Influence of subinhibitory concentrations of metronidazole, vancomycin, clindamycin and linezolid on toxin production in Clostridiumdifficile

Abstract number: 1134_04_135

Walch C., Gerber M., Ackermann G., Rodloff A.C.

Objective:  

Clostridiumdifficile is the major cause of hospital-acquired infectious diarrhoea. Several antimicrobials are known to induce and promote C. difficile-associated diarrhoea (CDAD). The impact of metronidazole (MET), vancomycin (VAN), clindamycin (CLI) and linezolid (LIN) on toxin-production in C. difficile was investigated in this study. Metronidzole and vancomycin are first line therapy drugs of CDAD. Clindamycin is strongly associated with the induction of CDAD. Linezolid is a new antimicrobial exhibiting activity mainly against gram-positive bacteria. A previous study showed enhanced and earlier toxin detection in the presence of vancomycin and metronidazole. This study intends to differentiate between toxin release and enhanced production.

Methods:  

C. difficile strain VPI10463 and three strains recovered from patients were tested for their susceptibility to MET, VAN, CLI and LIN using Etest. Toxigenicity was determined by PCR. Growth curves with and without subinhibitory concentrations of MET, VAN, CLI and LIN (0.5 × MIC) were determined for the C. difficile isolates. Growth was measured spectrophotometrically and by counting of plated cells. Toxin production was detected with ELISA (toxin A) and cytotoxicity assay (toxin B) from culture supernatant and from sonicated cell pellet also. Using real-time PCR mRNA for toxin A and B gene sequences was measured. Toxin production and mRNA copies were calculated in relation to the number of bacterial cells.

Results:  

The four strains showed very different growth and level of toxin production in the absence of antibiotics. Metronidazole and vancomycin stimulated toxin production in three of the four strains tested. Clindamycin did not stimulate and in some cases inhibited toxin production in all strains. Linezolid had variable effects on toxin A and B production in the four strains. Conclusion: The finding that our established drugs of choice for CDAD, MET and VAN, are able to enhance toxin-production in C. difficile indicates the nessicity to renew the discussion of the pathogenesis of severe CDAD, pseudomembranoese colitis and toxic megacolon, where MET and VAN cannot always change the clinical course. Animal studies are needed to confirm the in vitro results.