Long-Term Exposure to Benzalkonium Chloride Disinfectants Results in Change of Microbial Community Structure and Increased Antimicrobial Resistance

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

Phylogenetic (A) and gene-content (B) diversity among the <i>Escherichia</i> strains.

<p>(A) The phylogenetic analysis was carried out based on the concatenated sequence alignment of five gene sequences using the neighbor-joining algorithm in MEGA 4.0 with default settings. (B) <i>Escherichia</i> strains were clustered based on the conservation pattern of 5,978 genes by a binary system (1 = presence and 0 = absence), using the CLUSTER 3.0 software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047005#pone.0047005-Eisen1" target="_blank">[38]</a> with Euclidean distance. Clustering was visualized in Treeview <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047005#pone.0047005-Saldanha1" target="_blank">[39]</a>. The values on the nodes of the trees represent bootstrap support from 100 replicates in both (A) and (B).</p

Gene content and sequence relatedness between the <i>Escherichia</i> strains and the reference strain.

<p>Each data point represents the gANI value (evolutionary relatedness) and the gene content conservation (functional relatedness) between a tester and the reference (Sakai) strains. The shared gene content (%) was normalized by the total number of the reference (Sakai) genes represented on the microarray.</p

<i>Escherichia</i> strains used in this study.

<p><i>Escherichia</i> strains used in this study.</p

Gene signatures of human strains.

<p>The 98 genes differentially shared between G-II and G-III (p-values lower than 10⁻⁴ by Fisher’s exact test) are shown. Genes in blue boxes indicate hypothetical proteins and genes in red boxes are discussed in the text (associated with stress defense and adhesion). The color denotes gene presence (yellow) and absence (black).</p

Genomic Diversity of <em>Escherichia</em> Isolates from Diverse Habitats

<div><p>Our understanding of the <em>Escherichia</em> genus is heavily biased toward pathogenic or commensal isolates from human or animal hosts. Recent studies have recovered <em>Escherichia</em> isolates that persist, and even grow, outside these hosts. Although the environmental isolates are typically phylogenetically distinct, they are highly related to and phenotypically indistinguishable from their human counterparts, including for the coliform test. To gain insights into the genomic diversity of <em>Escherichia</em> isolates from diverse habitats, including freshwater, soil, animal, and human sources, we carried out comparative DNA-DNA hybridizations using a multi-genome <em>E. coli</em> DNA microarray. The microarray was validated based on hybridizations with selected strains whose genome sequences were available and used to assess the frequency of microarray false positive and negative signals. Our results showed that human fecal isolates share two sets of genes (n>90) that are rarely found among environmental isolates, including genes presumably important for evading host immune mechanisms (e.g., a multi-drug transporter for acids and antimicrobials) and adhering to epithelial cells (e.g., hemolysin E and fimbrial-like adhesin protein). These results imply that environmental isolates are characterized by decreased ability to colonize host cells relative to human isolates. Our study also provides gene markers that can distinguish human isolates from those of warm-blooded animal and environmental origins, and thus can be used to more reliably assess fecal contamination in natural ecosystems.</p> </div