Recombination drives evolution of the Clostridium difficile 16S-23S rRNA intergenic spacer region.

Sandra Janezic,Thomas Rattei,Thomas Weinmaier,Maja Rupnik,Alexander Indra,Michel R Popoff

doi:10.1371/journal.pone.0106545

Abstract

PCR-ribotyping, a typing method based on size variation in 16S-23S rRNA intergenic spacer region (ISR), has been used widely for molecular epidemiological investigations of C. difficile infections. In the present study, we describe the sequence diversity of ISRs from 43 C. difficile strains, representing different PCR-ribotypes and suggest homologous recombination as a possible mechanism driving the evolution of 16S-23S rRNA ISRs. ISRs of 45 different lengths (ranging from 185 bp to 564 bp) were found among 458 ISRs. All ISRs could be described with one of the 22 different structural groups defined by the presence or absence of different sequence modules; tRNAAla genes and different combinations of spacers of different lengths (33 bp, 53 bp or 20 bp) and 9 bp direct repeats separating the spacers. The ISR structural group, in most cases, coincided with the sequence length. ISRs that were of the same lengths had also very similar nucleotide sequence, suggesting that ISRs were not suitable for discriminating between different strains based only on the ISR sequence. Despite large variations in the length, the alignment of ISR sequences, based on the primary sequence and secondary structure information, revealed many conserved regions which were mainly involved in maturation of pre-rRNA. Phylogenetic analysis of the ISR alignment yielded strong evidence for intra- and inter-homologous recombination which could be one of the mechanisms driving the evolution of C. difficile 16S-23S ISRs. The modular structure of the ISR, the high sequence similarities of ISRs of the same sizes and the presence of homologous recombination also suggest that different copies of C. difficile 16S-23S rRNA ISR are evolving in concert.

Highlights

Clostridium difficile, an anaerobic, sporogenic bacterium, is one of the most important pathogens causing health care-associated infections
In C. difficile, the size variability of intergenic spacer region (ISR) seem to be greater than in other bacteria which is reflected in the good discriminatory power of PCR-ribotyping [10,12]
We describe the ISR sequence diversity and explore homologous recombination as a possible mechanism driving the evolution of C. difficile 16S-23S rRNA ISRs

Summary

Introduction

Clostridium difficile, an anaerobic, sporogenic bacterium, is one of the most important pathogens causing health care-associated infections. The incidence and severity of CDI has increased dramatically since 2004, partially due to the emergence of more virulent strains (i.e. PCR-ribotypes 027, 078, 017, 053) [4]. PCR-ribotyping targets the intergenic spacer region (ISR) between the 16S and 23S rRNA genes [10]. Like in many other bacteria, several copies of the rRNA operon are present in C. difficile genome [11]. The 16S23S rRNA ISRs of C. difficile differ in length and PCR amplification of ISRs with only a single primer pair results in a pattern of bands (ranging from < 200 – 700 bp) which is unique for a specific PCR-ribotype. In C. difficile, the size variability of ISRs seem to be greater than in other bacteria which is reflected in the good discriminatory power of PCR-ribotyping [10,12]

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Results