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

Dawson LF, Donahue EH, Cartman ST, Barton RH, Bundy J, McNerney R, Minton NP, Wren BW - BMC Microbiol. (2011)

Bottom Line: 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.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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious & Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK.

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.

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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.
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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.

Mentions: Initial growth dynamics and NMR spectroscopy analysis revealed that the hpdB, hpdC and hpdA mutants were indistinguishable in terms of the complete lack of p-cresol production in rich media supplemented with p-HPA (Figure 4A). Subsequent analysis was performed with the hpdC mutants as these were constructed in both parent strains R20291 and 630Δerm. Growth curves in minimal media (YP broth) revealed that the R20291ΔhpdC mutant grew significantly better than the parent strain R20291, however, no significant difference in in-vitro growth was observed between 630ΔermΔhpdC and the respective parent strain (Figure 4B). There were no significant differences between the tolerance of the mutants R20291ΔhpdC and 630ΔermΔhpdC to 0.1% p-cresol compared to their respective parent strains (Figure 4C), however, the R20291 strains (wild-type and R20291ΔhpdC) are significantly more tolerant to p-cresol than their 630 counterparts (wild-type and 630ΔermΔhpdC) (p < 0.01). The absence of p-cresol production observed in the R20291ΔhpdC and 630ΔermΔhpdC mutants by NMR spectroscopy in rich media supplemented with 0.1% p-HPA (Figure 4A), was reproducible in minimal media using zNose™ gas chromatography (data not shown).


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

Dawson LF, Donahue EH, Cartman ST, Barton RH, Bundy J, McNerney R, Minton NP, Wren BW - BMC Microbiol. (2011)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102038&req=5

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.
Mentions: Initial growth dynamics and NMR spectroscopy analysis revealed that the hpdB, hpdC and hpdA mutants were indistinguishable in terms of the complete lack of p-cresol production in rich media supplemented with p-HPA (Figure 4A). Subsequent analysis was performed with the hpdC mutants as these were constructed in both parent strains R20291 and 630Δerm. Growth curves in minimal media (YP broth) revealed that the R20291ΔhpdC mutant grew significantly better than the parent strain R20291, however, no significant difference in in-vitro growth was observed between 630ΔermΔhpdC and the respective parent strain (Figure 4B). There were no significant differences between the tolerance of the mutants R20291ΔhpdC and 630ΔermΔhpdC to 0.1% p-cresol compared to their respective parent strains (Figure 4C), however, the R20291 strains (wild-type and R20291ΔhpdC) are significantly more tolerant to p-cresol than their 630 counterparts (wild-type and 630ΔermΔhpdC) (p < 0.01). The absence of p-cresol production observed in the R20291ΔhpdC and 630ΔermΔhpdC mutants by NMR spectroscopy in rich media supplemented with 0.1% p-HPA (Figure 4A), was reproducible in minimal media using zNose™ gas chromatography (data not shown).

Bottom Line: 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.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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infectious & Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK.

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.

Show MeSH
Related in: MedlinePlus