Key role for efflux in the preservative susceptibility and adaptive resistance of Burkholderia cepacia complex bacteria

Abstract

Bacteria from the Burkholderia cepacia complex (Bcc) are encountered as industrial contaminants, and little is known about the species involved or their mechanisms of preservative resistance. Multilocus sequence typing (MLST) revealed that multiple Bcc species may cause contamination, with B. lata (n = 17) and B. cenocepacia (n = 11) dominant within the collection examined. At the strain level, 11 of the 31 industrial sequence types identified had also been recovered from either natural environments or clinical infections. Minimal inhibitory (MIC) and minimum bactericidal (MBC) preservative concentrations varied across 83 selected Bcc strains, with industrial strains demonstrating increased tolerance for dimethylol dimethyl hydantoin (DMDMH). Benzisothiazolinone (BIT), DMDMH, methylisothiazolinone (MIT), a blend of 3:1 methylisothiazolinone-chloromethylisothiazolinone (M-CMIT), methyl paraben (MP), and phenoxyethanol (PH), were all effective anti-Bcc preservatives; benzethonium chloride (BC) and sodium benzoate (SB) were least effective. Since B. lata was the dominant industrial Bcc species, the type strain, 383(T) (LMG 22485(T)), was used to study preservative tolerance. Strain 383 developed stable preservative tolerance for M-CMIT, MIT, BIT, and BC, which resulted in preservative cross-resistance and altered antibiotic susceptibility, motility, and biofilm formation. Transcriptomic analysis of the B. lata 383 M-CMIT-adapted strain demonstrated that efflux played a key role in its M-CMIT tolerance and elevated fluoroquinolone resistance. The role of efflux was corroborated using the inhibitor l-Phe-Arg-β-napthylamide, which reduced the MICs of M-CMIT and ciprofloxacin. In summary, intrinsic preservative tolerance and stable adaptive changes, such as enhanced efflux, play a role in the ability of Bcc bacteria to cause industrial contamination.

Fig 1

Susceptibility of Bcc species to M-CMIT. (A) MICs. (B) MBCs. The maximum level for use in rinse-off personal-care products in EU-regulated countries is 0.0015% (EU cosmetics directive 76/768/EEC annex VI).

Fig 2

Increased tolerance for dimethyol dimethyl hydantoin in Bcc isolates from industrial sources. The mean minimum inhibitory concentrations (A) and mean minimum bactericidal concentrations (B) of Bcc isolates from industrial sources were significantly higher than the values for Bcc isolates from clinical and environmental sources (*, P < 0.0001; two-tailed Mann-Whitney test). In EU-regulated countries, a maximum level of 0.3% DMDMH is regulated for use in rinse-off personal-care products (EU cosmetics directive 76/768/EEC annex VI). The error bars show standard errors.