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Genomic and Transcriptomic Insights into How Bacteria Withstand High Concentrations of Benzalkonium Chloride Biocides.

ABSTRACT: Benzalkonium chlorides (BAC) are commonly used biocides in broad-spectrum disinfectant solutions. How microorganisms cope with BAC exposure remains poorly understood, despite its importance for disinfection and disinfectant-induced antibiotic resistance. To provide insights into these issues, we exposed two isolates of an opportunistic pathogen, Pseudomonas aeruginosa, to increasing concentrations of BAC. One isolate was preadapted to BAC, as it originated from a bioreactor fed with subinhibitory concentrations of BAC for 3 years, while the other originated from a bioreactor that received no BAC. Replicated populations of both isolates were able to survive high concentrations of BAC, up to 1,200 and 1,600 mg/liter for the non- and preadapted strains, respectively, exceeding typical application doses. Transcriptome sequencing (RNA-seq) analysis revealed upregulation of efflux pump genes and decreased expression of porins related to BAC transport as well as reduced growth rate. Increased expression of spermidine (a polycation) synthase genes and mutations in the pmrB (polymyxin resistance) gene, which cause a reduction in membrane negative charge, suggested that a major adaptation to exposure to the cationic surfactant BAC was to actively stabilize cell surface charge. Collectively, these results revealed that P. aeruginosa adapts to BAC exposure by a combination of mechanisms and provided genetic markers to monitor BAC-resistant organisms that may have applications in the practice of disinfection.IMPORTANCE BAC are widely used as biocides in disinfectant solutions, food-processing lines, domestic households, and health care facilities. Due to their wide use and mode of action, there has been rising concern that BAC may promote antibiotic resistance. Consistent with this idea, at least 40 outbreaks have been attributed to infection by disinfectant- and antibiotic-resistant pathogens such as P. aeruginosa However, the underlying molecular mechanisms that bacteria use to deal with BAC exposure remain poorly elucidated. Elucidating these mechanisms may be important for monitoring and limiting the spread of disinfectant-resistant pathogens. Using an integrated approach that combined genomics and transcriptomics with physiological characterization of BAC-adapted isolates, this study provided a comprehensive understanding of the BAC resistance mechanisms in P. aeruginosa Our findings also revealed potential genetic markers to detect and monitor the abundance of BAC-resistant pathogens across clinical or environmental settings. This work contributes new knowledge about high concentrations of benzalkonium chlorides disinfectants-resistance mechanisms at the whole-cell genomic and transcriptomic level.

SUBMITTER: Kim M

PROVIDER: S-EPMC5981084 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Genomic and Transcriptomic Insights into How Bacteria Withstand High Concentrations of Benzalkonium Chloride Biocides.

Kim Minjae M Hatt Janet K JK Weigand Michael R MR Krishnan Raj R Pavlostathis Spyros G SG Konstantinidis Konstantinos T KT

Applied and environmental microbiology 20180531 12

Benzalkonium chlorides (BAC) are commonly used biocides in broad-spectrum disinfectant solutions. How microorganisms cope with BAC exposure remains poorly understood, despite its importance for disinfection and disinfectant-induced antibiotic resistance. To provide insights into these issues, we exposed two isolates of an opportunistic pathogen, <i>Pseudomonas aeruginosa</i>, to increasing concentrations of BAC. One isolate was preadapted to BAC, as it originated from a bioreactor fed with subin ...[more]

