乳源无乳链球菌的分离鉴定、耐药与毒力特征及基因组分析

doi:10.16431/j.cnki.1671-7236.2025.03.032

摘要/Abstract

摘要： 【目的】无乳链球菌是引起奶牛乳房炎的主要病原菌之一，明确无乳链球菌的耐药、毒力和基因组特性对无乳链球菌的耐药机制研究具有重要意义。【方法】本研究采用平板划线法将乳样接种于血平板上进行细菌分离纯化，采用形态学观察、革兰染色镜检及分子生物学方法对分离菌进行鉴定。采用微量肉汤稀释法检测分离菌的最小抑菌浓度(minimum inhibitory concentration，MIC)，采用PCR方法检测分离株菌的8种耐药基因和14种毒力基因，利用全基因组测序技术对分离菌进行耐药基因、毒力基因、基因组功能分析。【结果】本研究从36个样本中成功分离出2株菌，在血琼脂平板上表现为白色、圆形、光滑且凸起的菌落，镜检可见紫色球状革兰阳性菌体。生化鉴定结果显示，分离菌呈β-溶血，接触酶试验结果为阴性。2株分离菌16S rDNA PCR扩增均获得大小为1 500 bp的条带，测序结果显示分离菌与无乳链球菌ATCC 13813相似性均＞99.93%，从分子水平上鉴定2株分离菌为无乳链球菌，并将其分别命名为S1、S2。血清型和分子分型结果显示，菌株S1血清型为Ⅱ型，分子分型为ST312；菌株S2血清型为Ⅲ型，分子分型为ST309。药敏试验结果显示，菌株S1和S2对氨苄西林、阿莫西林/克拉维酸、苯唑西林、头孢噻呋、磺胺异噁唑和复方新诺明表现出高度敏感性。菌株S1对四环素和克林霉素耐药，而菌株S2则对四环素、红霉素和克林霉素耐药。此外，菌株S2中检测到tetO和ermB耐药基因，其他耐药基因未被发现。在毒力基因方面，菌株S1和S2均检测到了cfb、bca、iagA、bibA、cspA、pavA、sip、hylB和fbsA基因。基因组分析显示，菌株S1具有一条大小为2.44 Mb的环状染色体，GC含量为36.34%，预测含有2 448个基因。菌株S2的环状染色体大小为2.56 Mb，GC含量为36.59%，预测含有2 576个基因。通过构建系统发育树发现，菌株S1与英国无乳链球菌NCTC13947株一致性最高；S2与英国无乳链球菌NCTC8184亲缘性最近。【结论】本研究从36份奶牛乳房炎样本中分离得到2株无乳链球菌S1和S2，对多种抗菌药敏感，但同时也表现出耐药性。基因组分析揭示了其耐药和毒力基因，为进一步研究无乳链球菌的耐药性和毒力提供了数据支持。

