Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeongeup 56212, Republic of Korea.
BioMedical Sciences Graduate Program (BMSGP), Chonnam National University Medical School, Hwasun 58128, Republic of Korea.
J Microbiol Biotechnol. 2024 Nov 28;34(11):2385-2395. doi: 10.4014/jmb.2406.06003. Epub 2024 Sep 11.
With the recent stringent criteria for antibiotic susceptibility in probiotics, the presence of antibiotic resistance genes and plasmids associated with their transfer has become a limiting factor in the approval of probiotics. The need to remove genes related to antibiotic resistance and virulence through plasmid curing for the authorization of probiotics is increasing. In this study, we investigated the curing efficiency of ethidium bromide, acridine orange, and novobiocin at different concentrations and durations in five strains of plasmid-bearing lactic acid bacteria and examined the curing characteristics in each strain. and exhibited curing efficiencies ranging from 5% to 44% following treatment with ethidium bromide (10-50 μg/ml) for 24-72 h, while showed the highest efficiency at 14% following treatment with 10 μg/ml novobiocin for 24 h. , which harbors two or more plasmids, demonstrated curing efficiencies ranging from 1% to 8% after an additional 72-h treatment of partially cured strains with 10 μg/ml novobiocin. Plasmid curing in strains with larger plasmids exhibited lower efficiencies and required longer durations. In strains harboring two or more plasmids, a relatively low curing efficiency with a single treatment and a high frequency of false positives, wherein recovery occurred after curing, were observed. Although certain strains exhibited altered susceptibilities to specific antibiotics after curing, these outcomes could not be attributed to the loss of antibiotic resistance genes. Furthermore, the genomic data from the cured strains revealed minimal changes throughout the genome that did not lead to gene mutations.
随着益生菌抗生素药敏性标准的提高，抗生素耐药基因及其转移相关质粒的存在已成为益生菌批准的限制因素。为了获得益生菌的授权，需要通过质粒消除去除与抗生素耐药性和毒力相关的基因。在这项研究中，我们研究了溴化乙锭、吖啶橙和新生霉素在不同浓度和时间下对 5 株携带质粒的乳酸菌的消除效率，并检查了每种菌株的消除特征。溴化乙锭（10-50μg/ml）处理 24-72 小时后，5 株携带质粒的乳酸菌的消除效率在 5%-44%之间，而 10μg/ml 新生霉素处理 24 小时后， 的消除效率最高，为 14%。 该菌株携带两个或更多质粒，在部分消除的菌株中，用 10μg/ml 新生霉素再处理 72 小时后，消除效率为 1%-8%。带有较大质粒的菌株的质粒消除效率较低，需要更长的时间。在携带两个或更多质粒的菌株中，观察到单次处理的消除效率相对较低，假阳性率较高，在消除后恢复。尽管某些菌株在消除后对抗生素的敏感性发生了变化，但这些结果不能归因于抗生素耐药基因的丢失。此外，从消除的菌株中获得的基因组数据显示整个基因组的变化很小，不会导致基因突变。
