Diverse temperate bacteriophage carriage in Clostridium difficile 027 strains.

Janet Y Nale,Warren N Fawley,Mark H Wilcox,Peter T Hickenbotham,Martha R J Clokie,Jinyu Shan,Michel R Popoff

doi:10.1371/journal.pone.0037263

Janet Y Nale, Warren N Fawley + Show 5 more

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https://doi.org/10.1371/journal.pone.0037263

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Abstract

BackgroundThe hypervirulent Clostridium difficile ribotype 027 can be classified into subtypes, but it unknown if these differ in terms of severity of C. difficile infection (CDI). Genomic studies of C. difficile 027 strains have established that they are rich in mobile genetic elements including prophages. This study combined physiological studies, electron microscopy analysis and molecular biology to determine the potential role of temperate bacteriophages in disease and diversity of C. difficile 027.Methodology/Principal FindingsWe induced prophages from 91 clinical C. difficile 027 isolates and used transmission electron microscopy and pulsed-field gel electrophoresis to characterise the bacteriophages present. We established a correlation between phage morphology and subtype. Morphologically distinct tailed bacteriophages belonging to Myoviridae and Siphoviridae were identified in 63 and three isolates, respectively. Dual phage carriage was observed in four isolates. In addition, there were inducible phage tail-like particles (PT-LPs) in all isolates. The capacity of two antibiotics mitomycin C and norfloxacin to induce prophages was compared and it was shown that they induced specific prophages from C. difficile isolates. A PCR assay targeting the capsid gene of the myoviruses was designed to examine molecular diversity of C. difficile myoviruses. Phylogenetic analysis of the capsid gene sequences from eight ribotypes showed that all sequences found in the ribotype 027 isolates were identical and distinct from other C. difficile ribotypes and other bacteria species.Conclusion/SignificanceA diverse set of temperate bacteriophages are associated with C. difficile 027. The observed correlation between phage carriage and the subtypes suggests that temperate bacteriophages contribute to the diversity of C. difficile 027 and may play a role in severity of disease associated with this ribotype. The capsid gene can be used as a tool to identify C. difficile myoviruses present within bacterial genomes.

Highlights

Clostridium difficile is a nosocomial pathogen responsible for gut inflammation, with resultant diarrhoea or pseudomembranous colitis
Transmission electron microscopy (TEM) analysis following induction revealed a diverse set of phage morphologies associated with C. difficile ribotype 027, with a clear bias towards myoviruses, which were induced from 63 isolates (Figure 1, Table S1)
Given the excess morbidity and mortality associated with C. difficile ribotype 027, we investigated prophage content as a factor that may be contributing to its diversity and virulence

Summary

Introduction

Clostridium difficile is a nosocomial pathogen responsible for gut inflammation, with resultant diarrhoea or pseudomembranous colitis. Infection caused by hypervirulent strains such as C. difficile ribotype 027 is a serious global challenge [1,2]. A recent report showed that C. difficile 027 isolates could be divided into 23 and 5 subgroups using multiple-locus variable- number tandem repeat analysis (MLVA) and pulsed-field gel electrophoresis (PFGE), respectively [6]. These subtypes were associated with variable disease severity. It is important to establish factors that may contribute to the diversity and success of this hypervirulent ribotype and to the epidemic potential of new strains. This study combined physiological studies, electron microscopy analysis and molecular biology to determine the potential role of temperate bacteriophages in disease and diversity of C. difficile 027

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