Characterization of Proteobacterial Plasmid Integron-Encoded qac Efflux Pump Sequence Diversity and Quaternary Ammonium Compound Antiseptic Selection in Escherichia coli Grown Planktonically and as Biofilms.
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ABSTRACT:
SUBMITTER: Slipski CJ
PROVIDER: S-EPMC8448097 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A novel genomic island (LGI1) was discovered in Listeria monocytogenes isolates responsible for the deadliest listeriosis outbreak in Canada, in 2008. To investigate the functional role of LGI1, the outbreak strain 08-5578 was exposed to food chain-relevant stresses, and the expression of 16 LGI1 genes was measured. LGI1 genes with putative efflux (L. monocytogenes emrE [emrELm]), regulatory (lmo1851), and adhesion (sel1) functions were deleted, and the mutants were exposed to acid (HCl), cold (4°C), salt (10 to 20% NaCl), and quaternary ammonium-based sanitizers (QACs). Deletion of lmo1851 had no effect on the L. monocytogenes stress response, and deletion of sel1 did not influence Caco-2 and HeLa cell adherence/invasion, whereas deletion of emrE resulted in increased susceptibility to QACs (P < 0.05) but had no effect on the MICs of gentamicin, chloramphenicol, ciprofloxacin, erythromycin, tetracycline, acriflavine, and triclosan. In the presence of the QAC benzalkonium chloride (BAC; 5 ?g/ml), 14/16 LGI1 genes were induced, and lmo1861 (putative repressor gene) was constitutively expressed at 4 °C, 37 °C, and 52 °C and in the presence of UV exposure (0 to 30 min). Following 1 h of exposure to BAC (10 ?g/ml), upregulation of emrE (49.6-fold), lmo1851 (2.3-fold), lmo1861 (82.4-fold), and sigB (4.1-fold) occurred. Reserpine visibly suppressed the growth of the ?emrELm strain, indicating that QAC tolerance is due at least partially to efflux activity. These data suggest that a minimal function of LGI1 is to increase the tolerance of L. monocytogenes to QACs via emrELm. Since QACs are commonly used in the food industry, there is a concern that L. monocytogenes strains possessing emrE will have an increased ability to survive this stress and thus to persist in food processing environments.
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Project description:Staphylococcus aureus-associated infections can be difficult to treat due to multidrug resistance. Thus, infection prevention is critical. Cationic antiseptics, such as chlorhexidine (CHX) and benzalkonium chloride (BKC), are liberally used in health care and community settings to prevent infection. However, increased administration of antiseptics has selected for S. aureus strains that show reduced susceptibilities to cationic antiseptics. This increased resistance has been associated with carriage of specific efflux pumps (QacA, QacC, and NorA). Since prior published studies focused on different strains and on strains carrying only a single efflux gene, the relative importance of these various systems to antiseptic resistance is difficult to ascertain. To overcome this, we engineered a collection of isogenic S. aureus strains that harbored norA, qacA, and qacC, individually or in combination. MIC assays showed that qacA was associated with increased resistance to CHX, cetrimide (CT), and BKC, qacC was associated with resistance to CT and BKC, and norA was necessary for basal-level resistance to the majority of tested antiseptics. When all three pumps were present in a single strain, an additive effect was observed in the MIC for CT. Transcriptional analysis revealed that expression of qacA and norA was significantly induced following exposure to BKC. Alarmingly, in a strain carrying qacA and norA, preexposure to BKC increased CHX tolerance. Overall, our results reveal increased antiseptic resistance in strains carrying multiple efflux pumps and indicate that preexposure to BKC, which is found in numerous daily-use products, can increase CHX tolerance.IMPORTANCE S. aureus remains a significant cause of disease within hospitals and communities. To reduce the burden of S. aureus infections, antiseptics are ubiquitously used in our daily lives. Furthermore, many antiseptic compounds are dual purpose and are found in household products. The increased abundance of antiseptic compounds has selected for S. aureus strains that carry efflux pumps that increase resistance to antiseptic compounds; however, the effect of carrying multiple pumps within S. aureus is unclear. We demonstrated that an isogenic strain carrying multiple efflux pumps had an additive resistance phenotype to cetrimide. Moreover, in a strain carrying qacA and norA, increased chlorhexidine tolerance was observed after the strain was preexposed to subinhibitory concentrations of a different common-use antiseptic. Taken together, our findings demonstrate cooperation between antiseptic resistance efflux pumps and suggest that their protective phenotype may be exacerbated by priming with subinhibitory concentrations of household antiseptics.
