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

Abstract

Background: Clostridium 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.

Results: We 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.

Conclusions: C. 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.

Figure 1

Tolerance to p-cresol. Strains R20291 and 630 were tested for their in-vitro tolerance to 0.1% p-cresol. * indicates a significant difference p < 0.01 Student's T-test.

Figure 2

Detection and production of p-cresol by 630Δerm and R20291 using NMR spectroscopy and zNose™ gas chromatography. The relative production of p-cresol in rich media (BHI) supplemented with 0.1% p-HPA for strains 630Δerm and R20291 by A) NMR spectroscopy and B) zNose™ gas chromatography. The p-cresol peak is indicated with an arrow, at 6.7 seconds for zNose™ experiments

Figure 3

The hpdBCA operon and verification of mutant construction. The hpdBCA operon with insertion sites for the targeted ClosTron mutagenesis, the number refers to the insertion site (bp) and the s/a refers to sense/antisense orientation of the ClosTron insert. B) PCR screen of the mutants (M = mutant; W = wild type; P = plasmid and "-" negative control). Three PCR screens were performed, gene specific forward and reverse primers, intron specific with gene specific primers, and RAM specific primers (Heap et al., 2007). C) Southern blot using a probe specific to the inserted intron. HindIII digests were performed on DNA from M = mutant; W = wild type; P = plasmid. The strains and primer sets are indicated on each figure and in tables 1 and 2. The marker sizes are indicated on the figure and the expected band sizes are as follows for 630Δerm: hpdB 2.8Kb; hpdC 3.4 kb and for R20291: hpdA - 6.3 kb; hpdC - 3.4 kb.

Figure 4

Analysis of the decarboxylase mutants. A) NMR spectra showing p-cresol production in BHI broth supplemented with 0.1% p-HPA for parent and mutant strains, B) Growth curve of the R20291ΔhpdC and 630ΔhpdC mutants compared to respective parent strains. C) Tolerance to 0.1% p-cresol of ΔhpdC mutants and respective parent strains.

Figure 5

Temporal production of p-HPA and p-cresol in mutant and wild-type strains using NMR. A) NMR spectra showing an overview of the relative levels of tyrosine, p-HPA and p-cresol from all replicates and strains tested over a 24-hour time period, the colours define the 44 samples used in the time course experiment, over four strains and media controls. T = time of sampling (hours post inoculation). B) The relative production of p-HPA by mutant and patent strains over a 24-hour time period. C) The relative production of p-cresol by the parent strains over a 24-hour time period. (The levels of p-cresol by the ΔhpdC mutants were below the limits of detection by NMR and were not plotted).