山东地区隐性乳房炎牛乳中β-内酰胺酶blaZ基因与微生物相关性研究

doi:10.16431/j.cnki.1671-7236.2025.05.035

摘要/Abstract

摘要： 【目的】探究山东地区隐性乳房炎牛乳中β-内酰胺酶blaZ基因与微生物的关系，为防控奶牛隐性乳房炎和减少生牛乳中的耐药基因提供理论依据。【方法】2023年2月－2023年6月，随机取山东省15市33个奶牛场478头奶牛乳478份，分别用PCR方法检测blaZ基因和15种微生物的基因。基于R语言的pheatmap函数绘制blaZ基因与微生物的相对丰度热图，并用R语言的glm函数进行Logistic回归拟合。选取山东省5市506头隐性乳房炎奶牛乳506份，重复上述方案。【结果】山东地区15市478份随机抽样牛乳中blaZ基因阳性率为19.25%，小型牧场blaZ基因阳性率高；牛乳中的凝固酶阴性葡萄球菌、酵母菌和肠球菌属相对丰度较高，且与blaZ基因呈正相关(P＜0.01；P＜0.05)；而blaZ基因仅与相对丰度不高的克雷伯菌属呈负相关(P＜0.05)。隐性乳房炎牛乳中blaZ基因阳性率为52.17%，牛乳中酵母菌、肠球菌属和凝固酶阴性葡萄球菌的相对丰度较高，blaZ基因仅与凝固酶阴性葡萄球菌呈正相关(P＜0.01)，且仅与乳房链球菌呈负相关(P＜0.01)。【结论】山东地区隐性乳房炎牛乳中blaZ基因表达量高于随机抽样牛乳，在山东地区的牛乳中，blaZ基因主要源自于凝固酶阴性葡萄球菌，引起该地区隐性乳房炎的主要致病菌也是该菌。

关键词: 奶牛隐性乳房炎; β-内酰胺酶blaZ基因; Logistic回归; 微生物丰度

Abstract: 【Objective】 This study aimed to explore the relationship between β-lactase blaZ gene and microorganisms in milk of cows with latent mastitis in Shandong region,and provide theoretical basis for preventing and controlling latent mastitis in cows and reduce antibiotic resistance genes in raw milk.【Method】 From February to June 2023,478 milk samples from 478 cows randomly selected from 33 dairy farms in 15 cities were tested for the β-lactase blaZ gene and 15 microbial genes using PCR method.Heatmap of the relative abundance of blaZ genes and microorganisms based on the pheatmap function in R language was drew,Logistic regression fitting was performed using the glm function in R language.The above scheme was repeated with 506 milk samples from 506 cows with latent mastitis in 5 cities of Shandong province.【Result】 The positive rate of blaZ gene in randomly obtained milk samples from 15 cities in Shandong province was 19.25%,with a high positive rate in small ranches.The relative abundance of coagulase-negative Staphylococcus,yeast and Enterococcus spp.in milk was relatively high,and they were positively correlated with the blaZ gene (P＜0.01 or P＜0.05).blaZ gene was only negatively correlated with the relatively low abundance of Klebsiella spp.(P＜0.05).The positive rate of blaZ gene in milk of cows with latent mastitis was 52.17%.The relative abundance of yeast,Enterococcus spp.and coagulase-negative Staphylococcus in milk was relatively high.The blaZ gene was only positively correlated with coagulase-negative Staphylococcus (P＜0.01) and negatively correlated with Streptococcus uberis (P＜0.01).【Conclusion】 The expression level of blaZ gene in milk with latent mastitis in Shandong region was higher than that in randomly sampled milk. In milk from Shandong region, blaZ gene mainly originated from coagulase negative Staphylococcus aureus, which was also the main pathogenic bacterium causing latent mastitis in the region.

Key words: latent mastitis in dairy cows; β-lactase blaZ gene; Logistic regression; microbial abundance

中图分类号:

S852.61

刘志旭, 韩文昌, 李彦芹, 张利文, 刘燕, 苑昭伟, 李建斌, 王晓, 曹荣峰. 山东地区隐性乳房炎牛乳中β-内酰胺酶blaZ基因与微生物相关性研究[J]. 中国畜牧兽医, 2025, 52(5): 2318-2327.

LIU Zhixu, HAN Wenchang, LI Yanqin, ZHANG Liwen, LIU Yan, YUAN Shaowei, LIJianbin, WANG Xiao, CAO Rongfeng. Study on the Correlation Between β-lactase blaZ Gene and Microorganisms in Milk of Cows with Latent Mastitis in Shandong Region[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(5): 2318-2327.