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Project description:The qac genes of Staphylococcus species encode multidrug efflux pumps: membrane proteins that export toxic molecules and thus increase tolerance to a variety of compounds such as disinfecting agents, including quaternary ammonium compounds (for which they are named), intercalating dyes and some antibiotics. In Stapylococcus species, six different plasmid-encoded Qac efflux pumps have been described, and they belong to two major protein families. QacA and QacB are members of the Major Facilitator Superfamily, while QacC, QacG, QacH, and QacJ all belong to the Small Multidrug Resistance (SMR) family. Not all SMR proteins are called Qac and the reverse is also true, which has caused confusion in the literature and in gene annotations. The discovery of qac genes and their presence in various staphylococcal populations is briefly reviewed. A sequence comparison revealed that some of the PCR primers described in the literature for qac detection may miss particular qac genes due to lack of DNA conservation. Despite their resemblance in substrate specificity, the Qac proteins belonging to the two protein families have little in common. QacA and QacB are highly conserved in Staphylococcus species, while qacA was also detected in Enterococcus faecalis, suggesting that these plasmid-born genes have spread across bacterial genera. Nevertheless, these qacA and qacB genes are quite dissimilar to their closest homologues in other organisms. In contrast, SMR-type Qac proteins display considerable sequence variation, despite their short length, even within the Staphylococcus genus. Phylogenetic analysis of these genes identified similarity to a large number of other SMR members, found in staphylococci as well as in other genera. A number of phylogenetic trees of SMR Qac proteins are presented here, starting with genes present in S. aureus and S. epidermidis, and extending this to related genes found in other species of this genus, and finally to genes found in other genera.
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Project description:Biocides such as quaternary ammonium compounds (QACs) are potentially important contributors towards bacterial antimicrobial resistance development, however, their contributions are unclear due to a lack of internationally recognized biocide testing standards. Methods to detect QAC tolerance are limited to laborious traditional antimicrobial susceptibility testing (AST) methods. Here, we developed a rapid fluorescent dye-based membrane impermeant assay (RFDMIA) to discriminate QAC susceptibility among Gram-negative Enterobacterales and Pseudomonadales species. RFDMIA uses a membrane impermeant fluorescent dye, propidium iodide, in a 30-min 96-well fluorescent microplate-based assay where cell suspensions are exposed to increasing QAC concentrations. Our results demonstrate that RFDMIA can discriminate between QAC-susceptible and QAC-adapted Escherichia coli tolerant phenotypes and predict benzalkonium and cetrimide tolerance in all species tested except for intrinsically fluorescent Pseudomonas aeruginosa. RFDMIA identified a close association to minimum inhibitory concentration values determined by broth microdilution AST and increasing fluorescent dye emission values. RFDMIA emission values and scanning electron microscopy results also suggest that CET-adapted E. coli isolates have a CET dependence, where cells require sub-inhibitory CET concentrations to maintain bacilliform cell integrity. Overall, this study generates a new, rapid, sensitive fluorescent assay capable of detecting QAC-susceptible Gram-negative bacteria phenotypes and cell membrane perturbations.
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Project description:Antiseptics are widely used in dental practice and included in numerous over-the-counter oral care products. However, the effects of routine antiseptic use on microbial composition of oral biofilms and on the emergence of resistant phenotypes remain unclear. Microcosm biofilms were inoculated from saliva samples of four donors and cultured in the Amsterdam Active Attachment biofilm model for 3 days. Then, they were treated two times daily with chlorhexidine digluconate (CHX) or cetylpyridinium chloride (CPC) for a period of 7 days. Ecological changes upon these multiple antiseptic treatments were evaluated by semiconductor-based sequencing of bacterial 16S rRNA genes and identification of amplicon sequence variants (ASVs). Furthermore, culture-based approaches were used for colony-forming units (CFU) assay, identification of antiseptic-resistant phenotypes using an agar dilution method, and evaluation of their antibiotic susceptibilities. Both CHX and CPC showed only slight effects on CFU and could not inhibit biofilm growth despite the two times daily treatment for 7 days. Both antiseptics showed significant ecological effects on the microbial compositions of the surviving microbiota, whereby CHX led to enrichment of rather caries-associated saccharolytic taxa and CPC led to enrichment of rather gingivitis-associated proteolytic taxa. Antiseptic-resistant phenotypes were isolated on antiseptic-containing agar plates, which also exhibited phenotypic resistance to various antibiotics. Our results highlight the need for further research into potential detrimental effects of antiseptics on the microbial composition of oral biofilms and on the spread of antimicrobial resistance in the context of their frequent use in oral healthcare.
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Project description:There is a lack of comprehensive toxicity studies of QACs associated with pulmonary toxicity. Therefore, we aimed to elucidate the potential pulmonary toxic effects of QACs based on toxicogenomic approach. We conducted transcriptome analysis using RNA sequencing to identify alterations in gene expression associated with QACs exposure.
2024-04-08 | GSE263314 | GEO
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