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The molecular basis for preservative resistance in Burkholderia cepacia complex bacteria

Rushton, Laura 2011. The molecular basis for preservative resistance in Burkholderia cepacia complex bacteria. PhD Thesis, Cardiff University.
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Burkholderia cepacia complex bacteria can contaminate and survive in a variety of antimicrobial and preserved industrial products. Contamination may lead to economic loss for manufacturers and also potentially pose a risk to the health of vulnerable consumers. Understanding the interaction between Bcc bacteria and preservatives, and the molecular basis for their resistance, is essential in order to better target these organisms and to facilitate the implementation of improved preservative strategies which target resistance mechanisms. In the present study, multi-locus sequence typing analysis of a collection of 67 Bcc isolates from environmentalindustrial sources was used to expand the current knowledge of Bcc species diversity within this niche and identified B. lata (n=17) and B. cenocepacia (n=11) as predominant species groups. The relationship between Bcc species diversity, isolation source and preservative susceptibility was investigated using a collection of 83 genetically diverse Bcc strains from clinical, environmental and environmental-industrial isolation sources. Susceptibility to eight preservatives was not related to Bcc taxonomy, as susceptibility profiles varied both between and within species groups. However, Bcc isolates from environmental-industrial sources had a significantly higher minimum inhibitory and minimum bactericidal concentration (MIC and MBC) for the formaldehyde releasing agent DMDM hydantoin. This suggests that for this preservative agent, susceptibility was related to source and that the selection of highly tolerant Bcc bacteria had occurred within the niche. Isothiazolone, DMDM hydantoin, phenoxyethanol and methyl paraben preservatives were observed to be highly efficacious against Bcc bacteria when evaluated in growth medium at the maximum concentration regulated for use in rinse-off personal care products in EU-regulated countries. Benzethonium chloride and sodium benzoate preservatives had the weakest anti-Bcc activity at these levels but were effective against several strains. Combinations of preservatives, and preservatives with potentiating agents, were evaluated for synergistic anti- Bcc activity. The greatest anti-Bcc activity was observed when isothiazolone preservatives were combined with EDTA or phenoxyethanol, with each combination resulting in an additive effect. The competency of Bcc bacteria to adapt to preservatives was explored via the progressive sub-culture of B. lata strain 383 in subinhibitory preservative concentrations. This genome sequence strain represented a Bcc species commonly encountered in the environmental-industrial niche. Stable adaptive-resistance to isothiazolone and benzethonium chloride preservatives was developed. Phenoxyethanol, DMDM hydantoin and methyl paraben preservatives were recalcitrant to B. lata strain 383 adaptation. The preservative and antibiotic susceptibility profiles of the adapted B. lata strain 383 derivatives differed, suggesting the induction of agent-specific adaptive-resistance mechanisms had occurred. The B. lata 383-CMIT,-BIT, derivatives (adapted respectively to chloromethylisothiazolinone and benzisothiazolinone), demonstrated cross-resistance to isothiazolone preservatives and fluoroquinolone antibiotics. Sequence analysis of the topoisomerase genes in these derivatives revealed fluoroquinolone resistance was not mediated by target modification. Preservative-induced adaptive resistance was not associated with overall increased multi-drug resistance. The molecular basis for resistance to DMDM hydantoin and isothiazolone preservatives was investigated via the random transposon mutagenesis of B. lata strain 383 using pTnModOTp’. Several genetic pathways were identified as putative preservative resistance determinants, suggesting that resistance is multi-factorial. These included the detoxification of formaldehyde via a glutathione-dependent pathway; a type II general secretory system (A3244_A3233 genes); a homologue of an ABC-type efflux system involved in resistance to organic solvents (A3512_A3517 genes); homologues of multi-drug RND-type efflux systems EmrB/QacA-Emr-TolC; and bacterial defence mechanisms against oxidative stress. A transcriptomic microarray-based approach was used to profile global gene expression of B. lata strain 383 in response to sub-MIC of 0.00162% DMDM hydantoin and 0.00001498% of a methylisothiazolinone and CMIT blend, as well as isothiazolone-induced adaptive resistance. With a 1.5-fold change and P < 0.05 confidence level criterion applied, few significant changes were observed after a single sub-MIC exposure, and the differential expression of putative resistance determinants identified by transposon mutagenesis was not induced at these concentrations. Isothiazolone-induced adaptive-resistance involved a greater number of significant gene expression changes that were stable irrespective of the presence of the priming agent, with 126 up-regulated and 90 down-regulated genes. Transcriptomic analysis suggested that isothiazolone-induced adaptive resistance was multi-factorial in nature, and identified active efflux as a putative key resistance mechanism. A novel role for a RND-type efflux system (B1004_B1006 genes) was identified, and the up-regulation of the ABC-efflux system (A3512_A3517 genes) and bacterial defence mechanisms against oxidative stress corroborated the transposon mutagenesis findings. B. lata strain 383-CMIT demonstrated a four-fold reduction in MIC for the priming preservative (2.81E-04%) in the presence of 512 mg/L of the efflux inhibitor PAβN. Resistance mechanism targeted preservative strategies such as using efflux inhibitors may work to improve preservation.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Biosciences
Subjects: Q Science > QR Microbiology > QR180 Immunology
Uncontrolled Keywords: Preservative resistance; Burkholderia cepacia complex
Funders: BBSRC
Date of First Compliant Deposit: 30 March 2016
Last Modified: 11 Mar 2022 09:09

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