Horizontal gene transfer converts non-toxigenic Clostridium difficile strains into toxin producers

Abstract

Clostridium difficile is a major nosocomial pathogen and the main causative agent of antibiotic-associated diarrhoea. The organism produces two potent toxins, A and B, which are its major virulence factors. These are chromosomally encoded on a region termed the pathogenicity locus (PaLoc), which also contains regulatory genes, and is absent in non-toxigenic strains. Here we show that the PaLoc can be transferred from the toxin-producing strain, 630Δerm, to three non-toxigenic strains of different ribotypes. One of the transconjugants is shown by cytotoxicity assay to produce toxin B at a similar level to the donor strain, demonstrating that a toxigenic C. difficile strain is capable of converting a non-toxigenic strain to a toxin producer by horizontal gene transfer. This has implications for the treatment of C. difficile infections, as non-toxigenic strains are being tested as treatments in clinical trials.

Integration oferm(B)-containing elements through large chromosome fragment exchange inClostridium difficile

“…This is in accordance with observations done recently during PaLoc's transfer between C. difficile strains 10 suggesting that the mechanism involved can mobilise other regions of the genome. We also reported the peculiar in vitro behavior of the recipient strain CD13 that acquired genetic material through a transformation-like mechanism not yet observed in C. difficile, to our knowledge.…”

“…The length of the transferred fragments in each genome analyzed was determined by calculating the distance between the first SNP upstream and the last SNP downstream of the transferred element, as performed for PaLoc transfer. 10 Notably, a segment with a size comprised between 254458 and 256507 bp from C. difficile 630 was identified in the genome of 630xCD37A. In 630xCD13A, 2 segments of donor DNA covering a region comprised between 44869 and 46115 bp in length were detected, separated by a fragment with a length comprised between 10775 and 12064 bp corresponding to the recipient's sequence, suggesting that multiple events of homologous recombination occurred ( Fig.…”

“…The large size of the transferred fragment (>250 kb) identified in 630 £ CD37A makes it also improbable that generalized transduction occurred in this progeny, while conjugation cannot be excluded, as already hypothesized for the PaLoc transfer. 10 On the other hand, competence for transformation can be hypothesized for the CD13 recipient strain, because the transfer of the transposon was impaired by the treatment with DNAse. However, erythromycin-resistant transformants were not obtained when CD13 cells were mixed to 2-4 mg of genomic DNA from the donors and placed on filter for 24 hours, as performed in filter-mating assays (data not shown).…”

Clostridium difficile Infection

“…This concept was recently tested in humans; patients who could be colonized by a non-toxigenic C. difficile strain after receiving standard of care treatment for CDI had significantly reduced CDI recurrence rates 187 . However, further explorations of this approach should consider that nontoxinogenic strains can become toxinogenic by horizontal gene transfer 188 , although it is unknown whether this process also occurs in a human host.…”

Recent Technology of Clinical Microbiology; Whole Genome Sequencing (WGS) and Matrix-Assisted, Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS)

“…C. difficile, the most serious source of hospital-associated infections, is categorized as an asymptomatic carrier and causes diseases when normal neighboring bacteria are suppressed by antimicrobial drugs. An avirulent strain that has colonized the gastrointestinal tract can acquire the gene that encodes toxin b (tcdB) or an antimicrobialresistant gene from the same species or from a different species that is present nearby [19]. Streptococci or Neisseria species colonizing the respiratory tract can acquire specific virulence genes or cephalosporinresistant genes which are inserted into specific plasmids, prophages or mobile genomic islands [22].…”