Department of Food Science and Nutrition, College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China.
Biomedical Sciences Program, University of Science and Technology, Zewail City of Science and Technology, Giza, Egypt; Center for Genomics, Helmy Institute for Medical Sciences, Zewail City of Science and Technology, Giza, Egypt; Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine, Mansoura University, Mansoura, Egypt.
J Infect Public Health. 2023 Dec;16 Suppl 1:225-235. doi: 10.1016/j.jiph.2023.10.022. Epub 2023 Oct 28.
Although food-grade disinfectants are extensively used worldwide, it has been reported that the long-term exposure of bacteria to these compounds may represent a selective force inducing evolution including the emergence of antibiotic resistance. However, the mechanism underlying this correlation has not been elucidated. This study aims to investigate the genomic evolution caused by long-term disinfectant exposure in terms of antibiotic resistance in Salmonella enterica Typhimurium.
S. Typhimurium isolates were exposed to increasing concentrations of benzalkonium chloride (BAC) and variations of their antibiotic susceptibilities were monitored. Strains that survived BAC exposure were analyzed at whole genome perspective using comparative genomics, and Sanger sequencing-confirmed mutations in ramR gene were identified. Next, the efflux activity in ramR-mutated strains shown as bisbenzimide accumulation and expression of genes involved in AcrAB-TolC efflux pump using quantitative reverse transcriptase PCR were determined.
Mutation rates of evolved strains varied from 5.82 × 10 to 5.56 × 10, with fold increase from 18.55 to 1.20 when compared with strains evolved without BAC. Mutations in ramR gene were found in evolved strains. Upregulated expression and increased activity of AcrAB-TolC was observed in evolved strains, which may contribute to their increased resistance to clinically relevant antibiotics. In addition, several indels and point mutations in ramR were identified, including L158P, A37V, G42E, F45L, and R46H which have not yet been linked to antimicrobial resistance. Resistance and mutations were stable after seven consecutive cultivations without BAC exposure. These results suggest that strains with sequence type (ST) ST34 were the most prone to mutations in ramR among the three STs tested (ST34, ST19, ST36).
This work demonstrated that disinfectants, specifically BAC forces S. Typhimurium to enter a specific evolutionary trajectory towards antibiotic resistance illustrating the side effects of long-term exposure to BAC and probably also to other disinfectants. Most significantly, this study provides new insights in understanding the emergence of antibiotic resistance in modern society.
尽管食品级消毒剂在全球范围内被广泛使用，但据报道，细菌长期接触这些化合物可能会产生选择压力，导致包括抗生素耐药性在内的进化。然而，这种相关性的机制尚未阐明。本研究旨在探讨沙门氏菌鼠伤寒长期接触消毒剂引起的基因组进化与抗生素耐药性的关系。
用苯扎氯铵（BAC）逐渐增加浓度处理沙门氏菌鼠伤寒分离株，并监测其抗生素敏感性的变化。采用比较基因组学方法对 BAC 暴露后存活的菌株进行全基因组分析，并通过 Sanger 测序确认 ramR 基因突变。然后，通过吖啶橙积累和定量逆转录 PCR 测定参与 AcrAB-TolC 外排泵的基因表达，确定 ramR 突变株的外排活性。
与未用 BAC 进化的菌株相比，进化菌株的突变率为 5.82×10至 5.56×10，fold increase 为 18.55 至 1.20。在进化菌株中发现了 ramR 基因突变。在进化菌株中观察到 AcrAB-TolC 的表达上调和活性增加，这可能有助于它们对临床相关抗生素的耐药性增加。此外，还在 ramR 中鉴定出几个插入缺失和点突变，包括 L158P、A37V、G42E、F45L 和 R46H，这些突变尚未与抗菌药物耐药性相关。在没有 BAC 暴露的情况下连续培养七代后，耐药性和突变是稳定的。这些结果表明，在所测试的三个 ST 型（ST34、ST19、ST36）中，ST34 型菌株最容易在 ramR 中发生突变。
本研究表明，消毒剂，特别是 BAC，迫使鼠伤寒沙门氏菌进入特定的抗生素耐药进化轨迹，说明了长期暴露于 BAC 及可能其他消毒剂的副作用。最重要的是，本研究为理解现代社会抗生素耐药性的出现提供了新的见解。
