Abstract

The work aimed to estimate the potential of transfer of resistance genes from Lactobacillus fermentum 5-1 to the Gram-negative intestinal microbiota. The sensitivity of antibiotics was assessed using the gradient agar method and microdilution method. Antibiotic resistance (AR) genes in bacterial genomic DNA were detected by PCR, followed by Sanger sequencing of the amplicons and BLASTn sequence analysis. Acinetobacter baumannii, Citrobacter freundii, and Escherichia coli had neither phenotypic nor genotypic resistance to tetracycline and therefore were selected among nine Gram-negative strains as recipients of the AR genes. The transferability of the AR genes from L. fermentum 5-1 to these bacteria was studied under cocultivation in a simulated colonic environment, in filter mating experiments, and using transformation of bacteria by electroporation. Based on the values of minimum inhibitory concentration (MIC), we found that this strain was resistant to vancomycin (MIC = 256 μg/mL), ciprofloxacin (MIC = 64 μg/mL), aminoglycosides (MIC = 54–256 μg/mL), and chloramphenicol (MIC = 8 μg/mL); sensitive to ampicillin (MIC = 0.5 μg/mL), rifampicin (MIC = 2 μg/mL), and cefotaxime (MIC = 0.12 μg/mL); and showed intermediate (low) resistance to erythromycin (MIC = 1 μg/mL) and tetracycline (MIC = 8 μg/mL). Using PCR analysis in the chromosomal DNA of L. fermentum 5-1, we detected the erythromycin resistance gene ermB, while two tetracycline resistance genes, tetM and tetK, were identified in plasmid DNA. Moreover, we demonstrated that L. fermentum 5-1 could transfer their tetK gene to C. freundii via electroporation and transformation with the plasmid DNA of the lactobacilli. Our results demonstrate that lactobacilli can negatively contribute to the problem of antibiotic resistance and warrant stricter control over the practical use of LAB.

中文翻译：

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发酵乳杆菌中的抗生素抗性及其遗传决定因素的迁移

摘要

这项工作旨在评估将抗性基因从发酵乳杆菌5-1转移至革兰氏阴性肠道菌群的潜力。使用梯度琼脂法和微量稀释法评估抗生素的敏感性。通过PCR检测细菌基因组DNA中的抗生素抗性（AR）基因，然后对Sanger进行扩增子测序和BLASTn序列分析。鲍曼不动杆菌，弗氏柠檬酸杆菌和大肠杆菌对四环素均无表型和基因型抗性，因此被选为9种革兰氏阴性菌株中的AR基因受体。发酵乳杆菌AR基因的可转移性在模拟的结肠环境中，在滤器交配实验中，在共培养条件下研究这些细菌的5-1-1，并使用通过电穿孔转化细菌的方法。根据最小抑菌浓度（MIC）的值，我们发现该菌株对万古霉素（MIC = 256μg/ mL），环丙沙星（MIC = 64μg/ mL），氨基糖苷（MIC = 54–256μg/ mL）有抗药性）和氯霉素（MIC = 8μg/ mL）；对氨苄西林（MIC = 0.5μg/ mL），利福平（MIC = 2μg/ mL）和头孢噻肟（MIC = 0.12μg/ mL）敏感; 并显示出对红霉素（MIC = 1μg/ mL）和四环素（MIC = 8μg/ mL）的中等（低）耐药性。通过对发酵乳杆菌5-1的染色体DNA进行PCR分析，我们检测到了红霉素抗性基因erm B，而两个四环素抗性基因在质粒DNA中鉴定出tet M和tetK。此外，我们证明发酵乳杆菌5-1可以通过电穿孔和用乳杆菌的质粒DNA转化将其tet K基因转移至弗氏梭菌。我们的结果表明，乳酸菌可能会对抗生素耐药性产生负面影响，并需要对LAB的实际使用进行更严格的控制。