一株绵羊链球菌的分离鉴定及石榴皮对其抑菌效果评价

Isolation and identification of a strain of Streptococcus ovis and evaluation of inhibitory effect of pomegranate peel on this strain

摘要:

背景 近年来，随着养羊业快速发展，羊链球菌病呈上升趋势，主要发生于绵羊，山羊次之，潜伏期短感染性强，对养殖安全造成极大威胁。目的 从患病湖羊呼吸道中进行病原分离，对分离到的一株绵羊链球菌(Streptococcus ovis)进行鉴定，通过基因组测序明确其基因组基本特征及毒力因子和耐药基因，并测定石榴皮水提物对其抑菌活性与生物膜(biofilm, BF)的影响。方法 对湖羊的鼻拭子样品进行细菌分离培养，通过形态学观察、革兰氏染色、16S rRNA基因测序初步鉴定为S. ovis，通过Illumina二代测序技术构建基因组框架图，通过NR、GO、KEGG、COG、VFDB、CARD数据库获得基因组注释；采用肉汤稀释法测定最小抑菌浓度(minimal inhibitory concentration, MIC)和最小杀菌浓度(minimum bactericidal concentration, MBC)，并绘制在MIC、1/2 MIC、1/4 MIC下的生长曲线，研究石榴皮的体外抑菌效果；采用结晶紫法和3-(4,5-二甲基噻唑基-2)-2,5二苯基四氮唑溴盐[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, MTT]法，探究其对生物膜的抑制与清除效果，以及在生物膜清除后对细菌存活量的影响。结果 根据16S rRNA基因测序和NR数据库注释结果，确定分离株为S. ovis，全基因组的大小为2 206 748 bp (2.21 Mb)，GO、KEGG、COG数据库分别注释到1 599、1 208、1 997个基因，毒力相关基因88种，耐药相关基因51种；石榴皮对S. ovis的MIC和MBC分别为62.5 mg/mL和250 mg/mL，并且在MIC浓度下能显著控制细菌的生长；石榴皮能显著抑制生物膜的形成(P<0.01)，清除初始和成熟生物膜(P<0.01)，减少生物膜内的活菌数(P<0.01)，并且在本研究剂量范围内随着浓度增大效果增强。结论 分离鉴定出一株绵羊链球菌，挖掘出系列毒力因子和耐药基因，石榴皮对该菌株的体外抑制作用显著，对生物膜有显著的破坏作用，为进一步研究绵羊链球菌的致病性和耐药性奠定了基础，同时为开发替抗中草药产品提供了依据。

Abstract:

Background In recent years, the rapid expansion of sheep farming has been accompanied by an increasing incidence of streptococcosis in sheep and goats. This disease has a short incubation period and poses serious risks to the farming industry.Objective We aimed to isolate and identify a strain of Streptococcus ovis from the respiratory tract of Hu sheep, elucidate its genomic features, virulence factors, and antibiotic resistance genes through genome sequencing, and evaluate the inhibitory activities of pomegranate peel against the growth and biofilm formation of this strain.Methods We isolated bacteria from nasal swabs of Hu sheep. The strain was preliminarily identified via morphological observation, Gram staining, and 16S rRNA gene sequencing. We performed whole-genome sequencing with Illumina technology and annotated the genome in the NR, GO, KEGG, COG, VFDB, and CARD databases. We adopted the broth dilution method to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of pomegranate peel. The growth curves were plotted at MIC, 1/2 MIC, and 1/4 MIC of the extract. Furthermore, we employed crystal violet and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays to assess the inhibition and clearance effects of pomegranate peel on the biofilm and viable bacterial count in the biofilm removed.Results The isolate was identified as S. ovis. It had a genome size of 2 206 748 bp (2.21 Mb), with 1 599, 1 208, and 1 997 genes annotated in the GO, KEGG, and COG databases, respectively. We identified 88 virulence genes and 51 antibiotic resistance genes. The pomegranate peel at MIC significantly inhibited the growth of S. ovis, with MIC and MBC of 62.5 mg/mL and 250 mg/mL, respectively. In addition, the pomegranate peel inhibited the biofilm formation (P<0.01), disrupted both initial and mature biofilms (P<0.01), and reduced viable bacterial counts within biofilms (P<0.01). These effects were concentration-dependent within the tested range.Conclusion We successfully isolated and identified a strain of S. ovis and uncovered multiple virulence and antibiotic resistance genes. The pomegranate peel showed significant inhibitory activities against the growth and biofilm formation of this strain. The findings provide a basis for further research into the pathogenesis and resistance mechanisms of S. ovis, as well as the development of herbal agents as alternatives of antibiotics.