参考文献

[1] 岳婷婷,闫磊,闫跃飞,等.牛奶中β-内酰胺类药物检测研究进展[J].中国奶牛,2022,1:43-46.YUE T T,YAN L,YAN Y F,et al.Research progress on detection of β-lactam drugs in milk[J].China Dairy Cattle,2022,1:43-46.(in Chinese)
[2] 宋淑英.抚顺某奶牛场隐性乳房炎病原菌耐药性的调查研究[J].现代畜牧兽医,2022,3:56-59.SONG S Y.Investigation and research on antibiotics resistance of bacteria obtained from subclinical mastitis in a dairy farm in Fushun[J].Modern Journal of Animal Husbandry and Veterinary Medicine,2022,3:56-59.(in Chinese)
[3] SZTACHA AN＇G SKA M,BARA AN＇G SKI W,JANOWSKI T,et al.Prevalence and etiological agents of subclinical mastitis at the end of lactation in nine dairy herds in North-East Poland[J].Polish Journal of Veterinary Sciences,2016,19(1):119-124.
[4] TENHAGEN B A,KÖSTER G,WALLMANN J,et al.Prevalence of mastitis pathogens and their resistance against antimicrobial agents in dairy cows in Brandenburg,Germany[J].Journal of Dairy Research,2006,89(7):2542-2551.
[5] 孙垚,贾坤,平晓坤,等.广东省部分地区奶牛乳房炎凝固酶阴性葡萄球菌的毒力基因和耐药性分析[J].中国兽医学报,2017,37(8):1495-1500.SUN Y,JIA K,PING X K,et al.Analysis of virulence genes and antibiotics resistance of Staphylococci isolated from mastitis cows in parts of Guangdong province[J].Chinese Journal of Veterinary Science,2017,37(8):1495-1500.(in Chinese)
[6] 孙健,孙先枝,安朋朋,等.上海地区奶牛乳房炎致病菌基因诊断结果分析[J].中国奶牛,2018,2:28-31.SUN J,SUN X Z,AN P P,et al.Study on pathogenic bacteria gene diagnosis of cow mastitis in Shanghai area[J].China Dairy Cattle,2018,2:28-31.(in Chinese)
[7] 罗小凤,刘盼盼,陈晓慧,等.枸杞槲皮素对金黄色葡萄球菌产β-内酰胺酶的抑制作用[J].中国畜牧兽医,2024,51(1):382-391.LUO X F,LIU P P,CHEN X H,et al.Inhibitory effect of Lycium barbarum quercetin on β-lactamase from Staphylococcus aureus[J].China Animal Husbandry & Veterinary Medicine,2024,51(1):382-391.(in Chinese)
[8] ZHANG Z,CHEN Y,LI X,et al.Detection of antibiotic resistance,virulence gene,and drug resistance gene of Staphylococcus aureus isolates from bovine mastitis[J].Microbiology Spectrum,2022,31,10(4):e0047122.
[9] 张行.奶牛乳房炎凝固酶阴性葡萄球菌耐药性及其相关基因研究[D].北京：中国农业科学院,2020.ZHANG X.Antimicrobial resistance and associated genes of coagulase-negative Staphylococci isolated from bovine mastitis[D].Beijing:Chinese Academy of Agricultural Sciences, 2020. (in Chinese)
[10] 马强,杨蕊,万佳宏,等.宁夏地区牛源金黄色葡萄球菌β-内酰胺酶的分析及作用方式的研究[J].畜牧兽医学报,2020,51(5):1138-1148.MA Q,YANG R,WAN J H,et al.Study on β-lactamase and its action mode of Staphylococcus aureus isolated from cows in Ningxia[J].Acta Veterinaria et Zootechnica Sinica,2020,51(5):1138-1148. (in Chinese)
[11] WANG K,CHA J,LIU K,et al.The prevalence of bovine mastitis-associated Staphylococcus aureus in China and its antimicrobial resistance rate:A Meta-analysis[J].Frontiers in Veterinary Science,2022,9:1006676.
[12] SIPAHI N,KAYA E,ÇELIK C,et al.The characterization and β-lactam resistance of Staphylococcal community recovered from raw bovine milk[J].Nature Compounds as Antibiotics Agents,2023,12(3):556.
[13] 李托,李陇平,贺绥乐,等.奶牛乳房炎源葡萄球菌的分离鉴定及药敏试验[J].中国兽医杂志,2020,56(4):87-90.LI T,LI L P,HE S L,et al.Isolation,identification and drug resistance of Staphylococci from bovine mastitis of dairy cows[J].Chinese Journal of Veterinary Medicine, 2020,56(4):87-90.(in Chinese)
[14] 郭佳禾,唐程,王梦琦,等.江苏某奶牛场乳房炎致病菌的分离鉴定及药敏试验[J].中国兽医杂志,2019,55(9):67-70.GUO J H,TANG C,WANG M Q,et al.Isolation and identification of pathogenic bacteria for mastitis in a dairy farm in Jiangsu province and drug sensitivity testing[J].Chinese Journal of Veterinary Medicine, 2019,55(9):67-70.(in Chinese)