关键词: 无乳链球菌; 耐药基因; 毒力因子; 基因组

Abstract: 【Objective】 Streptococcus agalactiae was one of the main pathogens causing mastitis in dairy cows,and it was of great significance to clarify the resistance,virulence and genomic properties of Streptococcus agalactiae for the study of the resistance mechanism of Streptococcus agalactiae.【Method】 The test was carried out by inoculating milk samples on blood plates for isolation and purification by plate scribing method,and the isolates were identified by morphological observation,Gram staining microscopy and molecular biology methods.The minimum inhibitory concentration (MIC) of the isolates was detected by micro broth dilution method,8 drug resistance genes and 14 virulence genes of the isolates were detected by PCR method,and the isolates were analyzed for drug resistance,virulence genes,and genome function using whole-genome sequencing technology.【Result】 In this study,two strains of bacteria were successfully isolated from 36 samples,which showing white,round,smooth and raised colonies on blood agar plates,and purple spherical Gram-positive organisms were seen on microscopic examination.The biochemical identification showed that the isolates were β-hemolytic and the contact enzyme test was negative,and the PCR amplification showed two bands of 1 500 bp in size as expected,and the sequencing result showed that the similarity with Streptococcus agalactiae ATCC 13813 was ＞99.93%,so the isolates were identified as Streptococcus agalactiae at the molecular level,and were designated as S1 and S2.The results of serotyping and molecular typing showed that the serotype of S1 strain was type Ⅱ and molecular typing was ST312,whereas the serotype of strain S2 was type Ⅲ and molecular typing was ST309.Drug sensitivity tests showed that strains S1 and S2 exhibited high sensitivity to ampicillin,amoxicillin/clavulanic acid,oxacillin,cefphalothin,sulfisoxazole and compound sulfamethoxazole.However,strain S1 was resistant to tetracycline and clindamycin,whereas S2 was resistant to tetracycline,erythromycin and clindamycin.In addition,tetO and ermB resistance genes were detected in strain S2,whereas other resistance genes were not found.In terms of virulence genes,cfb,bca,iagA,bibA,cspA,pavA,sip, hylB and fbsA were detected in both strains S1 and S2.Genomic analysis showed that strain S1 had a circular chromosome of 2.44 Mb in size,with a GC content of 36.34%,and were predicted to contain 2 448 genes.Strain S2 had a ring chromosome of 2.56 Mb in size with a GC content of 36.59% and was predicted to contain 2 576 genes.By constructing a homologous phylogenetic tree,it was found that strain S1 had the highest consistency with the Streptococcus agalactiae NCTC13947 strain from UK; S2 was most closely related to Streptococcus agalactiae NCTC8184 strain from UK.【Conclusion】 In this study,two strains of Streptococcus agalactiae S1 and S2 were isolated from 36 samples of dairy cows with mastitis and found to be sensitive to a variety of antibiotics,but also showed resistance.Genomic analysis revealed their resistance and virulence genes,providing valuable data for further studies on the resistance and virulence of Streptococcus agalactiae.

Key words: Streptococcus agalactiae; drug resistance genes; virulence factors; genomics

中图分类号:

S852.61

高姣姣, 郑楠, 邵伟, 陈贺, 马宪兰, 赵艳坤. 乳源无乳链球菌的分离鉴定、耐药与毒力特征及基因组分析[J]. 中国畜牧兽医, 2025, 52(3): 1301-1316.

GAO Jiaojiao, ZHENG Nan, SHAO Wei, CHEN He, MA Xianlan, ZHAO Yankun. Isolation and Identification of Lactogenic Streptococcus agalactiae and Characterisation of Drug Resistance and Virulence and Genome Analysis[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(3): 1301-1316.

