While 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, a different class of biocides than antibiotics such as benzalkonium chlorides (BAC), has contributed to this problem. Here, we provide evidence that exposure to BAC co-selects for antibiotic-resistant bacteria, and describe the underlying genetic mechanisms. BAC-fed bioreactors inoculated with river sediment selected for several bacterial taxa, including the opportunistic pathogen , that were more resistant to several antibiotics compared to their counterparts in a control (no BAC) bioreactor. 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. Further, exposure of the isolates to increasing concentrations of BAC selected for mutations in (polymyxin resistance) and physiological adaptations that contributed to higher tolerance to polymyxin B and other antibiotics. Physiological adaptations included, for example, the overexpression of multidrug efflux pump genes when BAC was added in the growth medium at sub-inhibitory concentrations. Collectively, our results demonstrated that disinfectants can promote antibiotic resistance via several mechanisms, and highlight the need to remediate (degrade) disinfectants in non-target environments to further restrain the spread of antibiotic resistant bacteria. Benzalkonium chlorides (BAC) are broadly used biocides in disinfectant solutions. Disinfectants are widely used in food processing lines, domestic households, and pharmaceuticals products, and are typically designed to have a different mode of action than antibiotics in order to not interfere with the use of the latter. Whether or not exposure to BAC makes bacteria more resistant to antibiotics remains, however, an unresolved issue of obvious practical consequences for public health. Using an integrated approach that combined metagenomics of natural microbial communities with gene cloning experiments with isolates and experimental evolution assays, we show that the widely used benzalkonium chloride disinfectants can promote clinically-relevant antibiotic resistance. Therefore, more attention should be given to the usage of these disinfectants, and their fate in non-target environments should be more tightly monitored.

Lake Lanier is an important freshwater lake for the southeast United States, as it represents the main source of drinking water for the Atlanta metropolitan area and is popular for recreational activities. Temperate freshwater lakes such as Lake Lanier are underrepresented among the growing number of environmental metagenomic data sets, and little is known about how functional gene content in freshwater communities relates to that of other ecosystems. To better characterize the gene content and variability of this freshwater planktonic microbial community, we sequenced several samples obtained around a strong summer storm event and during the fall water mixing using a random whole-genome shotgun (WGS) approach. Comparative metagenomics revealed that the gene content was relatively stable over time and more related to that of another freshwater lake and the surface ocean than to soil. However, the phylogenetic diversity of Lake Lanier communities was distinct from that of soil and marine communities. We identified several important genomic adaptations that account for these findings, such as the use of potassium (as opposed to sodium) osmoregulators by freshwater organisms and differences in the community average genome size. We show that the lake community is predominantly composed of sequence-discrete populations and describe a simple method to assess community complexity based on population richness and evenness and to determine the sequencing effort required to cover diversity in a sample. This study provides the first comprehensive analysis of the genetic diversity and metabolic potential of a temperate planktonic freshwater community and advances approaches for comparative metagenomics.

The effect of benzalkonium chlorides (BACs), a widely used class of quaternary ammonium disinfectants, on microbial community structure and antimicrobial resistance was investigated using three aerobic microbial communities: BACs-unexposed (DP, fed a mixture of dextrin/peptone), BACs-exposed (DPB, fed a mixture of dextrin/peptone and BACs), and BACs-enriched (B, fed only BACs). Long-term exposure to BACs reduced community diversity and resulted in the enrichment of BAC-resistant species, predominantly Pseudomonas species. Exposure of the two microbial communities to BACs significantly decreased their susceptibility to BACs as well as three clinically relevant antibiotics (penicillin G, tetracycline, ciprofloxacin). Increased resistance to BACs and penicillin G of the two BACs-exposed communities is predominantly attributed to degradation or transformation of these compounds, whereas resistance to tetracycline and ciprofloxacin is largely due to the activity of efflux pumps. Quantification of several key multidrug resistance genes showed a much higher number of copies of these genes in the DPB and B microbial communities compared to the DP community. Collectively, our findings indicate that exposure of a microbial community to BACs results in increased antibiotic resistance, which has important implications for both human and environmental health.

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