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Depletion of antibiotic targets has widely varying effects on growth.

ABSTRACT: It is often assumed that antibiotics act on the most vulnerable cellular targets, particularly those that require limited inhibition to block growth. To evaluate this assumption, we developed a genetic method that can inducibly deplete targeted proteins and that mimics their chemical inactivation. We applied this system to current antibiotic targets in mycobacteria. Although depleting some antibiotic targets significantly perturbs bacterial growth, surprisingly, we found that reducing the levels of other targets by more than 97% had little or no effect on growth. For one of these targets, dihydrofolate reductase, metabolic analysis suggested that depletion mimics the use of subinhibitory concentrations of the antibiotic trimethroprim. These observations indicate that some drug targets can exist at levels much higher than are needed to support growth. However, protein depletion can be used to identify promising drug targets that are particularly vulnerable to inhibition.

SUBMITTER: Wei JR

PROVIDER: S-EPMC3053961 | biostudies-literature | 2011 Mar

REPOSITORIES: biostudies-literature

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Depletion of antibiotic targets has widely varying effects on growth.

Wei Jun-Rong JR Krishnamoorthy Vidhya V Murphy Kenan K Kim Jee-Hyun JH Schnappinger Dirk D Alber Tom T Sassetti Christopher M CM Rhee Kyu Y KY Rubin Eric J EJ

Proceedings of the National Academy of Sciences of the United States of America 20110222 10

It is often assumed that antibiotics act on the most vulnerable cellular targets, particularly those that require limited inhibition to block growth. To evaluate this assumption, we developed a genetic method that can inducibly deplete targeted proteins and that mimics their chemical inactivation. We applied this system to current antibiotic targets in mycobacteria. Although depleting some antibiotic targets significantly perturbs bacterial growth, surprisingly, we found that reducing the levels ...[more]

PMID: 21368134

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Project description:While the misuse of antibiotics has clearly contributed to the emergence and proliferation of resistant bacterial pathogens, with major health consequences, it remains less clear if the widespread use of disinfectants, such as benzalkonium chlorides (BAC), a different class of biocides than antibiotics, has contributed to this problem. Here, we provide evidence that exposure to BAC coselects for antibiotic-resistant bacteria and describe the underlying genetic mechanisms. After inoculation with river sediment, BAC-fed bioreactors selected for several bacterial taxa, including the opportunistic pathogen Pseudomonas aeruginosa, that were more resistant to several antibiotics than their counterparts in a control (no BAC) bioreactor. A metagenomic analysis of the bioreactor microbial communities, confirmed by gene cloning experiments with the derived isolates, suggested that integrative and conjugative elements encoding a BAC efflux pump together with antibiotic resistance genes were responsible for these results. Furthermore, the exposure of the P. aeruginosa isolates to increasing concentrations of BAC selected for mutations in pmrB (polymyxin resistance) and physiological adaptations that contributed to a higher tolerance to polymyxin B and other antibiotics. The physiological adaptations included the overexpression of mexCD-oprJ multidrug efflux pump genes when BAC was added in the growth medium at subinhibitory concentrations. Collectively, our results demonstrated that disinfectants promote antibiotic resistance via several mechanisms and highlight the need to remediate (degrade) disinfectants in nontarget environments to further restrain the spread of antibiotic-resistant bacteria.IMPORTANCE Benzalkonium chlorides (BAC) are biocides broadly used in disinfectant solutions. Disinfectants are widely used in food processing lines, domestic households, and pharmaceutical products and are typically designed to have a different mode of action than antibiotics to avoid interfering with the use of the latter. Whether exposure to BAC makes bacteria more resistant to antibiotics remains an unresolved issue of obvious practical consequences for public health. Using an integrated approach that combines metagenomics of natural microbial communities with gene cloning experiments with isolates and experimental evolution assays, we show that the widely used benzalkonium chloride disinfectants promote clinically relevant antibiotic resistance. Therefore, more attention should be given to the usage of these disinfectants, and their fate in nontarget environments should be monitored more tightly.

Project description:Extensive use of colistin in food animals is deemed a major driving force for the emergence and transmission of mcr-1 However, a non-colistin usage factor(s) contributing to mobile colistin resistance may also exist in animal production systems. Given that polymyxin, a bacterium-derived peptide antibiotic, has been successfully used as a surrogate to study bacterial resistance to antimicrobial peptides (AMPs), acquisition of MCR-1 may confer cross-resistance to the unrelated AMPs implicated in practical applications. To test this, we first constructed Escherichia coli recombinant strains differing only in the presence or absence of functional MCR-1. Among diverse tested AMPs, MCR-1 was observed to confer cross-resistance to bacitracin, an in-feed antibiotic widely used in animal industry. The significantly (2-fold) increased bacitracin MIC was confirmed by using different bacitracin products, broth media, and laboratory host strains for susceptibility tests. Subsequently, an original mcr-1 gene-bearing plasmid, pSLy21, was conjugatively transferred to eight clinical E. coli recipient strains isolated from diarrheic pigs, which also led to significantly increased MICs of both colistin (4-fold to 8-fold) and bacitracin (2-fold). Growth curve examination further demonstrated that MCR-1 provides a growth advantage to various E. coli strains in the presence of bacitracin. Given that bacitracin, a feed additive displaying low absorption in the intestine, can be used in food animals with no withdrawal required, imprudent use of bacitracin in food animals may serve as a risk factor to enhance the ecological fitness of MCR-1-positive E. coli strains, consequently facilitating the persistence and transmission of plasmid-mediated colistin resistance in agricultural ecosystem.IMPORTANCE Polymyxins (e.g., colistin) are the drugs of last resort to treat multidrug-resistant infections in humans. To control mobile colistin resistance, there is a worldwide trend to limit colistin use in animal production. However, simply limiting colistin use in animal production may still not effectively mitigate colistin resistance due to an overlooked non-colistin usage factor(s). Using controlled systems, in this study, we observed that MCR-1 confers cross-resistance to bacitracin, a popular in-feed antibiotic used in food animals. Thus, imprudent and extensive usage of bacitracin in food animals may serve as a non-colistin usage risk factor for the transmissible colistin resistance. Further comprehensive in vitro and in vivo studies are highly warranted to generate science-based information for risk assessment and risk management of colistin resistance, consequently facilitating the development of proactive and effective strategies to mitigate colistin resistance in animal production system and protect public health.

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Depletion of antibiotic targets has widely varying effects on growth.

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Depletion of antibiotic targets has widely varying effects on growth.

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