鹅源芽孢杆菌和乳酸菌耐药基因及生物学特性分析

doi:10.16431/j.cnki.1671-7236.2025.01.032

摘要/Abstract

摘要： 【目的】研究从鹅肠道分离出的8株潜在益生菌的生物学特性,为后续合理开发和利用菌株提供参考。【方法】通过形态学观察、生化鉴定、PCR鉴定、16S rRNA基因测序对菌株进行鉴定。通过菌株产酸性能、最适生长温度、耐酸性能、耐胆盐性能、抑菌活性、产酶性能等试验对菌株进行生物学特性检测。通过药敏试验和耐药基因检测分析菌株耐药情况。【结果】将从鹅肠道分离的8株菌分别命名为A1~A5和X1~X3。通过形态学观察、生化鉴定初步判断菌株A1~A5为芽孢杆菌,菌株X1~X3为乳酸菌。分离株16S rRNA基因PCR扩增均获得大小约为1 500 bp的条带。16S rRNA基因序列相似性比对结果鉴定菌株A1、A2为枯草芽孢杆菌,菌株A3~A5分别为贝莱斯芽孢杆菌、解淀粉芽孢杆菌和地衣芽孢杆菌,菌株X1~X3分别为发酵乳杆菌、罗伊氏乳杆菌和副干酪乳杆菌。产酸性能结果显示,菌株X1~X3具有较强的产酸性能,菌株X1产酸性能最好,36 h时pH可达到3.84。8株菌最适生长温度均为37 ℃。耐酸耐胆盐性能试验结果显示,8株菌均具有良好的耐受性,其中菌株A2耐受性最好,当pH为4.0,胆盐浓度为0.9%时,存活率可达70%以上。抑菌试验结果显示,8株菌均具有良好的抑菌效果,抑菌圈直径均在10.00 mm以上,其中菌株A2抑菌性最强。产酶性能试验结果显示,菌株A1~A5具有较强的产酶性能,菌株A2产酶效果最好,淀粉酶圈直径可达15.5 mm,纤维素酶圈直径可达14.3 mm。药敏试验结果显示,8株菌对大部分抗菌药都保持中度以上敏感。耐药基因分析表明,四环素类基因TetA(25.0%)、喹诺酮类基因GyrA(12.5%)和大环内酯类基因ErmF(12.5%)为主要耐药基因,其中菌株A3携带四环素类基因TetA(480 bp),菌株A4菌株携带喹诺酮类基因GyrA(382 bp)和四环素类基因TetA(480 bp),菌株X1携带大环内酯类基因ErmF(306 bp)。【结论】从鹅肠道分离的8株菌中枯草芽孢杆菌A2有较强的益生性,具有作为鹅源微生态制剂的潜力。

关键词: 芽孢杆菌; 乳酸菌; 生长特性; 抑菌性能; 耐药基因

Abstract: 【Objective】 The aim of this experiment was to study the biological characteristics of eight potential probiotic strains isolated from the intestinal tract of geese,and provide a reference for the subsequent development and utilization of the strains in a rational way. 【Method】 The strains were characterized by morphological observation,biochemical identification,PCR identification and 16S rRNA gene sequencing.Biological characterization of the strains included acid production,optimum growth temperature,acid resistance,bile salt resistance,bacteriostatic activity and enzyme production were tested.The drug resistance of the strains was analyzed by drug sensitivity test and drug resistance gene detection. 【Result】 Eight strains of bacteria isolated from goose intestines were named A1-A5 and X1-X3,respectively.Through morphological observation and biochemical identification,it was preliminarily determined that A1-A5 were Bacillus and X1-X3 were lactic acid bacteria.PCR amplification of 16S rRNA gene obtained bands of about 1 500 bp in all isolates.The sequence similarity alignment results of 16S rRNA gene showed that A1 and A2 were Bacillus subtilis,A3-A5 were Bacillus velezensis,Bacillus amyloliquefaciens and Bacillus licheniformis respectively,X1-X3 were Lactobacillus fermentum, Lactobacillus reuteri and Lacticaseibacillus paracase,respectively.The results of acid production performance showed that X1-X3 had strong acid production performance,and the acid production performance of X1 was the best,with the pH reaching 3.84 at 36 h.The optimal growth temperature of all eight strains of bacteria was 37 ℃.The results of acid and bile salt resistance test showed that all eight strains of bacteria had good tolerance,among which A2 had the best tolerance,with a survival rate of more than 70% when the pH was 4.0 and the bile salt concentration was 0.9%.The results of the bacterial inhibition test showed that all eight strains of bacteria had good bacterial inhibition,and the inhibition zone diameters were all above 10.00 mm,among which A2 had the best bacterial inhibition effect.The results of the enzyme production performance test showed that A1-A5 had strong enzyme production performance,and the enzyme production effect of A2 was the best,with the diameter of the amylase ring reaching 15.5 mm,and the diameter of the cellulase ring reaching 14.3 mm.The results of the drug sensitivity test showed that the eight strains of bacteria were more than moderately sensitive to most antibacterials.Resistance gene analysis showed that tetracycline gene TetA (25.0%),quinolone gene GyrA (12.5%) and macrolide gene ErmF (12.5%) were the major resistance genes,among which strain A3 carried tetracycline gene TetA (480 bp),strain A4 carried quinolone gene GyrA (382 bp) and tetracycline gene TetA (480 bp),and strain X1 carried the macrolide gene ErmF (306 bp). 【Conclusion】 Among the eight strains of bacteria isolated from the intestinal tract of geese,Bacillus subtilis A2 had strong probiotic properties and had the potential to be used as a microecological agent of goose origin.

Key words: Bacillus; lactic acid bacteria; growth characteristics; antibacterial properties; drug resistance gene

中图分类号:

S852.61

宛宝霞, 孟令滢, 孙思雨, 赵玉洁, 王佳奇, 王秋菊. 鹅源芽孢杆菌和乳酸菌耐药基因及生物学特性分析[J]. 中国畜牧兽医, 2025, 52(1): 351-363.

WAN Baoxia, MENG Lingying, SUN Siyu, ZHAO Yujie, WANG Jiaqi, WANG Qiuju. Analysis of Drug Resistance Genes and Biological Characterization of Goose Derived Bacillus and Lactic Acid Bacteria[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(1): 351-363.

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