Dataset Information

Intrinsic Resistance of Burkholderia cepacia Complex to Benzalkonium Chloride.

ABSTRACT: 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.

Importance

Benzyldimethylalkylammonium 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.

SUBMITTER: Ahn Y

PROVIDER: S-EPMC5120141 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Intrinsic Resistance of Burkholderia cepacia Complex to Benzalkonium Chloride.

Ahn Youngbeom Y Kim Jeong Myeong JM Kweon Ohgew O Kim Seong-Jae SJ Jones Richard C RC Woodling Kellie K Gamboa da Costa Gonçalo G LiPuma John J JJ Hussong David D Marasa Bernard S BS Cerniglia Carl E CE

mBio 20161122 6

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 (C<sub>14</sub>BDMA-Cl), dodecyldimethylbenzylammonium chloride (C<sub>12</sub>BDMA-Cl), dec ...[more]

PMID: 27879334

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Project description:Burkholderia pseudomallei, the founding member of the B. pseudomallei complex (Bpc), is a biothreat agent and causes melioidosis, a disease whose treatment mainly relies on ceftazidime and meropenem. The concern is that B. pseudomallei could enhance its drug resistance repertoire by the acquisition of DNA from resistant near-neighbor species. Burkholderia ubonensis, a member of the B. cepacia complex (Bcc), is commonly coisolated from environments where B. pseudomallei is present. Unlike B. pseudomallei, in which significant primary carbapenem resistance is rare, it is not uncommon in B. ubonensis, but the underlying mechanisms are unknown. We established that carbapenem resistance in B. ubonensis is due to an inducible class A PenB ?-lactamase, as has been shown for other Bcc bacteria. Inducibility is not sufficient for high-level resistance but also requires other determinants, such as a PenB that is more robust than that present in susceptible isolates, as well as other resistance factors. Curiously and diagnostic for the two complexes, both Bpc and Bcc bacteria contain distinct annotated PenA class A ?-lactamases. However, the protein from Bcc bacteria is missing its essential active-site serine and, therefore, is not a ?-lactamase. Regulated expression of a transcriptional penB'-lacZ (?-galactosidase) fusion in the B. pseudomallei surrogate B. thailandensis confirms that although Bpc bacteria lack an inducible ?-lactamase, they contain the components required for responding to aberrant peptidoglycan synthesis resulting from ?-lactam challenge. Understanding the diversity of antimicrobial resistance in Burkholderia species is informative about how the challenges arising from potential resistance transfer between them can be met.IMPORTANCE Burkholderia pseudomallei causes melioidosis, a tropical disease that is highly fatal if not properly treated. Our data show that, in contrast to B. pseudomallei, B. ubonensis ?-lactam resistance is fundamentally different because intrinsic resistance is mediated by an inducible class A ?-lactamase. This includes resistance to carbapenems. Our work demonstrates that studies with near-neighbor species are informative about the diversity of antimicrobial resistance in Burkholderia and can also provide clues about the potential of resistance transfer between bacteria inhabiting the same environment. Knowledge about potential adverse challenges resulting from the horizontal transfer of resistance genes between members of the two complexes enables the design of effective countermeasures.

Project description:Antibiotic resistance is a threat to our modern society, and new strategies leading to the identification of new molecules or targets to combat multidrug-resistant pathogens are needed. Species of the genus Burkholderia, including the Burkholderia cepacia complex (Bcc), Burkholderia pseudomallei, and Burkholderia mallei, can be highly pathogenic and are intrinsically resistant to multiple classes of antibiotics. Bcc species are nonetheless sensitive to extracellular products released by Pseudomonas aeruginosa in interspecies competition. We screened for Burkholderia transposon mutants with increased sensitivity to P. aeruginosa spent medium and identified multiple mutants in genes sharing homology with the Mla pathway. Insertional mutants in representative genes of the Bcc Mla pathway had a compromised cell membrane and were more sensitive to various extracellular stresses, including antibiotics and human serum. More precisely, mla mutants in the Bcc species Burkholderia cenocepacia and Burkholderia dolosa were more susceptible to Gram-positive antibiotics (i.e., macrolides and rifampin), fluoroquinolones, tetracyclines, and chloramphenicol. Genetic complementation of mlaC insertional mutants restored cell permeability and resistance to Gram-positive antibiotics. Importantly, Bcc mla mutants were not universally weaker strains since their susceptibilities to other classes of antibiotics were unaffected. Although cell permeability of homologous mla mutants in Escherichia coli or P. aeruginosa was also impaired, they were not more sensitive to Gram-positive antibiotics or other antimicrobials as was observed in Bcc mla mutants. Together, the data suggest that the Mla pathway in Burkholderia may play a different biological role, which could potentially represent a Burkholderia-specific drug target in combination therapy with antibiotic adjuvants.IMPORTANCE The outer membrane of Gram-negative bacteria acts as an effective barrier against toxic compounds, and therefore compromising this structure could increase sensitivity to currently available antibiotics. In this study, we show that the Mla pathway, a system involved in maintaining the integrity of the outer membrane, is genetically and functionally different in Burkholderia cepacia complex species compared to that in other proteobacteria. Mutants in mla genes of Burkholderia cenocepacia or Burkholderia dolosa were sensitive to Gram-positive antibiotics, while this effect was not observed in Escherichia coli or Pseudomonas aeruginosa The Mla pathway in Burkholderia species may represent an ideal genus-specific target to address their intrinsic antimicrobial resistances.

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.

Dataset Information

Intrinsic Resistance of Burkholderia cepacia Complex to Benzalkonium Chloride.

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Publications

Intrinsic Resistance of Burkholderia cepacia Complex to Benzalkonium Chloride.

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