参考文献

[1] CHOI Y,HAN H S,CHONG G O,et al.Updates on group B Streptococcus infection in the field of obstetrics and gynecology[J].Microorganisms,2022,10(12):2398.
[2] MOHAMMAD O D,ALBERTD V,FIONA P M,et al.Epidemiologic and economic analyses of pregnancy loss attributable to mastitis in primiparous Holstein cows[J].Journal of Dairy Science,2018,101(11):10142-10150.
[3] LIANG T,HUO G,CHEN L,et al.Antibacterial activity and metabolomic analysis of linalool against bovine mastitis pathogen Streptococcus agalactiae[J]. Life Science,2023,313:121299.
[4] DOBRUT A,WÓJCIK-GRZYBEK D,MŁODZIŃSKA A,et al.Detection of immunoreactive proteins of Escherichia coli,Streptococcus uberis,and Streptococcus agalactiae isolated from cows with diagnosed mastitis[J].Frontiers in Cellular and Infection Microbiology,2023,13:987842.
[5] BONSAGLIA E C R,ROSSI R S,LATOSINSKI G,et al.Relationship between biofilm production and high somatic cell count in Streptococcus agalactiae isolated from milk of cows with subclinical mastitis[J].Pathogens,2023,12(2):311.
[6] WANG N,ZHOU C H,BASANG W D,et al.Mechanisms by which mastitis affects reproduction in dairy cow:A review[J].Reproduction in Domestic Animals,2021,56(9):1165-1175.
[7] LOHRMANN F,HUFNAGEL M,KUNZE M,et al.Neonatal invasive disease caused by Streptococcus agalactiae in Europe:The DEVANI multi-center study[J]. Infection,2023,51(4):981-991.
[8] FURFARO L L,CHANG B J,PAYNE M S,et al.Perinatal Streptococcus agalactiae epidemiology and surveillance targets[J].Clinical Microbiology Reviews,2018,31(4):e00049-18.
[9] MOHAMED N A,HINGE M,LARSEN O H,et al.Streptococcus agalactiae do not penetrate human chorioamniotic membranes in vitro but alter their biomechanical properties[J]. Acta Obstetricia et Gynecologica Scandinavica,2021,100(10):1814-1821.
[10] ESPINAL E R,MATTHEWS T,HOLDER B M,et al.Group B Streptococcus-induced macropinocytosis contributes to bacterial invasion of brain endothelial cells[J]. Pathogens,2022,11(4):474.
[11] ARMISTEAD B,WHIDBEY C,LYER L M,et al.The cyl genes reveal the biosynthetic and evolutionary origins of the group B Streptococcus hemolytic lipid,granadaene[J].Frontiers in Microbiology,2020,10:3123.
[12] KAWASAKI M,DELAMARE-DEBOUTTEVILLE J,BOWATER R O,et al.Microevolution of aquatic Streptococcus agalactiae ST-261 from Australia indicates dissemination via imported tilapia and ongoing adaptation to marine hosts or environment[J]. Applied and Environmental Microbiology,2018,84(16):e00859-18.
[13] TORRES G,MACIAS D,REYES-VÉLEZ J,et al. Streptococcus agalactiae virulence factors isolated from bovine mastitis and antibiotic treatment response[J]. Journal of Applied Microbiology,2023,134(6):lxad116.
[14] FARZANA Z,SAHA A,SIDDIKI A Z.Molecular characterization of Streptococcus agalactiae and Streptococcus dysgalactiae causing bovine mastitis in the Southern region of Bangladesh[J].Journal of Advanced Veterinary and Animal Research,2023,10(2):178-184.
[15] 张保海,罗梓丹,芦彪,等.四川部分地区奶牛源无乳链球菌的分离鉴定、毒力基因检测及耐药性分析[J].西北农业学报,2020,29(3):327-333. ZHANG B H,LUO Z D,LU B,et al.Isolation and identification of Streptococcus agalactiae from dairy cattle in some areas of Sichuan,virulence gene detection and drug resistance analysis[J]. Northwest Journal of Agriculture,2020,29(3):327-333.(in Chinese)
[16] 陈寰,董方,吕志勇,等.儿童侵袭性无乳链球菌血清型和耐药性分析[J].检验医学,2024,39(3):260-264. CHEN H,DONG F,LYU Z Y,et al.Analysis of serotypes and drug resistance of invasive Streptococcus agalactiae in children[J].Laboratory Medicine,2024,39(3):260-264.(in Chinese)
[17] LEGHARI A,LAKHO S A,KHAND F M,et al.Molecular epidemiology,characterization of virulence factors and antibiotic resistance profile of Streptococcus agalactiae isolated from dairy farms in China and Pakistan[J].Journal of Integrative Agriculture,2023,22(5):1514-1528.
