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The contribution of antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates: an emphasis on efflux pump activity.

ABSTRACT: Due to the limited information of the contribution of various antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates, Antibiotic resistance mechanisms, including integron analysis, identification of quinolone resistance-determining region mutations, measurement of efflux pump activity, and sequence analysis of efflux pump regulators, were investigated in 66 clinical B. cepacia complex isolates. Species were identified via recA-RFLP and MALDI-TOF. Four genomovars were identified by recA-RFLP. B. cenocepacia (genomovar III) was the most prevalent genomovar (90.1%). Most isolates (60/66, 90.9%) were correctly identified by MALDI-TOF analysis. Clonal relatedness determined by PFGE analysis revealed 30 pulsotypes, including two major pulsotypes that comprised 22.7% and 18.2% of the isolates, respectively. Seventeen (25.8%) isolates harboured class 1 integron with various combinations of resistance genes. Among six levofloxacin-resistant isolates, five had single-base substitutions in the gyrA gene and three demonstrated efflux pump activities. Among the 42 isolates exhibiting resistance to at least one antimicrobial agent, 94.4% ceftazidime-resistant isolates (17/18) and 72.7% chloramphenicol-resistant isolates (16/22) demonstrated efflux pump activity. Quantitation of efflux pump RNA level and sequence analysis revealed that over-expression of the RND-3 efflux pump was attributable to specific mutations in the RND-3 efflux pump regulator gene. In conclusion, high-level expression of efflux pumps is prevalent in B. cepacia complex isolates. Mutations in the RND-3 efflux pump regulator gene are the major cause of efflux pump activity, resulting in the resistance to antibiotics in clinical B. cepacia complex isolates.

SUBMITTER: Tseng SP

PROVIDER: S-EPMC4143217 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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The contribution of antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates: an emphasis on efflux pump activity.

Tseng Sung-Pin SP Tsai Wan-Chi WC Liang Chih-Yuan CY Lin Yin-Shiou YS Huang Jun-Wei JW Chang Chung-Yu CY Tyan Yu-Chang YC Lu Po-Liang PL

PloS one 20140825 8

Due to the limited information of the contribution of various antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates, Antibiotic resistance mechanisms, including integron analysis, identification of quinolone resistance-determining region mutations, measurement of efflux pump activity, and sequence analysis of efflux pump regulators, were investigated in 66 clinical B. cepacia complex isolates. Species were identified via recA-RFLP and MALDI-TOF. Four genomovars were ...[more]

PMID: 25153194

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Project description:Members of the Burkholderia cepacia complex (Bcc) are an important cause of opportunistic or nosocomial infections that may be hard to treat due to a high incidence of multidrug resistance. We characterised a collection of 51 clinical isolates from this complex, assigning them to 18 sequence types using multi-locus sequence type analysis. Resistance to eight commonly used antibiotics was assessed using by using agar-dilution assays to calculate MICs and widespread and heterogeneous multidrug resistance was confirmed, with eight strains proving resistant to all antibiotics tested. Disc diffusion screening of antimicrobial activity of a range of plant essential oils against these Bcc isolates identified six oils with significant activity (lavender, lemongrass, marjoram, peppermint, tea tree and rosewood) and broth microdilution assays indicated that of these lemongrass and rosewood oils had the highest activity, with MIC50 values of 0.5% and MIC90 values of 1%. Comparison of MIC and MBC values showed that four of these six oils, including lemongrass and rosewood, were bacteriocidal rather than bacteriostatic in their effects. Qualitative analysis of the four bacteriocidal essential oils via GC/MS indicated the presence of 55 different component compounds, mostly monoterpenes. We assessed selected essential oil components as anti-Bcc agents and demonstrated that terpinen-4-ol and geraniol were effective with MICs of 0.125-0.5% (v/v) and 0.125-1% (v/v), respectively. Time-kill studies indicate that these two alcohols are effective against non-growing cells in an efflux-dependent manner. Analysis of bacterial leakage of potassium ions and 260 nm UV-absorbing material on treatment with terpinen-4-ol and geraniol suggested that the observed anti-Bcc activity was a consequence of membrane disruption. This finding was supported by a gas chromatography analysis of bacterial fatty acid methyl esters, which indicated changes in membrane fatty acid composition caused by terpinen-4-ol and geraniol. These essential oils or oil components may ultimately prove useful as therapeutic drugs, for example to treat Bcc infections in CF patients.

Project description:Burkholderia cepacia complex (Bcc) infection, associated with cystic fibrosis (CF) is intrinsically multidrug resistant to antibiotic treatment making eradication from the CF lung virtually impossible. Infection with Bcc leads to a rapid decline in lung function and is often a contraindication for lung transplant, significantly influencing morbidity and mortality associated with CF disease. Standard treatment frequently involves antibiotic combination therapy. However, no formal strategy has been adopted in clinical practice to guide successful eradication. A new class of direct-acting, large molecule polycationic glycopolymers, derivatives of a natural polysaccharide poly-N-acetyl-glucosamine (PAAG), are in development as an alternative to traditional antibiotic strategies. During treatment, PAAG rapidly targets the anionic structural composition of bacterial outer membranes. PAAG was observed to permeabilize bacterial membranes upon contact to facilitate potentiation of antibiotic activity. Three-dimensional checkerboard synergy analyses were used to test the susceptibility of eight Bcc strains (seven CF clinical isolates) to antibiotic combinations with PAAG or ceftazidime. Potentiation of tobramycin and meropenem activity was observed in combination with 8-128 ?g/mL PAAG. Treatment with PAAG reduced the minimum inhibitory concentration (MIC) of tobramycin and meropenem below their clinical sensitivity breakpoints (?4 ?g/mL), demonstrating the ability of PAAG to sensitize antibiotic resistant Bcc clinical isolates. Fractional inhibitory concentration (FIC) calculations showed PAAG was able to significantly potentiate antibacterial synergy with these antibiotics toward all Bcc species tested. These preliminary studies suggest PAAG facilitates a broad synergistic activity that may result in more positive therapeutic outcomes and supports further development of safe, polycationic glycopolymers for inhaled combination antibiotic therapy, particularly for CF-associated Bcc infections.

Dataset Information

The contribution of antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates: an emphasis on efflux pump activity.

Publications

The contribution of antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates: an emphasis on efflux pump activity.

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