Distribution of triclosan-resistant genes in major pathogenic microorganisms revealed by metagenome and genome-wide analysis

The substantial use of triclosan (TCS) has been aimed to kill pathogenic bacteria, but TCS resistance seems to be prevalent in microbial species and limited knowledge exists about TCS resistance determinants in a majority of pathogenic bacteria. We aimed to evaluate the distribution of TCS resistance determinants in major pathogenic bacteria (N = 231) and to assess the enrichment of potentially pathogenic genera in TCS contaminated environments. A TCS-resistant gene (TRG) database was constructed and experimentally validated to predict TCS resistance in major pathogenic bacteria. Genome-wide in silico analysis was performed to define the distribution of TCS-resistant determinants in major pathogens. Microbiome analysis of TCS contaminated soil samples was also performed to investigate the abundance of TCS-resistant pathogens. We experimentally confirmed that TCS resistance could be accurately predicted using genome-wide in silico analysis against TRG database. Predicted TCS resistant phenotypes were observed in all of the tested bacterial strains (N = 17), and heterologous expression of selected TCS resistant genes from those strains conferred expected levels of TCS resistance in an alternative host Escherichia coli. Moreover, genome-wide analysis revealed that potential TCS resistance determinants were abundant among the majority of human-associated pathogens (79%) and soil-borne plant pathogenic bacteria (98%). These included a variety of enoyl-acyl carrier protein reductase (ENRs) homologues, AcrB efflux pumps, and ENR substitutions. FabI ENR, which is the only known effective target for TCS, was either co-localized with other TCS resistance determinants or had TCS resistance-associated substitutions. Furthermore, microbiome analysis revealed that pathogenic genera with intrinsic TCS-resistant determinants exist in TCS contaminated environments. We conclude that TCS may not be as effective against the majority of bacterial pathogens as previously presumed. Further, the excessive use of this biocide in natural environments may selectively enrich for not only TCS-resistant bacterial pathogens, but possibly for additional resistance to multiple antibiotics.

三氯生（triclosan, TCS）被大量应用于病原菌杀灭，但微生物物种对三氯生的耐药性已日趋普遍，而目前针对绝大多数病原菌的三氯生耐药决定因子的相关研究仍较为匮乏。本研究旨在明确三氯生耐药决定因子在231株主要病原菌中的分布特征，并评估三氯生污染环境中潜在致病菌属的富集情况。我们构建了三氯生耐药基因（triclosan-resistant gene, TRG）数据库，并通过实验验证其可用于预测主要病原菌的三氯生耐药性。通过全基因组生物信息学分析，明确了三氯生耐药决定因子在主要病原菌中的分布规律；同时对三氯生污染土壤样本开展微生物组分析，以探究耐药病原菌的丰度水平。本研究通过实验证实，基于TRG数据库的全基因组生物信息学分析可准确预测三氯生耐药性。在17株受试细菌菌株中均观测到了预测的三氯生耐药表型，且从这些菌株中筛选得到的三氯生耐药基因的异源表达，可在异源宿主大肠杆菌（Escherichia coli）中赋予预期水平的三氯生耐药性。全基因组分析进一步显示，潜在三氯生耐药决定因子在多数人体共生病原菌（79%）和土传植物病原菌（98%）中广泛分布，涵盖多种烯酰-酰基载体蛋白还原酶（enoyl-acyl carrier protein reductase, ENRs）同源物、AcrB外排泵以及ENR突变位点。作为目前已知唯一对三氯生有效的作用靶点，FabI ENR要么与其他三氯生耐药决定因子共定位，要么携带与三氯生耐药性相关的突变位点。此外，微生物组分析结果表明，携带固有三氯生耐药决定因子的致病菌属存在于三氯生污染环境中。本研究表明，三氯生对绝大多数病原菌的杀灭效果可能不如此前预期。此外，在自然环境中过度使用该杀菌剂，不仅会选择性富集三氯生耐药病原菌，还可能诱导其获得对多种抗生素的交叉耐药性。