[18] IMPERI M,PATARACCHIA M,ALFARONE G,et al.A multiplex PCR assay for the direct identification of the capsular type (Ⅰa to Ⅸ) of Streptococcus agalactiae[J]. Journal of Microbiological Methods,2010,80(2):212-214.
[19] 张雨薇.不同动物源无乳链球菌荚膜多糖血清型及分子分型研究[D].成都：四川农业大学,2017. ZHANG Y W.Serotyping and molecular typing study of different animal sources of non-lactating Streptococcus pyogenes podocarboxylic polysaccharides[D].Chengdu:Sichuan Agricultural University,2017.(in Chinese)
[20] CLINICAL AND LABORATORY STANDARDS INSTITUTE.Performance standards for antimicrobial susceptibility testing[S].28th ed.CLSI supplement M100.Wayne,PA:Clinical and Laboratory Standards Institute,2018.
[21] POYART C,JARDY L,QUESNE G,et al.Genetic basis of antibiotic resistance in Streptococcus agalactiae strains isolated in a French hospital[J]. Antimicrobial Agents and Chemotherapy,2003,47(2):794-797.
[22] LOPARDO H A,VIDAL P,JERIC P,et al.Six-month multicenter study on invasive infections due to group B Streptococci in Argentina[J]. Journal of Clinical Microbiology,2003,41(10):4688-4694.
[23] BOLUKAOTO J Y,MONYAMA C M,CHUKWU M O,et al.Antibiotic resistance of Streptococcus agalactiae isolated from pregnant women in Garankuwa,South Africa[J]. BMC Research Notes,2015,8:364.
[24] KACZOREK E，MAŁACZEWSKA J，WÓJCIK R，et al.Biofilm production and other virulence factors in Streptococcus spp.isolated from clinical cases of bovine mastitis in Poland[J].BMC Veterinary Research，2017,13(1):398.
[25] 范雪,邵伟,赵艳坤,等.牛源无乳链球菌耐药性与毒力基因的表达量差异分析[J].新疆农业科学,2022,59(9):2310-2317. FAN X,SHAO W,ZHAO Y K,et al.Differential analysis of the expression of drug resistance and virulence genes of bovine-derived Streptococcus agalactiae[J]. Xinjiang Agricultural Science,2022,59(9):2310-2317.(in Chinese)
[26] ZHANG H M,JIANG H,FAN Y L,et al.Transcriptomics and iTRAQ-proteomics analyses of bovine mammary tissue with Streptococcus agalactiae-induced mastitis[J]. Journal of Agricultural and Food Chemistry,2018,66(42):11188-11196.
[27] 秦平伟,张艳,黄宝银,等.奶牛无乳链球菌乳房炎研究进展[J].畜牧兽医杂志,2021,40(6):105-108. QIN P W,ZHANG Y,HUANG B Y,et al.Progress in the study of milk-free streptococcal mastitis in dairy cows[J].Journal of Animal Husbandry and Veterinary Medicine,2021,40(6):105-108.(in Chinese)
[28] 徐晶晶,蔡扩军,马亚珍,等.乌鲁木齐及周边地区奶牛乳房炎无乳链球菌的分离鉴定及耐药性分析[J].养殖与饲料,2024,23(8):53-56. XU J J,CAI K J,MA Y Z,et al.Isolation and identification of Streptococcus agalactiae in dairy cows with mastitis in Urumqi and surrounding areas and analysis of drug resistance[J]. Breeding and Feed,2024,23(8):53-56.(in Chinese)
[29] 尹金花,李玫毅,吴兴,等.牛源无乳链球菌分离鉴定与耐药性分析[J].动物医学进展,2022,43(9):40-43. YIN J H,LI M Y,WU X,et al.Isolation and characterization of bovine-derived Streptococcus lactis and analysis of drug resistance[J]. Progress in Animal Medicine,2022,43(9):40-43.(in Chinese)
[30] RAABE V N,SHANE A L.Group B Streptococcus (Streptococcus agalactiae)[J].Microbiology Spectrum,2019,7(2):10.1128/microbiolspec.gpp3-0007-2018.
[31] 宋倩,达举云,李怡娜,等.宁夏吴忠地区奶牛临床型乳房炎主要病原菌的分离鉴定及耐药性分析[J].动物医学进展,2022,43(2):70-75. SONG Q,DA J Y,LI Y N,et al.Isolation and identification of major pathogenic bacteria of clinical mastitis in dairy cows in Wuzhong,Ningxia and analysis of drug resistance[J].Progress in Animal Medicine,2022,43(2):70-75.(in Chinese)
[32] MA X,CHEN H,WANG F,et al.Molecular characterisation and antimicrobial resistance of Streptococcus agalactiae isolates from dairy farms in China[J].Journal of Veterinary Research,2023,67(2):161-167.
[33] AI W,ZHOU Y,WANG B,et al.First report of coexistence of bla_SFO-1 and bla_NDM-1 β-lactamase genes as well as colistin resistance gene mcr-9 in a transferrable plasmid of a clinical isolate of Enterobacter hormaechei[J].Frontiers in Microbiology,2021,12:676113.
