一株沙门氏菌噬菌体的分离鉴定及其与抗生素联用抑菌效果

Isolation and identification of a Salmonella phage and antibacterial effect evaluation of the phage combined with antibiotics

摘要:

近几十年来，由于抗生素广泛使用甚至滥用，抗生素耐药(antimicrobial resistance, AMR)细菌对人类健康构成巨大威胁。噬菌体疗法在防治耐药细菌感染方面具有巨大潜力，已成为新的研究热点。然而，噬菌体抗性细菌的快速出现使得噬菌体疗法的疗效难以保障，“噬菌体转向”策略有望解决这一难题。目的 分离适用于“噬菌体转向”疗法的沙门氏菌(Salmonella)烈性噬菌体。方法 以Salmonella S503作为宿主菌，利用双层平板法从农贸市场污水样品中分离纯化烈性噬菌体，分析该噬菌体的基本生物学特性、抑菌能力、基因组信息及体外生物安全性；利用细菌-噬菌体共培养法分离出噬菌体抗性菌株并进行验证，随后采用生长曲线测定、细菌毒力测试和抗生素敏感性测试等实验，系统比较野生型菌株和噬菌体抗性菌株的特性差异。结果 将分离得到的1株沙门氏菌烈性噬菌体命名为HK-1，该噬菌体抑菌能力强、稳定性好，且在体外具有良好的生物安全性。与野生型菌株Salmonella S503相比，噬菌体抗性菌株Salmonella S503-R生长速度放缓，毒力显著降低，对11种抗生素的敏感性增强。噬菌体HK-1与利福平、氨苄西林、磷霉素和庆大霉素存在协同杀菌作用，其中噬菌体分别与氨苄西林、磷霉素和庆大霉素联用均能在24 h内完全抑制Salmonella S503的生长。结论 本研究从污水样品中分离得到1株沙门氏菌烈性噬菌体，该噬菌体适用于“噬菌体转向”疗法，可用于AMR沙门氏菌的防治。

Abstract:

In recent decades, the extensive and inappropriate use of antibiotics has led to the emergence of antibiotic-resistant bacteria, posing a serious threat to human health. Phage therapy has emerged as a promising approach for preventing and treating infections caused by drug-resistant bacteria, garnering considerable research interest. However, the rapid development of phage-resistant bacterial strains complicates the effectiveness of phage therapy. The phage steering strategy holds promise for addressing this challenge.Objective To isolate virulent phages specific to Salmonella that are suitable for phage steering therapy.Methods Specific virulent phages for Salmonella S503 were isolated and purified from wastewater samples collected from a wet market via the double agar overlay method. Their fundamental biological characteristics, antibacterial efficacy, genomic information, and in vitro biological safety were analyzed. Phage-resistant strains were generated through co-culturing Salmonella S503 with the phages. Subsequently, growth curve analysis, bacterial virulence testing, and antibiotic sensitivity assays were employed to systematically compare the characteristics of the wild-type strain and its phage-resistant counterpart.Results The isolated Salmonella phage was designated HK-1. This phage exhibited strong antibacterial properties, high stability, and confirmed biological safety in vitro. Compared with the wild-type strain Salmonella S503, the phage-resistant strain Salmonella S503-R displayed slow growth, significantly reduced virulence, and increased susceptibility to 11 different antibiotics. Furthermore, phage HK-1 demonstrated synergistic bactericidal effects when being combined with rifampicin, ampicillin, fosfomycin, and gentamicin. Notably, the combinations of HK-1 with ampicillin, fosfomycin, and gentamicin effectively inhibited the growth of Salmonella S503 within 24 h.Conclusion We successfully isolated a virulent phage from wastewater samples. This phage is suitable for phage steering therapy and offers potential for the prevention and treatment of antibiotic-resistant Salmonella.