairy cows[J] China Animal Industry,2023,22:115-116.(in Chinese)
[2] GAO J,BARKEMA H W,ZHANG L,et al.Incidence of clinical mastitis and distribution of pathogens on large Chinese dairy farms[J].Journal of Dairy Science,2017,100(6):4797-4806.
[3] CHENG J,QU W,BAEKEMA H W,et al.Antimicrobial resistance profiles of 5 common bovine mastitis pathogens in large Chinese dairy herds[J].Journal of Dairy Science,2019,102(3):2416-2426.
[4] SUGIYAMA M,WATANABE M,SONOBE T,et al.Efficacy of antimicrobial therapy for bovine acute Klebsiella pneumoniae mastitis[J].Journal of Veterinary Medical Science,2022,84(7):1023-1028.
[5] TOMAZI T,TOMAZI A C C H,SILVA J C C,et al.Immunization with a novel recombinant protein (YidR) reduced the risk of clinical mastitis caused by Klebsiella spp.and decreased milk losses and culling risk after Escherichia coli infections[J]. Journal of Dairy Science,2021,104(4):4787-4802.
[6] SCHUKKEN Y,CHUFF M,MORONI P,et al.The "other" Gram-negative bacteria in mastitis:Klebsiella,serratia,and more[J].Veterinary Clinics of North America,2012,28(2):239-256.
[7] GRÖHN Y T,WILSON D J,GONZÁLEZ R N,et al.Effect of pathogen-specific clinical mastitis on milk yield in dairy cows[J]. Journal of Veterinary Medical Science,2004,87(10):3358-3374.
[8] CHENG J,ZHANG J,YANG J,et al.Klebsiella pneumoniae infection causes mitochondrial damage and dysfunction in bovine mammary epithelial cells[J].Veterinary Research,2021,52(1):17.
[9] CHENG J,ZHANG J,HAN B,et al.Klebsiella pneumoniae isolated from bovine mastitis is cytopathogenic for bovine mammary epithelial cells[J]. Journal of Veterinary Medical Science,2020,103(4):3493-3504.
[10] 马晓敏.肺炎克雷伯菌诱导奶牛乳腺上皮细胞损伤机制及硒的保护作用研究[D].扬州:扬州大学,2023. MA X M.Study on the mechanism of breast epithelial cell injury induced by Klebsiella pneumoniae and the protective effect of selenium in dairy cows[D].Yangzhou:Yangzhou University,2023.(in Chinese)
[11] 席健峰,刘效伊,姜良勇,等.高毒力肺炎克雷伯菌的研究进展[J].山东化工,2019,48(24):54-56. XI J F,LIU X Y,JIANG L Y,et al.Research progress of Klebsiella pneumoniae with high virulence[J].Shandong Chemical Industry,2019,48(24):54-56.(in Chinese)
[12] KO K S.The contribution of capsule polysaccharide genes to virulence of Klebsiella pneumoniae[J]. Virulence,2017,5:485-486.
[13] KRZY AZ EWSKA-DUDEK E,DULIPATI V,KAPCZY ANŹSKA K,et al.Lipopolysaccharide with long O-antigen is crucial for Salmonella Enteritidis to evade complement activity and to facilitate bacterial survival in vivo in the Galleria mellonella infection model[J].Medical Microbiology and Immunology,2024,213(1):8.
[14] PACZOSA M K,MECSAS J.Klebsiella pneumoniae:Going on the offense with a strong defense[J].Microbiology and Molecular Biology Reviews,2016,80(3):629-661.
[15] WANG G,ZHAO G,CHAO X,et al.The characteristic of virulence,biofilm and antibiotic resistance of Klebsiella pneumoniae[J].International Journal of Environmental Research and Public Health,2020,17(17):6278.
[16] BACHMAN M A,LENIO S,SCHMIDT L,et al.Interaction of lipocalin 2,transferrin,and siderophores determines the replicative niche of Klebsiella pneumoniae during pneumonia[J].mBio,2012,3(6):e00224-11.