PMID: 29654181

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Project description:Assessing the risk of resistance associated with biocide exposure commonly involves exposing microorganisms to biocides at concentrations close to the MIC. With the aim of representing exposure to environmental biocide residues, Escherichia coli MG1655 was grown for 20 passages in the presence or absence of benzalkonium chloride (BAC) at 100 ng/liter and 1,000 ng/liter (0.0002% and 0.002% of the MIC, respectively). BAC susceptibility, planktonic growth rates, motility, and biofilm formation were assessed, and differentially expressed genes were determined via transcriptome sequencing. Planktonic growth rate and biofilm formation were significantly reduced (P < 0.001) following BAC adaptation, while BAC minimum bactericidal concentration increased 2-fold. Transcriptomic analysis identified 289 upregulated and 391 downregulated genes after long-term BAC adaptation compared with the respective control organism passaged in BAC-free medium. When the BAC-adapted bacterium was grown in BAC-free medium, 1,052 genes were upregulated and 753 were downregulated. Repeated passage solely in biocide-free medium resulted in 460 upregulated and 476 downregulated genes compared with unexposed bacteria. Long-term exposure to environmentally relevant BAC concentrations increased the expression of genes associated with efflux and reduced the expression of genes associated with outer-membrane porins, motility, and chemotaxis. This was manifested phenotypically through the loss of function (motility). Repeated passage in a BAC-free environment resulted in the upregulation of multiple respiration-associated genes, which was reflected by increased growth rate. In summary, repeated exposure of E. coli to BAC residues resulted in significant alterations in global gene expression that were associated with minor decreases in biocide susceptibility, reductions in growth rate and biofilm formation, and loss of motility.IMPORTANCE Exposure to very low concentrations of biocides in the environment is a poorly understood risk factor for antimicrobial resistance. Repeated exposure to trace levels of the biocide benzalkonium chloride (BAC) resulted in loss of function (motility) and a general reduction in bacterial fitness but relatively minor decreases in susceptibility. These changes were accompanied by widespread changes in the Escherichia coli transcriptome. These results demonstrate the importance of including phenotypic characterization in studies designed to assess the risks of biocide exposure.

Project description:INTRODUCTION:The preservative benzalkonium chloride (BAK) is used to preserve several topical, intraocular pressure (IOP)-lowering glaucoma medications but can cause tolerability concerns that may lead to decreased adherence to treatment and ultimately diminish the effectiveness of IOP control. The study aimed to determine the efficacy and tolerability of BAK-free travoprost preserved with polyquaternium-1 in glaucoma patients switched from BAK-preserved latanoprost or bimatoprost. METHODS:This 12-week, open-label study was conducted in Europe between December 2011 and February 2013. We enrolled adult patients with open-angle glaucoma or ocular hypertension who were receiving BAK-preserved latanoprost 0.005% or bimatoprost 0.01% and, in the opinion of the investigator, would benefit from transition to BAK-free travoprost 0.004% preserved with polyquaternium-1 because of tolerability concerns. Assessments included IOP, proportion of patients with IOP ?18 mmHg, ocular surface status, hyperemia, patient treatment preference, and adherence. Adverse events were recorded throughout the study. RESULTS:Of the 202 patients screened, 187 patients were included in the intent-to-treat population (mean age, 66.6 years; range, 19-90 years). The mean IOP significantly reduced from baseline (17.0 mmHg) to week 6 (mean change, -1.17 mmHg; P<0.001) and week 12 (-1.16 mmHg; P<0.001). At week 12, more patients achieved IOP ?18 mmHg (81.2% versus 73.3% at baseline), and ocular surface disease severity improved from baseline to week 12. Most patients preferred BAK-free travoprost (74.9%) versus their previous medication and were very confident in their adherence (84.1%). Reduced visual acuity and eye pruritus were the most common adverse events (2.5% each). CONCLUSION:BAK-free travoprost 0.004% preserved with polyquaternium-1 was efficacious and well tolerated and may be an advantageous prostaglandin analog option for patients with open-angle glaucoma or ocular hypertension who are intolerant to BAK-preserved latanoprost or bimatoprost.

