Abstract

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.

Highlights

  • Clostridium difficile is a spore-forming Gram-positive anaerobic bacterium and a major cause of nosocomial and community-acquired diarrhoea (Wistrom et al, 2001)

  • Rifampicin Etests on these 184 C. difficile strains yielded Minimal inhibitory concentration (MIC) ranging from ¡0.002 (n5117) to ¢32 mg ml21 (n559); 59 strains had reduced susceptibility (MIC ¢32 mg ml21)

  • No discordant results were observed among these 67 isolates with reduced susceptibility using an MIC of ¢32 mg ml21 as breakpoint for reduced rifampicin susceptibility and a,38 mm inhibition zone as breakpoint for reduced rifaximin susceptibility

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Summary

Introduction

Clostridium difficile is a spore-forming Gram-positive anaerobic bacterium and a major cause of nosocomial and community-acquired diarrhoea (Wistrom et al, 2001). Metronidazole and oral vancomycin are the main antibiotics used to treat C. difficile infection (CDI). In 2005, in Austria, oral rifaximin was licensed for ‘treatment of Abbreviation: CDI, Clostridium difficile infection. Gastrointestinal diseases caused or partially caused by rifaximin-susceptible bacteria, e.g. gastrointestinal infections, pseudomembranous colitis due to C. difficile, hepatic encephalopathy, small bowel bacterial overgrowth and diverticulitis’ (Willerroider, 2009). To our knowledge, no guidelines have yet been published to test in vitro susceptibility of C. difficile to rifaximin. Jiang et al (2000) reported high rifaximin concentrations (4000 to 8000 mg g21) in stools 3 days after a single oral administration. Johnson et al (2007) reported on the possible prevention of recurrence of CDI by administering rifaximin immediately after completion of the last course of vancomycin therapy of CDI G 2011 SGM Printed in Great Britain clinical isolates. Jiang et al (2000) reported high rifaximin concentrations (4000 to 8000 mg g21) in stools 3 days after a single oral administration. Johnson et al (2007) reported on the possible prevention of recurrence of CDI by administering rifaximin immediately after completion of the last course of vancomycin therapy of CDI

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