School of Environmental Science and Engineering, Zhejiang Gongshang University, Hangzhou 310012, China; Zhejiang Provincial Key Laboratory of Solid Waste Treatment and Recycling, Hangzhou 310012, China.
Department of Infectious Diseases, Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310009, China.
Environ Int. 2021 Nov;156:106619. doi: 10.1016/j.envint.2021.106619. Epub 2021 May 11.
The rapid increase of antibiotic resistance is a serious challenge around the world. Antibiotics are present in various environments at sub-lethal concentrations, but how resistance emerges under sub-lethal conditions is not fully clear. In this study, we evolved Pseudomonas aeruginosa PAO1 under sub-lethal conditions, in the presence of either 15-30 μg/mL or 150-300 μg/mL of ampicillin. We found a ~ 5-6 fold increase in the minimum inhibitory concentration (MIC) among evolved isolates exposed to 15-30 μg/mL of ampicillin, and more than a 19-fold of increase in 150-300 μg/mL of ampicillin exposure. DNA sequencing revealed that mpl and ampD were frequently mutated in these resistant strains. We performed a transcriptome analysis of deletion mutations of mpl or ampD, compared to PAO1. Both showed a two-fold increase in expression of quorum sensing (QS) genes including lasR and rhlI/R; the heightened expression was positively correlated with the expression of the ampicillin resistance gene ampC. We queried if quorum sensing contributes to the increase in the ampicillin MIC. After adding the quorum quencher acylase I, the growth yield both decreased by roughly 50% for Δmpl in 2000 μg/mL of ampicillin and ΔampD in 4000 μg/mL of ampicillin. Addition of the QS signals into synthase mutants restored the higher MIC, but only for the rhlI/R circuit. This study highlights the involvement of QS in antibiotic resistance evolution, and shows the multifactorial contributors to the observed phenotypes.
抗生素耐药性的迅速增加是全球面临的一个严重挑战。抗生素以亚致死浓度存在于各种环境中，但在亚致死条件下耐药性是如何产生的还不完全清楚。在这项研究中，我们在亚致死条件下，用 15-30μg/ml 或 150-300μg/ml 的氨苄青霉素对铜绿假单胞菌 PAO1 进行了进化。我们发现，在暴露于 15-30μg/ml 氨苄青霉素的进化分离株中，最低抑菌浓度（MIC）增加了约 5-6 倍，在暴露于 150-300μg/ml 氨苄青霉素的分离株中增加了 19 倍以上。DNA 测序显示，mpl 和 ampD 在这些耐药株中经常发生突变。我们对 mpl 或 ampD 的缺失突变与 PAO1 进行了转录组分析。两者的群体感应（QS）基因包括 lasR 和 rhlI/R 的表达都增加了两倍；表达的增加与氨苄青霉素耐药基因 ampC 的表达呈正相关。我们询问了群体感应是否有助于增加氨苄青霉素的 MIC。在用群体感应淬灭酶 I 处理后，在 2000μg/ml 氨苄青霉素中的Δmpl 和在 4000μg/ml 氨苄青霉素中的ΔampD 的生长产量都降低了约 50%。将 QS 信号添加到合成突变体中恢复了更高的 MIC，但仅适用于 rhlI/R 回路。本研究强调了群体感应在抗生素耐药性进化中的作用，并显示了观察到的表型的多种因素贡献。
