【作者】 张婷；

【导师】 周紫峣；

【作者基本信息】 四川农业大学 ， 临床兽医学， 2025， 硕士

【摘要】 抗生素是小动物临床中的常用药物,是细菌性肠炎治疗的首选药物,但对非细菌性腹泻等肠道疾病无法起到有效的治疗作用。过度使用抗生素可能干扰肠道菌群的稳定性,进一步引起慢性腹泻。益生菌是非细菌性腹泻的有效治疗措施之一,临床常被用于使用抗生素后的菌群重建,但其活性可能被抗生素所影响。本研究在模拟抗生素残留的情况下筛选并研究了犬源唾液乳酸杆菌LSbg3的肠道益生作用和机制,主要内容和结果如下:试验一:采集20只健康比格犬的粪便,均质化后与临床上常用的四种抗生素共同厌氧培养8 h,通过16S rRNA测序和气相色谱技术检测探究抗生素残留对肠道菌群及短链脂肪酸的影响。结果显示:333μg/ml甲硝唑处理组肠道菌群的变化与对照组最相似;在4种抗生素((333μg/ml甲硝唑、333μg/mL阿莫西林、125μg/mL万古霉素、125μg/mL头孢噻呋)处理后肠道菌群中的乳酸菌比例增加了5.96±5.7%。试验二:在333μg/ml甲硝唑存在的条件下分离培养出了24株乳酸菌,通过抑菌试验、模拟胃肠道试验、表面性质试验、药敏试验和溶血试验评估乳酸菌的益生特性及安全性。结果显示:在24株乳酸菌中筛选出5株具有较好益生潜能的菌株(LRbg2、LSbg3、WCbg1、LSbg12和LSbg13)。进一步安全性评估结果表明5株乳酸菌均不溶血,对8种常见抗生素高度敏感(>60%),不具有多重耐药性。其中,唾液乳杆菌LSbg3在所有指标中都具有较好的益生效果。试验三:采用2 mL/kg番泻叶灌喂法构建比格犬腹泻模型,以8 mg/kg甲硝唑作为抗生素残留组,使用5.8×10~8 CFU/mL浓度唾液乳杆菌LSbg3治疗,验证LSbg3在抗生素残留条件下对比格犬腹泻的治疗效果。结果显示,唾液乳杆菌LSbg3能够有效改善比格犬腹泻症状、肠道菌群结构和多样性。8 mg/kg甲硝唑不影响LSbg3的益生作用。试验四:通过全基因组测序,在GO、CAZy和Swiss-Prot等数据库中进行基因注释,然后进行益生基因簇和细菌素基因簇分析,从基因水平层面进一步探究唾液乳杆菌LSbg3的益生机制。结果显示:唾液乳杆菌LSbg3基因组大小为2.25Mb,平均GC含量为37.05%。基因组中含有dltA、lmrA和ribBA等功能基因,在基因组中鉴定到了一个细菌素合成基因簇T3PKS类物质和一种以Enterolysin＿A为核心的细菌素基因簇。同时唾液乳杆菌LSbg3基因组中不具有多重耐药基因和编码溶血素、肠毒素的毒力基因,表明唾液乳杆菌LSbg3具有良好安全性、益生潜力和抗逆性。综上所述,本研究在甲硝唑残留条件下筛选出了1株具有良好益生特性和安全性的唾液乳杆菌LSbg3,其改善腹泻的能力可能通过dltA、lmrA和ribBA等功能基因表达产生。本研究为研发替抗益生菌提供了一定的理论和实践支持。更多还原

【Abstract】 Antibiotics are commonly used in small animal clinics and are the first choice for the treatment of bacterial enteritis,but they cannot play an effective role in the treatment of intestinal diseases such as non-bacterial diarrhea.Overuse of antibiotics may interfere with the stability of the gut microbiota,further causing chronic diarrhea.Probiotics are one of the effective treatments for non-bacterial diarrhea,and are often used in clinical practice to rebuild microbiota after antibiotics,but their activity may be affected by antibiotics.In this study,the intestinal probiotic effects and mechanisms of Lactobacillus canine salivarius LSbg3 were screened and studied under the condition of simulated antibiotic residues,and the main contents and results are as follows:Test 1:The feces of 20 healthy beagle dogs were collected,homogenized and anaerobic culture with four commonly used antibiotics in clinical practice for 8 h,and the effects of antibiotic residues on intestinal flora and short-chain fatty acids were detected by 16S rRNA sequencing and gas chromatography.The results showed that the changes of intestinal microbiota in the 333μg/ml metronidazole treatment group were the most similar to those in the control group.The proportion of lactic acid bacteria in the intestinal microbiota increased by 5.96±5.7%after treatment with four antibiotics(333μg/mL metronidazole,333μg/mL amoxicillin,125μg/mL vancomycin,125μg/mL cefotaxifur).Test 2:Twenty-four strains of lactic acid bacteria were isolated and cultured in the presence of 333μg/ml metronidazole,and the probiotic properties and safety of lactic acid bacteria were evaluated by antibacterial test,simulated gastrointestinal test,surface property test,antimicrobial susceptibility test and hemolysis test.The results showed that 5 strains with good probiotic potential(LRbg2,LSbg3,WCbg1,LSbg12 and LSbg13)were screened out of 24 strains of lactic acid bacteria.Further safety evaluation results showed that all five strains of lactic acid bacteria were hemolytic,highly sensitive(>60%)to 8 common antibiotics,and did not have multi-drug resistance.Among them,Lactobacillus salivarius LSbg3 had good probiotic effects in all indicators.Tset 3:A model of diarrhea in beagle dogs was constructed by 2 mL/kg senna leaf irrigation,and 8 mg/kg metronidazole was used as the antibiotic residue group,and Lactobacillus salivarius LSbg3 at a concentration of 5.8×108 CFU/mL was used to verify the therapeutic effect of LSbg3 on diarrhea in beagle dogs under antibiotic residue conditions.The results showed that Lactobacillus salivarius LSbg3 could effectively improve the diarrhea symptoms,intestinal microbiota structure and diversity of beagle dogs.8mg/kg metronidazole did not affect the probiotic effect of LSbg3.Test 4:The probiotic mechanism of Lactobacillus salivarius LSbg3 was further explored at the gene level by whole genome sequencing,gene annotation in GO,CAZy and Swiss-Prot databases,and then analysis of probiotic gene clusters and bacteriocin gene clusters.The results showed that the genome size of Lactobacillus salivarius LSbg3 was2.25Mb,and the average GC content was 37.05%.The genome contained functional genes such as dltA,lmrA and ribBA,and a bacteriocin synthesis gene cluster T3PKS and a Enterolysin＿A-centered bacteriocin gene cluster were identified in the genome.At the same time,the genome of Lactobacillus salivarius LSbg3 did not contain multiple drug resistance genes and virulence genes encoding hemolysin and enterotoxin,indicating that Lactobacillus salivarius LSbg3 had good safety,probiotic potential and stress resistance.In summary,in this study,a strain of Lactobacillus salivarius LSbg3 with good probiotic properties and safety was screened under metronidazole residue conditions,and its ability to improve diarrhea may be produced through the expression of functional genes such as dltA,lmrA and ribBA.This study provides some theoretical and practical support for the development of substitute probiotics.更多还原