[15] 蒋梦娜,程方俊,范才良,等.重庆部分地区奶牛乳房炎葡萄球菌菌种的鉴定及毒力基因研究[J].黑龙江畜牧兽医,2020,12:77-81.JIANG M N,CHENG F J,FAN C L,et al.Identification and virulence gene study of Staphylococcus aureus strains in dairy cows with mastitis in some areas of Chongqing[J].Heilongjiang Animal Science and Veterinary Medicine, 2020,12:77-81.(in Chinese)
[16] 李婷,尹伊娅,唐鑫,等.miRNA在奶牛乳腺炎疾病中的研究进展[J].中国兽医学报,2020,40(8):1660-1664.LI T,YIN Y Y,TANG X,et al.Research progress on miRNA in dairy cow mastitis[J].Chinese Journal of Veterinary Science,2020,40(8):1660-1664.(in Chinese)
[17] KACZOREL-ŁUKOWSKA E,MALACZEWSKA J,WOJCIK R,et al.Streptococci as the new dominant aetiological factors of mastitis in dairy cows in North-Eastern Poland:Analysis of the results obtained in 2013-2019[J].Irish Veterinary Journal,2021,74(1):2.
[18] 王强,黄攀,王倩倩,等.江苏地区奶牛源葡萄球菌耐药性分析与红霉素诱导的克林霉素耐药检测[J].中国动物传染病学报，2021,29(2):77-81.WANG Q,HUANG P,WANG Q Q,et al.Antibiotic resistance of Staphylococcus strains isolated from cows in Jiangsu province and detection of clindamycin-induced resistance by erythromycin[J].Chinese Journal of Animal Infectious Diseases, 2021,29(2):77-81.(in Chinese)
[19] 于泳鑫,吴钰楠,齐富磊,等.苏北地区奶牛乳房炎乳汁中微生物菌群结构及多样性调查分析[J].畜牧与兽医，2024,56(5):64-72.YU Y X,WU Y N,QI F L,et al.Investigation and analysis of microflora structure and diversity in milk of dairy cows with mastitis in Northern Jiangsu province[J].Animal Husbandry & Veterinary Medicine, 2024,56(5):64-72.(in Chinese)
[20] 吴钰楠.苏北地区奶牛隐性乳房炎凝固酶阴性葡萄球菌的分离鉴定与耐药性分析[D].南京：南京农业大学,2022.WU Y N.Isolation,identification and antimicrobial resistance analysis of coagulase-negative Staphylococci from bovine subclinical mastitis in Northern Jiangsu[D].Nanjing:Nanjing Agricultural University,2022.(in Chinese)
[21] 代梦迪.黑龙江某规模化牛场奶牛隐性乳房炎发病规律调查及病原菌检测[D].哈尔滨：东北农业大学,2022.DAI M D.Investigation of the incidence pattern of subclinical mastitis in dairy cows and detection of pathogenic bacteria in a large-scale cattle farm in Heilongjiang[D].Harbin:Northeast Agricultural University,2022.(in Chinese)
[22] 刘冬霞,张潇,李尧,等.陕西省奶牛乳房炎乳中凝固酶阴性葡萄球菌的分离及耐药性分析[J].动物医学进展,2021,42(10):134-138.LIU D X,ZHANG X,LI Y,et al.Isolation and drug resistance of coagulase-negative Staphylococcus from mastitis milk of dairy cows in Shaanxi province[J].Progress in Veterinary Medicine,2021,42(10):134-138.(in Chinese)
[23] 林伟东,宰栩生,于恩琪,等.江苏地区奶牛乳房炎凝固酶阴性葡萄球菌耐药基因分析[J].中国兽医学报,2015,35(7):1130-1135.LIN W D,ZAI X S,YU E Q,et al.Analysis of antibiotic resistance and resistance genes in coagulase-negative Staphylococci isolated from bovine mastitis in Jiangsu area[J].Chinese Journal of Veterinary Science,2015,35(7):1130-1135.(in Chinese)
[24] 王赞嘉,锡林高娃,吴金花,等.牛乳腺炎粪肠球菌cylA基因对牛外周血单核巨噬细胞完全吞噬功能的影响[J].中国动物传染病学报,2023,31(3):178-183.WANG Z J,XI L G W,WU J H,et al.Effect of the bovine Enterococcus feacalia cylA gene on cow mononuclear macrophages phagocytes[J].Chinese Journal of Animal Infectious Diseases,2023,31(3):178-183.(in Chinese)
[25] 许淑珍,童乐艳,于艳华.耐氨苄西林肠球菌β-内酰胺酶及基因的检测[J].中华医院感染学杂志,2008,18(7):928-930.XU S Z,DONG L Y,YU Y H.Detection of β-lactamase and blaZ gene of ampicillin-resistant Enterococcus[J].Chinese Journal of Nosocomiology,2008,18(7):928-930.(in Chinese)
[26] DWORECKA-KASZAK B,KRUTKIEWICZ A,SZOPA D,et al.High prevalence of Candida yeast in milk samples from cows suffering from mastitis in Poland[J].Scientific World Journal,2012,2012:196347.
[27] 张峥臻.头孢噻呋治疗隐性乳房炎对乳汁中主要致病菌区系的影响[J].畜牧与兽医,2019,51(1):107-113.ZHANG Z Z.Effects of treatment with ceftiofur on the main pathogenic microflora in milk from cows with subclinical mastitis[J].Animal Husbandry & Veterinary Medicine,2019,51(1):107-113.(in Chinese)