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Clostridium difficile transcriptome analysis using pig ligated loop model reveals modulation of pathways not modulated in vitro.

ABSTRACT: A pig ligated loop model was used to analyze the in vivo transcriptome response of Clostridium difficile. Bacterial RNA from the loops was retrieved at different times and was used for microarray analysis. Several virulence-associated genes and genes involved in sporulation cascade were differentially expressed (DE). In concordance with observed upregulation of toxin genes in microarray, enzyme-linked immunosorbent assay estimation of total toxin showed high amounts of toxin in the loops. Several genes that were absent in primary annotation of C. difficile 630 but annotated in a secondary annotation were found to be DE. Pathway comparison of DE genes in vitro and in vivo showed that when several pathways were expressed in all conditions, several of the C. difficile pathways were uniquely expressed only in vivo. The pathways observed to be modulated only in this study could be targets of new therapeutic agents against C. difficile infection.

SUBMITTER: Scaria J

PROVIDER: S-EPMC3096783 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Clostridium difficile transcriptome analysis using pig ligated loop model reveals modulation of pathways not modulated in vitro.

Scaria Joy J Janvilisri Tavan T Fubini Susan S Gleed Robin D RD McDonough Sean P SP Chang Yung-Fu YF

The Journal of infectious diseases 20110601 11

A pig ligated loop model was used to analyze the in vivo transcriptome response of Clostridium difficile. Bacterial RNA from the loops was retrieved at different times and was used for microarray analysis. Several virulence-associated genes and genes involved in sporulation cascade were differentially expressed (DE). In concordance with observed upregulation of toxin genes in microarray, enzyme-linked immunosorbent assay estimation of total toxin showed high amounts of toxin in the loops. Severa ...[more]

PMID: 21592991

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Project description:Rifaximin is a rifampicin derivative, poorly absorbed by the gastro-intestinal tract. We studied the in vitro susceptibility to rifamixin of 1082 Clostridium difficile isolates; among these, 184 isolates from a strain collection were tested by an in-house rifaximin disc (40 µg) diffusion test, by an in-house rifaximin broth microdilution test, by rifampicin Etest and by rpoB gene sequencing. In the absence of respective CLSI or EUCAST MIC breakpoints for rifaximin and rifampicin against C. difficile we chose MIC ≥32 µg ml(-1) as criterion for reduced in vitro susceptibility. To further validate the disc diffusion test 898 consecutive clinical isolates were analysed using the disc diffusion test, the Etest and rpoB gene sequence analysis for all resistant strains. Rifaximin broth microdilution tests of the 184 reference strains yielded rifaximin MICs ranging from 0.001 (n = 1) to ≥1024 µg ml(-1) (n = 61); 62 isolates showed a reduced susceptibility (MIC ≥32 µg ml(-1)). All of these 62 strains showed rpoB gene mutations producing amino acid substitutions; the rifampicin- and rifaximin-susceptible strains showed either a wild-type sequence or silent amino acid substitutions (19 strains). For 11 arbitrarily chosen isolates with rifaximin MICs of >1024 µg ml(-1), rifaximin end-point MICs were determined by broth dilution: 4096 µg ml(-1) (n = 2), 8192 µg ml(-1) (n = 6), 16,384 µg ml(-1) (n = 2) and 32,678 µg ml(-1) (n = 1). Rifampicin Etests on the 184 C. difficile reference strains yielded MICs ranging from ≤0.002 (n = 117) to ≥32 µg ml(-1) (n = 59). Using a 38 mm inhibition zone as breakpoint for reduced susceptibility the use of rifaximin disc diffusion yielded 59 results correlating with those obtained by use of rifaximin broth microdilution in 98.4 % of the 184 strains tested. Rifampicin Etests performed on the 898 clinical isolates revealed that 67 isolates had MICs of ≥32 µg ml(-1). There were no discordant results observed among these isolates with reduced susceptibility using an MIC of ≥32 µg ml(-1) as breakpoint for reduced rifampicin susceptibility and a <38 mm inhibition zone as breakpoint for reduced rifaximin susceptibility. The prevalence of reduced susceptibility was 7.5 % for all isolates tested. However, for PCR ribotype 027 the prevalence of reduced susceptibility was 26 %. Susceptibility testing in the microbiology laboratory therefore could have an impact on the care and outcome of patients with infection. Our results show that rifaximin--despite its water-insolubility--may be a suitable candidate for disc diffusion testing.

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Clostridium difficile transcriptome analysis using pig ligated loop model reveals modulation of pathways not modulated in vitro.

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Clostridium difficile transcriptome analysis using pig ligated loop model reveals modulation of pathways not modulated in vitro.

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