[34] LI P,WEI Y,LI G,et al.Comparison of antimicrobial efficacy of eravacycline and tigecycline against clinical isolates of Streptococcus agalactiae in China:In vitro activity,heteroresistance,and cross-resistance[J]. Microbial Pathogenesis,2020,149:104502.
[35] DUARTE R S,BELLEI B C,MIRANDA O P,et al.Distribution of antimicrobial resistance and virulence-related genes among Brazilian group B Streptococci recovered from bovine and human sources[J].Antimicrobial Agents and Chemotherapy,2005,49(1):97-103.
[36] VIDAL AMARAL J R,JUCA RAMOS R T,ALMEIDA ARAUJO F,et al.Bacteriocin producing Streptococcus agalactiae strains isolated from bovine mastitis in Brazil[J]. Microorganisms,2022,10(3):588.
[37] 刘泽梁.克林霉素耐药无乳链球菌分子特征及耐药机制[D].唐山：华北理工大学,2023. LIU Z L.Molecular characterization and resistance mechanism of clindamycin-resistant Streptococcus lactis[D].Tangshan:North China University of Science and Technology,2023.(in Chinese)
[38] 王宗海.无乳链球菌特性及毒力相关因子研究进展[J].中国乳业,2023，8:63-68. WANG Z H.Progress in the characterization and virulence-related factors of Streptococcus agalactiae[J]. China Dairy Industry,2023,8:63-68.(in Chinese)
[39] SHI H Z,ZHOU M X,ZHANG Z T,et al.Molecular epidemiology,drug resistance,and virulence gene analysis of Streptococcus agalactiae isolates from dairy goats in backyard farms in China[J].Frontiers in Cellular and Infection Microbiology,2023,12:1049167.
[40] 陈杰,魏勇,王晨豫,等.新疆部分地区牛源无乳链球菌主要毒力基因和耐药基因的检测与序列分析[J].江苏农业科学,2022,50(22):171-179. CHEN J,WEI Y,WANG C Y,et al.Detection and sequence analysis of major virulence genes and resistance genes of bovine-derived Streptococcus agalactiae in some regions of Xinjiang[J]. Jiangsu Agricultural Science,2022,50(22):171-179.(in Chinese)
[41] YUAN X Y,LIU H Z,LIU J F,et al.Pathogenic mechanism,detection methods and clinical significance of group B Streptococcus[J].Future Microbiology,2021,16:671-685.
[42] ZASTEMPOWSKA E,TWARUZEK M,GRAJEWSKI J,et al.Virulence factor genes and cytotoxicity of Streptococcus agalactiae isolated from bovine mastitis in Poland[J]. Microbiology Spectrum,2022,10(3):e0222421.
[43] LIU Y,LIU J.Group B Streptococcus:Virulence factors and pathogenic mechanism[J]. Microorganisms,2022,10(12):2483.
[44] 施金谷,马振花,王志强,等.一株罗非鱼无乳链球菌Streptococcus agalactiae CS2108的全基因组测序及功能分析[J].南方农业,2024,18(5):1-7. SHI J G,MA Z H,WANG Z Q,et al.Whole genome sequencing and functional analysis of a strain of Streptococcus agalactiae CS2108 from tilapia[J]. Southern Agriculture,2024,18(5):1-7.(in Chinese)
[45] ZHANG D,KE X,LIU Z,et al.Capsular polysaccharide of Streptococcus agalactiae is an essential virulence factor for infection in Nile tilapia (Oreochromis niloticus Linn.)[J].Journal of Fish Diseases,2018,42(2):293-302.
[46] 刘泽梁,董爱英,李娟.无乳链球菌毒力因子的研究进展[J].微生物学免疫学进展,2023,51(5):74-79. LIU Z L,DONG A Y,LI J.Progress in the study of virulence factors of Streptococcus lactis[J].Advances in Microbiology Immunology,2023,51(5):74-79.(in Chinese)
[47] 马晓姣.牛源无乳链球菌耐药性及全基因组测序分析研究[D].乌鲁木齐：新疆农业大学,2023. MA X J.Study on drug resistance and whole genome sequencing analysis of bovine-derived Streptococcus lactis[D].Urumqi:Xinjiang Agricultural University,2023.(in Chinese)
[48] KANNIKA K,PISUTTHARACHAI D,SRISAPOOME P,et al.Molecular serotyping,virulence gene profiling and pathogenicity of Streptococcus agalactiae isolated from tilapia farms in Thailand by multiplex PCR[J].Journal of Applied Microbiology,2017,122(6):1497-1507.
[49] SLAVETINSKY C J,HAUSER J N,GEKELER C,et al.Sensitizing Staphylococcus aureus to antibacterial agents by decoding and blocking the lipid flippase MprF[J].eLife,2022,11:e66376.