Project description:Pharmaceutical products that are contaminated with Burkholderia cepacia complex (BCC) bacteria may pose serious consequences to vulnerable patients. Benzyldimethylalkylammonium chloride (BZK) cationic surfactants are extensively used in medical applications and have been implicated in the coselection of antimicrobial resistance. The ability of BCC to degrade BZK, tetradecyldimethylbenzylammonium chloride (C14BDMA-Cl), dodecyldimethylbenzylammonium chloride (C12BDMA-Cl), decyldimethylbenzylammonium chloride (C10BDMA-Cl), hexyldimethylbenzylammonium chloride, and benzyltrimethylammonium chloride was determined by incubation in 1/10-diluted tryptic soy broth (TSB) to determine if BCC bacteria have the ability to survive and inactivate these disinfectants. With BZK, C14BDMA-Cl, and C12BDMA-Cl, inhibition of the growth of 20 BCC strains was observed in disinfectant solutions that ranged from 64 to 256 µg/ml. The efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone increased the sensitivity of bacteria to 64 µg/ml BZK. The 20 BCC strains grew well in 1/10-diluted TSB medium with BZK, C12BDMA-Cl, and C10BDMA-Cl; they absorbed and degraded the compounds in 7 days. Formation of benzyldimethylamine and benzylmethylamine as the initial metabolites suggested that the cleavage of the C alkyl-N bond occurred as the first step of BZK degradation by BCC bacteria. Proteomic data confirmed the observed efflux activity and metabolic inactivation via biodegradation in terms of BZK resistance of BCC bacteria, which suggests that the two main resistance mechanisms are intrinsic and widespread.ImportanceBenzyldimethylalkylammonium chloride is commonly used as an antiseptic in the United States. Several recent microbial outbreaks were linked to antiseptics that were found to contain strains of the Burkholderia cepacia complex. Burkholderia species survived in antiseptics, possibly because of the degradation of antiseptic molecules or regulation of relevant gene expression. In this study, we assessed the efflux pump and the potential of B. cepacia complex bacteria to degrade benzyldimethylalkylammonium chloride and improved our understanding of the resistance mechanisms, by using proteomic and metabolic information. To our knowledge, this is the first systematic report of the intrinsic mechanisms of B. cepacia complex strain resistance to benzyldimethylalkylammonium chloride, based on the metabolic and proteomic evidence for efflux pumps and the complete biodegradation of benzyldimethylalkylammonium chloride.

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:BackgroundBiocide disinfectants are essential tools in infection control, but their use can inadvertently contribute to emergence of antibiotic-resistant bacteria. In this study we systematically examine the effect of the biocide benzalkonium chloride, which is primarily used for surface disinfection but is also present as a preservative in many consumer products, on the activity of aminoglycoside antibiotics in Acinetobacter baumannii.MethodsThe effect of subinhibitory BAC on aminoglycoside treatment of A. baumannii ATCC17978 was investigated using time-to-kill assays, MIC determination, directed evolution experiments, fluctuation tests and labelled gentamicin accumulation assays. Further MIC determinations and directed evolution experiments were performed with additional Gram-negative ESKAPE pathogens.FindingsIn A. baumannii ATCC17978, BAC prevents gentamicin killing and drastically increases the frequency at which resistant mutants emerge, through reducing intracellular antibiotic accumulation. BAC also increases the MIC of multiple aminoglycoside antibiotics (kanamycin, tobramycin, streptomycin, gentamicin and amikacin). BAC promotes the emergence of mutants with reduced gentamicin susceptibility in other Gram-negative ESKAPE pathogens but does not always alter the MIC. These effects occur at BAC concentrations which are similar to residual levels in high-use environments, and just below the concentration range for BAC when used as a preservative in eye drops and ear drops.InterpretationOur results suggest that subinhibitory BAC has the potential to antagonise aminoglycoside activity and promote the emergence of bacterial mutants with reduced susceptibility. We suggest that the extremely widespread use of BAC in clinical and home settings and its long half-life mean there is potential for these interactions to occur in the environment, or in patients who use BAC-containing products while taking aminoglycosides to treat skin, eye or ear infections, although such co-exposure is likely to be rare. We suggest that biocide stewardship is needed to prevent the types of exposure that can contribute to antibiotic resistance.FundingThis work was funded by the National Health and Medical Research Council of Australia. The funders had no role in study design, interpretation or decision to publish.

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Genomic and Transcriptomic Insights into How Bacteria Withstand High Concentrations of Benzalkonium Chloride Biocides.

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Genomic and Transcriptomic Insights into How Bacteria Withstand High Concentrations of Benzalkonium Chloride Biocides.

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