Contribution of phenotypic heterogeneity to adaptive antibiotic resistance
Contribution of phenotypic heterogeneity to adaptive antibiotic resistance
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DOI：
10.1073/pnas.1316084111
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发表时间：
2014-01-07
影响因子：
11.1
通讯作者：
Casadesus, Josep
中科院分区：
文献类型：
--
作者：
Antonia Sanchez-Romero, Maria;Casadesus, Josep
在含有致死浓度的利福平、卡那霉素和奈利地酸的平板上筛选出耐药的肠炎沙门氏菌。非选择性生长后对抗性表型的稳定性进行评分。利福平耐药株(Rif(R))是稳定的，这表明它们是通过突变产生的。在rifr突变体中确实检测到了rpoB基因的突变。相反，部分卡那霉素耐药(Km(R))和耐奈利地酸(Nal(R))菌株在非选择性生长后表现出较低的耐药性，这表明在致死选择时，除突变外的机制有助于细菌存活。单细胞分析表明，孔蛋白基因OMPC的表达存在异质性，亚群分离提供了证据，表明降低OMPC的表达赋予了对卡那霉素的适应性抗性。在NALR分离株中，gyrA基因突变存在于大多数耐药株中。然而，外排泵抑制剂Phe-Arg-β-naphtylama(PAβN)降低了NalR突变体的抗性水平，表明主动外排对奈利地酸的总体抗性水平有贡献。在单个细胞和集落中检测到异质性外排泵活性，并发现高外排与对奈立地酸的耐药性增加有关。这些观察表明，细菌细胞关键功能的表达和活性的波动，无论是单独的还是与突变结合在一起，都可能导致对抗生素的适应性耐药性。
Antibiotic-resistant isolates of Salmonella enterica were selected on plates containing lethal concentrations of rifampicin, kanamycin, and nalidixic acid. The stability of the resistance phenotype was scored after nonselective growth. Rifampicin-resistant (Rif(r)) isolates were stable, suggesting that they had arisen by mutation. Mutations in the rpoB gene were detected indeed in Rifr mutants. In contrast, a fraction of kanamycin-resistant (Km(r)) and nalidixic acid-resistant (Nal(r)) isolates showed reduced resistance after nonselective growth, suggesting that mechanisms other than mutation had contributed to bacterial survival upon lethal selection. Single-cell analysis revealed heterogeneity in expression of the porin gene ompC, and subpopulation separation provided evidence that reduced ompC expression confers adaptive resistance to kanamycin. In the case of Nalr isolates, mutations in the gyrA gene were present in most nalidixic acid-resistant isolates. However, the efflux pump inhibitor Phe-Arg-beta-naphtylamide (PA beta N) reduced the level of resistance in Nalr mutants, indicating that active efflux contributes to the overall level of nalidixic acid resistance. Heterogeneous efflux pump activity was detected in single cells and colonies, and a correlation between high efflux and increased resistance to nalidixic acid was found. These observations suggest that fluctuations in the expression and the activity of critical functions of the bacterial cell, alone or combined with mutations, can contribute to adaptive resistance to antibiotics.