[17] 赵杭,何知恩,张慧敏,等.高毒力肺炎克雷伯菌临床感染特征及毒力因子研究进展[J].国外医药(抗生素分册),2023,44(2):83-90. ZHAO H,HE Z E,ZHANG H M,et al.Research progress on clinical infection characteristics and virulence factors of highly virulent Klebsiella pneumoniae[J].World Notes on Antibiotics,2023,44(2):83-90.(in Chinese)
[18] KRISHNAMOORTHY P,SATYANARAYANA M L,SHOME B R,et al.Intramammary infection with different Staphylococcus species in mice[J].Journal of Microscopy and Ultrastructure,2018,6(2):93-98.
[19] CANI P D,AMAR J,IGLESIAS M A,et al.Metabolic endotoxemia initiates obesity and insulin resistance[J]. Diabetes,2007,56(7):1761-1772.
[20] SAMPSON T R,CHALLIS C,JAIN N,et al.A gut bacterial amyloid promotes α-synuclein aggregation and motor impairment in mice[J]. eLife,2020,9:e53111.
[21] TU P C,PAN Y L,LIANG Z Q,et al.Mechanical stretch promotes macrophage polarization and inflammation via the RhoA-ROCK/NF-κB pathway[J]. BioMed Research International,2022,2022:6871269.
[22] SUN M Z,ZHANG Y J,SONG Y F,et al.Electroacupuncture alleviates water avoidance stress-induced irritable bowel syndrome in mice by improving intestinal barrier functions and suppressing the expression of inflammatory cytokines[J]. Journal of Traditional Chinese Medicine,2023,43(3):494-500.
[23] HUANG S,DONG S,LIN L,et al.Inulin ameliorates metabolic syndrome in high-fat diet-fed mice by regulating gut microbiota and bile acid excretion[J].Frontiers in Pharmacology,2023,14:1226448.
[24] DENG C,LI J,LI L,et al.Effects of hypoxia ischemia on Caspase-3 expression and neuronal apoptosis in the brain of neonatal mice[J].Experimental and Therapeutic Medicine,2019,17(6):4517-4521.
[25] ALMEIDA F,FERREIRA I L,NAIA L,et al.Mitochondrial dysfunction and decreased cytochrome C in cell and animal models of Machado-Joseph disease[J].Cells,2023,12(19):2397.
[26] CARLEY E,STEWART R M,ZIEMAN A,et al.The LINC complex transmits integrin-dependent tension to the nuclear lamina and represses epidermal differentiation[J]. eLife,2021,10:e58541.
[27] 盛席静,张凡,刘方佳,等.动物源肺炎克雷伯菌致病性与耐药性研究进展[J].中国畜牧兽医,2023,50(7):2966-2973. SHENG X J,ZHANG F,LIU F J,et al.Research progress on pathogenicity and drug resistance of Klebsiella pneumoniae of animal origin[J].China Animal Husbandry & Veterinary,2023,50(7):2966-2973.(in Chinese)
[28] ZHENG Z,GORDEN P J,XIA X,et al.Whole-genome analysis of Klebsiella pneumoniae from bovine mastitis milk in the U.S[J]. Applied and Environmental Microbiology,2022,24(3):1183-1199.
[29] FU S,WEN C,WANG Z,et al.Molecular epidemiology and antimicrobial resistance of outbreaks of Klebsiella pneumoniae clinical mastitis in Chinese dairy farms[J].Microbiology Spectrum,2022,10(6):e0299722.
[30] 朱珂萱.四种主要ST型猪源沙门菌的小鼠致病性研究[D].武汉:华中农业大学,2023. ZHU K X.Pathogenic study of four major sequence types of porcine-derived Salmonella species in mice[D].Wuhan:Huazhong Agricultural University,2023.(in Chinese)
[31] TAJ A,JIA L,SHA S,et al.Functional analysis and enzyme characterization of mannose-L-phosphate guanylyl transferase (ManB) from Mycobacterium tuberculosis[J].Microbiological Research,2022,173(1-2):103884.
[32] SUOMALAINEN M,HAIKO J,RAMU P,et al.Using every trick in the book:The Pla surface protease of Yersinia pestis[J]. Advances in Experimental Medicine and Biology,2007,603:268-278.
[33] CARBONNELLE E,HÉLAINE S,PROUVENSIER L,et al.Type Ⅳ pilus biogenesis in Neisseria meningitidis:PilW is involved in a step occurring after pilus assembly,essential for fibre stability and function[J].Molecular Microbiology,2005,55(1):54-64.