DataSheet_2_Impact of mutagenesis and lateral gene transfer processes in bacterial susceptibility to phage in food biocontrol and phage therapy.docx

IntroductionThe emergence of resistance and interference mechanisms to phage infection can hinder the success of bacteriophage-based applications, but the significance of these mechanisms in phage therapy has not been determined. This work studies the emergence of Salmonella isolates with reduced susceptibility to a cocktail of three phages under three scenarios: i) Salmonella cultures (LAB), ii) biocontrol of cooked ham slices as a model of food safety (FOOD), and iii) oral phage therapy in broilers (PT).
MethodsS. Typhimurium ATCC 14028 RifR variants with reduced phage susceptibility were isolated from the three scenarios and conventional and molecular microbiology techniques were applied to study them.
Results and discussionIn LAB, 92% of Salmonella isolates lost susceptibility to all three phages 24 h after phage infection. This percentage was lower in FOOD, with 4.3% of isolates not susceptible to at least two of the three phages after seven days at 4°C following phage treatment. In PT, 9.7% and 3.3 % of isolates from untreated and treated broilers, respectively, displayed some mechanism of interference with the life cycle of some of the phages. In LAB and FOOD scenarios, resistant variants carrying mutations in rfc and rfaJ genes involved in lipopolysaccharide synthesis (phage receptor) were identified. However, in PT, the significant decrease of EOP, ECOI, and burst size observed in isolates was prompted by lateral gene transfer of large IncI1 plasmids, which may encode phage defense mechanisms. These data indicate that the acquisition of specific conjugative plasmids has a stronger impact than mutagenesis on the emergence of reduced phage-susceptibility bacteria in certain environments. In spite of this, neither mechanism seems to significantly impair the success of Salmonella biocontrol and oral phage therapy.

引言
噬菌体感染抗性与干扰机制的出现可能会阻碍基于噬菌体的应用效果，但此类机制在噬菌体治疗中的意义尚未明确。本研究针对三种场景下，对三种噬菌体混合制剂敏感性降低的沙门氏菌（Salmonella）分离株的出现情况展开探究：①沙门氏菌培养体系（LAB），②以熟火腿片为模型的食品安全生物防治（FOOD），③肉鸡口服噬菌体治疗（PT）。

方法
本研究从上述三种场景中分离得到噬菌体敏感性降低的鼠伤寒沙门氏菌（S. Typhimurium）ATCC 14028利福平抗性（RifR）变异株，并采用常规微生物学与分子微生物学技术对其进行研究。

结果与讨论
在LAB场景中，噬菌体感染24小时后，92%的沙门氏菌分离株丧失了对三种噬菌体的敏感性。FOOD场景中的该比例相对更低：经噬菌体处理后，于4℃培养7天的样本中，仅有4.3%的分离株对至少两种噬菌体不敏感。在PT场景中，未处理与经噬菌体处理的肉鸡来源分离株中，分别有9.7%与3.3%的菌株表现出可干扰部分噬菌体生命周期的机制。
在LAB与FOOD场景中，研究人员鉴定出携带参与脂多糖（噬菌体受体）合成的rfc与rfaJ基因突变的抗性变异株。而在PT场景中，分离株的噬斑形成效率（EOP, Efficiency of Plating）、感染中心效率（ECOI, Efficiency of Infection Center）与裂解量（burst size）出现显著下降，该现象由大型IncI1质粒的水平基因转移所介导，此类质粒或编码噬菌体防御机制。
上述数据表明，在特定环境中，特定接合性质粒的获得对噬菌体敏感性降低的沙门氏菌的出现的促进作用，强于诱变作用。尽管如此，这两种机制似乎均未显著削弱沙门氏菌生物防治与口服噬菌体治疗的效果。