The analysis of para-cresol production and tolerance in Clostridium difficile 027 and 012 strains

Abstract

BackgroundClostridium difficile is the major cause of antibiotic associated diarrhoea and in recent years its increased prevalence has been linked to the emergence of hypervirulent clones such as the PCR-ribotype 027. Characteristically, C. difficile infection (CDI) occurs after treatment with broad-spectrum antibiotics, which disrupt the normal gut microflora and allow C. difficile to flourish. One of the relatively unique features of C. difficile is its ability to ferment tyrosine to para-cresol via the intermediate para-hydroxyphenylacetate (p-HPA). P-cresol is a phenolic compound with bacteriostatic properties which C. difficile can tolerate and may provide the organism with a competitive advantage over other gut microflora, enabling it to proliferate and cause CDI. It has been proposed that the hpdBCA operon, rarely found in other gut microflora, encodes the enzymes responsible for the conversion of p-HPA to p-cresol.ResultsWe show that the PCR-ribotype 027 strain R20291 quantitatively produced more p-cresol in-vitro and was significantly more tolerant to p-cresol than the sequenced strain 630 (PCR-ribotype 012). Tyrosine conversion to p-HPA was only observed under certain conditions. We constructed gene inactivation mutants in the hpdBCA operon in strains R20291 and 630Δerm which curtails their ability to produce p-cresol, confirming the role of these genes in p-cresol production. The mutants were equally able to tolerate p-cresol compared to the respective parent strains, suggesting that tolerance to p-cresol is not linked to its production.ConclusionsC. difficile converts tyrosine to p-cresol, utilising the hpdBCA operon in C. difficile strains 630 and R20291. The hypervirulent strain R20291 exhibits increased production of and tolerance to p-cresol, which may be a contributory factor to the virulence of this strain and other hypervirulent PCR-ribotype 027 strains.

The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes

“…Related outbreak clusters are immediately visible from the phyletic patterns of the alignment, confirming the primary clades of the tree. In addition, the SNP heatmap highlights the phyletic signature of several subclades, in this case within the known hpdBCA operon [95] that is extremely well conserved across all 826 genomes. Figure 4 shows a zoomed view of the 826 P. difficile genome alignment in Gingr, highlighting a single annotated gene.…”

In-situ Product Recovery as a Strategy to Increase Product Yield and Mitigate Product Toxicity

“…It was this toxicity that attributed C. difficile its virulent characteristics [6], and rendered the bacteria a competitive advantage over other microorganisms in its natural habitat [7]. This study explored the possibilities of mitigating the toxicity imposed by p-cresol by implementing an ISPR strategy in order to improve product yield and productivity.…”

Metabolite profiling of Clostridium difficile ribotypes using small molecular weight volatile organic compounds

“…Another study, which concentrated on the production of end products from the metabolism of aromatic acids of phenylalanine, tyrosine and tryptophan by growing Clostridia cultures, found that only C. difficile emitted p-cresol and this compound was not detected in 22 other Clostridia bacteria (Elsden et al 1976). More recently, Dawson et al (Dawson et al 2011) have shown R027 produce more p-cresol that R012 ribotype. Consequently, a signal at m/z 109 was tentatively assigned to p-cresol and was detected in R014/R020, R002 and R013.…”

“…Again a substantial signal at m/z 109 from the blank medium made it difficult to identify p-cresol from the other ribotype emissions. Further, it is worth noting that C. difficile doesn't produce p-cresol in BHIS liquid broth (Dawson et al 2011). …”