Characterization of a Stable, Metronidazole-Resistant Clostridium difficile Clinical Isolate

Tarah Lynch,Michael R Mulvey,Morag R Graham,Romeo Hizon,Pamela Kibsey,Timothy F Booth,Tim Du,Patrick Chong,Canadian Nosocomial Infection Surveillance Program (Cnisp) ,Jason Zhang,Denise Gravel,Daniel R Beniac,Mark Miller

doi:10.1371/journal.pone.0053757

Abstract

Background Clostridium difficile are Gram-positive, spore forming anaerobic bacteria that are the leading cause of healthcare-associated diarrhea, usually associated with antibiotic usage. Metronidazole is currently the first-line treatment for mild to moderate C. difficile diarrhea however recurrence occurs at rates of 15–35%. There are few reports of C. difficile metronidazole resistance in the literature, and when observed, the phenotype has been transient and lost after storage or exposure of the bacteria to freeze/thaw cycles. Owing to the unstable nature of the resistance phenotype in the laboratory, clinical significance and understanding of the resistance mechanisms is lacking.Methodology/Principal FindingsGenotypic and phenotypic characterization was performed on a metronidazole resistant clinical isolate of C. difficile. Whole-genome sequencing was used to identify potential genetic contributions to the phenotypic variation observed with molecular and bacteriological techniques. Phenotypic observations of the metronidazole resistant strain revealed aberrant growth in broth and elongated cell morphology relative to a metronidazole-susceptible, wild type NAP1 strain. Comparative genomic analysis revealed single nucleotide polymorphism (SNP) level variation within genes affecting core metabolic pathways such as electron transport, iron utilization and energy production.Conclusions/SignificanceThis is the first characterization of stable, metronidazole resistance in a C. difficile isolate. The study provides an in-depth genomic and phenotypic analysis of this strain and provides a foundation for future studies to elucidate mechanisms conferring metronidazole resistance in C. difficile that have not been previously described.

Highlights

Clostridium difficile infection (CDI) is the most common cause of healthcare-associated infectious diarrhea
VLOO13 were typed as NAP1, pattern 001 by pulse-field gel electrophoresis (PFGE)
We have presented four phenotypes associated with the stable, metronidazole-resistant CD26A54_R subpopulation: (i) aberrant growth in liquid media, (ii) attenuated cell wall separation (iii) lack of spore production by 48 hours and (iv) heteroresistance or more accurately, a slower growing subpopulation that increases the metronidazole minimum inhibitory concentrations (MIC)

Summary

Introduction

Clostridium difficile infection (CDI) is the most common cause of healthcare-associated infectious diarrhea. Disease severity can range from self-limiting diarrhea to pseudomembranous colitis which can be associated with additional complications leading to increased mortality rates [1,2]. Laboratory diagnosis involves detection of toxin A and B in stool samples by enzyme immunoassay and/or molecular detection [7]. Stool culture remains the most sensitive diagnostic owing to the slow turnaround time it is more often used for epidemiological studies rather than patient diagnosis [7]. Clostridium difficile are Gram-positive, spore forming anaerobic bacteria that are the leading cause of healthcare-associated diarrhea, usually associated with antibiotic usage. Owing to the unstable nature of the resistance phenotype in the laboratory, clinical significance and understanding of the resistance mechanisms is lacking

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