Identification and quantitation of mucosal and faecal desulfovibrios using real time polymerase chain reaction

Abstract

Background: Desulfovibrios produce sulphide, which is toxic to colonic epithelial cells. These bacteria have previously been linked to ulcerative colitis. Traditional methods of culturing these organisms are slow, and often unreliable, while molecular approaches are either non-quantitative or lack sensitivity.

Aims: To develop a sensitive method for quantitating desulfovibrios in stools and biopsy tissue, and to investigate the effects of age and disease on these bacteria.

Methods: Rectal biopsies were taken from 10 colitis patients and 10 healthy controls. Stool samples were obtained from 10 healthy infants (mean age 1.01 (0.18) years), 10 healthy young adults (26.7 (1.2) years), and 10 healthy elderly people (71.7 (1.2) years). Primers were designed and developed for analysing Desulfovibrio populations in the bowel using real time polymerase chain reaction (PCR).

Results: The PCR primers were highly specific for desulfovibrios. Large numbers (approximately 10(6)-10(7)/g) occurred in biopsies in colitis patients and healthy subjects, and no disease related differences were observed. Measurements of mucosal desulfovibrios over 12 months showed marked changes in some patients. Infants (10(6)-10(7)/g) and elderly people (10(7)-10(8)/g) had significantly higher numbers of desulfovibrios in stools compared with young adults (10(5)/g).

Conclusions: Real time PCR analysis of desulfovibrios was an efficient and accurate method for studying these potentially harmful microorganisms. Desulfovibrios were ubiquitous in the bowel, irrespective of age. As rectal mucosae were heavily colonised in health and disease, if these bacteria play a role in colitis, some host defect, possibly in sulphide detoxication pathways or in bacterial antigen handling, is required for manifestations of pathogenicity.

Figures

Figure 1

Specificity of the desulfovibrio polymerase chain reaction (PCR) assay. Electrophoresis was done on an agarose (2% w/v) gel with PCR products obtained with primer pair DSV691-F and DSV826-R, and genomic DNA from various bacterial templates. DNA ladder (100 bp) (lane 1), Bacteroides fragilis NCTC 9343 (lane 2), Faecalibacterium prausnitzii ATCC 27768 (lane 3), Clostridium difficile NTCC 11223 (lane 4), C histolyticum DSM 2158 (lane 5), Lactobacillus acidophilus DSM 20079 (lane 6), E coli ATCC 11775 (lane 7), Desulfovibrio desulfuricans NCIMB 12833 (lane 8), Bifidobacterium catenulatum NCIMB 702246 (lane 9), Bif bifidum NCIMB 702715 (lane 10), Bif breve NCTC 11815 (lane 11), Faecalibacterium prausnitzii NCIMB 13872 (lane 12), Desulfovibrio vulgaris NCIMB 8303 (lane 13), Eubacterium limosum DUN-112 (lane 14), C clostridioforme DUN-120 (lane 15), C malenominatum DUN-102 (lane 16), Eubacterium aerofaciens DUN-207 (lane 17), Clostridium perfringens NCTC 8346 (lane 18). Desulfovibrio desulfuricans NCIMB 12833 (lane 21), Dsv vulgaris NCIMB 8303 (lane 22), Dsv desulfuricans NCIMB 8307 (lane 23), Desulfotomaculum ruminis NCIMB 8452 (lane 24), Desulfococcus multivorans NCIMB 12965 (lane 25), Desulfobacter vibrioformis NCIMB 13525 (lane 26), Dsb vibrioformis NCIMB 13525 (obtained as DNA) (lane 27), Desulfotomaculum ruminis DSM 2154 (lane 28), Dsm ruminis-Reading (lane 29), negative control (lane 30). PCR positivity is indicated by the presence of a 135 bp PCR product.

Figure 2

Post-amplification melt curve analysis of desulfovibrios. Fluorescence of SYBR Green with the progress of amplicon cycles is shown in (A). Values are duplicates of different dilutions of the plasmid used as standard. 16S rRNA gene copy numbers ranging from log10 1.0 to 7.0 are shown in duplicate. The rate of change of fluorescence against temperature is shown in (B). The peak (T = 89 (1)) is specific to the genus Desulfovibrio. Symbols and lines correspond to amplifications shown in (A). PCR, polymerase chain reaction; CF, curve fit; RFU, relative fluorescent unit.

Figure 3

Quantitation of desulfovibrios. A standard curve for desulfovibrios was constructed with 10-fold dilution of plasmid from 101–107 copies. Threshold cycle (CT) is plotted against the starting copy number. Values on the line represent the result of duplicates. The equation for the curve is: CT = −3.425logSQ+33.16945 (r = 0.997). The efficiency of the determination was calculated to be 97%.

Figure 4

Recovery of desulfovibrio DNA added to tissue samples by real time polymerase chain reaction (PCR). The target bacterium (Desulfovibrio desulfuricans) was added in 10-fold dilutions from a culture grown in Postgate’s medium B to colonic tissue. The tissue was cut into six subsamples and each was treated with the bacterial preparation before extraction of genomic DNA. Means of triplicate copy number of 16S rRNA genes were plotted against bacterial cell numbers (mean (SEM)). A sample without added sulphate reducing bacteria was used to determine whether the original tissue samples contained desulfovibrios.

Figure 5

Quantitation of desulfovibrios from biopsies (A) and different stool specimens (B). Bacterial density is expressed as copy number of 16S rRNA genes for desulfovibrios (SQ) per g of biopsy or stool. Biopsies from patients with ulcerative colitis (UC) and biopsies from healthy normal adults (HN), and faeces from healthy infants (HI), healthy young adults (YA), and healthy elderly people (HE) are shown. Values are mean (SEM) for each group. Significant difference, irrespective of sex: *p = 0.008 compared with stool YA; †p = 0.021 compared with stool HI; and ‡p = 0.000 compared with stool HE.

Figure 6

Stability of desulfovibrio populations on the rectal mucosa in four patients with ulcerative colitis.