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Spontaneous and evolutionary changes in the antibiotic resistance of Burkholderia cenocepacia observed by global gene expression analysis.

ABSTRACT: BACKGROUND: Burkholderia cenocepacia is a member of the Burkholderia cepacia complex group of bacteria that cause infections in individuals with cystic fibrosis. B. cenocepacia isolate J2315 has been genome sequenced and is representative of a virulent, epidemic CF strain (ET12). Its genome encodes multiple antimicrobial resistance pathways and it is not known which of these is important for intrinsic or spontaneous resistance. To map these pathways, transcriptomic analysis was performed on: (i) strain J2315 exposed to sub-inhibitory concentrations of antibiotics and the antibiotic potentiator chlorpromazine, and (ii) on spontaneous mutants derived from J2315 and with increased resistance to the antibiotics amikacin, meropenem and trimethoprim-sulfamethoxazole. Two pan-resistant ET12 outbreak isolates recovered two decades after J2315 were also compared to identify naturally evolved gene expression changes. RESULTS: Spontaneous resistance in B. cenocepacia involved more gene expression changes and different subsets of genes than those provoked by exposure to sub inhibitory concentrations of each antibiotic. The phenotype and altered gene expression in the resistant mutants was also stable irrespective of the presence of the priming antibiotic. Both known and novel genes involved in efflux, antibiotic degradation/modification, membrane function, regulation and unknown functions were mapped. A novel role for the phenylacetic acid (PA) degradation pathway genes was identified in relation to spontaneous resistance to meropenem and glucose was found to repress their expression. Subsequently, 20 mM glucose was found to produce greater that 2-fold reductions in the MIC of multiple antibiotics against B. cenocepacia J2315. Mutation of an RND multidrug efflux pump locus (BCAM0925-27) and squalene-hopene cyclase gene (BCAS0167), both upregulated after chlorpromazine exposure, confirmed their role in resistance. The recently isolated outbreak isolates had altered the expression of multiple genes which mirrored changes seen in the antibiotic resistant mutants, corroborating the strategy used to model resistance. Mutation of an ABC transporter gene (BCAS0081) upregulated in both outbreak strains, confirmed its role in B. cenocepacia resistance. CONCLUSIONS: Global mapping of the genetic pathways which mediate antibiotic resistance in B. cenocepacia has revealed that they are multifactorial, identified potential therapeutic targets and also demonstrated that putative catabolite repression of genes by glucose can improve antibiotic efficacy.

SUBMITTER: Sass A

PROVIDER: S-EPMC3155924 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Spontaneous and evolutionary changes in the antibiotic resistance of Burkholderia cenocepacia observed by global gene expression analysis.

Sass Andrea A Marchbank Angela A Tullis Elizabeth E Lipuma John J JJ Mahenthiralingam Eshwar E

BMC genomics 20110722

<h4>Background</h4>Burkholderia cenocepacia is a member of the Burkholderia cepacia complex group of bacteria that cause infections in individuals with cystic fibrosis. B. cenocepacia isolate J2315 has been genome sequenced and is representative of a virulent, epidemic CF strain (ET12). Its genome encodes multiple antimicrobial resistance pathways and it is not known which of these is important for intrinsic or spontaneous resistance. To map these pathways, transcriptomic analysis was performed ...[more]

PMID: 21781329

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Project description:Bacteria from the Burkholderia cepacia complex (Bcc) are the only group of cystic fibrosis (CF) respiratory pathogens that may cause death by an invasive infection known as cepacia syndrome. Their large genome (> 7000 genes) and multiple pathways encoding the same putative functions make virulence factor identification difficult in these bacteria.A novel microarray was designed to the genome of Burkholderia cenocepacia J2315 and transcriptomics used to identify genes that were differentially regulated when the pathogen was grown in a CF sputum-based infection model. Sputum samples from CF individuals infected with the same B. cenocepacia strain as genome isolate were used, hence, other than a dilution into a minimal growth medium (used as the control condition), no further treatment of the sputum was carried out.A total of 723 coding sequences were significantly altered, with 287 upregulated and 436 downregulated; the microarray-observed expression was validated by quantitative PCR on five selected genes. B. cenocepacia genes with putative functions in antimicrobial resistance, iron uptake, protection against reactive oxygen and nitrogen species, secretion and motility were among the most altered in sputum. Novel upregulated genes included: a transmembrane ferric reductase (BCAL0270) implicated in iron metabolism, a novel protease (BCAL0849) that may play a role in host tissue destruction, an organic hydroperoxide resistance gene (BCAM2753), an oxidoreductase (BCAL1107) and a nitrite/sulfite reductase (BCAM1676) that may play roles in resistance to the host defenses. The assumptions of growth under iron-depletion and oxidative stress formulated from the microarray data were tested and confirmed by independent growth of B. cenocepacia under each respective environmental condition.Overall, our first full transcriptomic analysis of B. cenocepacia demonstrated the pathogen alters expression of over 10% of the 7176 genes within its genome when it grows in CF sputum. Novel genetic pathways involved in responses to antimicrobial resistance, oxidative stress, and iron metabolism were revealed by the microarray analysis. Virulence factors such as the cable pilus and Cenocepacia Pathogenicity Island were unaltered in expression. However, B. cenocepacia sustained or increased expression of motility-associated genes in sputum, maintaining a potentially invasive phenotype associated with cepacia syndrome.

Project description:BACKGROUND: Burkholderia cenocepacia is an opportunistic pathogen causing life-threatening infections in patients with cystic fibrosis. The bacterium survives within macrophages by interfering with endocytic trafficking and delaying the maturation of the B. cenocepacia-containing phagosome. We hypothesize that B. cenocepacia undergoes changes in gene expression after internalization by macrophages, inducing genes involved in intracellular survival and host adaptation. RESULTS: We examined gene expression by intracellular B. cenocepacia using selective capture of transcribed sequences (SCOTS) combined with microarray analysis. We identified 767 genes with significantly different levels of expression by intracellular bacteria, of which 330 showed increased expression and 437 showed decreased expression. Affected genes represented all aspects of cellular life including information storage and processing, cellular processes and signaling, and metabolism. In general, intracellular gene expression demonstrated a pattern of environmental sensing, bacterial response, and metabolic adaptation to the phagosomal environment. Deletion of various SCOTS-identified genes affected bacterial entry into macrophages and intracellular replication. We also show that intracellular B. cenocepacia is cytotoxic towards the macrophage host, and capable of spread to neighboring cells, a role dependent on SCOTS-identified genes. In particular, genes involved in bacterial motility, cobalamin biosynthesis, the type VI secretion system, and membrane modification contributed greatly to macrophage entry and subsequent intracellular behavior of B. cenocepacia. CONCLUSIONS: B. cenocepacia enters macrophages, adapts to the phagosomal environment, replicates within a modified phagosome, and exhibits cytotoxicity towards the host cells. The analysis of the transcriptomic response of intracellular B. cenocepacia reveals that metabolic adaptation to a new niche plays a major role in the survival of B. cenocepacia in macrophages. This adaptive response does not require the expression of any specific virulence-associated factor, which is consistent with the opportunistic nature of this microorganism. Further investigation into the remaining SCOTS-identified genes will provide a more complete picture of the adaptive response of B. cenocepacia to the host cell environment.

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Spontaneous and evolutionary changes in the antibiotic resistance of Burkholderia cenocepacia observed by global gene expression analysis.

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Spontaneous and evolutionary changes in the antibiotic resistance of Burkholderia cenocepacia observed by global gene expression analysis.

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