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Real-time PCR analysis of enteric pathogens from fecal samples of irritable bowel syndrome subjects.

Rinttilä T, Lyra A, Krogius-Kurikka L, Palva A - Gut Pathog (2011)

Bottom Line: The S. aureus -positive IBS samples were confirmed by sequencing of the PCR amplicons.S. aureus has not been previously reported to be associated with the onset of IBS.Although we discovered significant differences in the prevalence of S. aureus between the study groups, its importance in giving rise to IBS symptoms requires further studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Biosciences, Faculty of Veterinary Medicine, University of Helsinki, PO Box 66, FIN-00014, Helsinki, Finland. airi.palva@helsinki.fi.

ABSTRACT

Background: Growing amount of scientific evidence suggests that microbes are involved in the pathophysiology of irritable bowel syndrome (IBS). The predominant fecal microbiota composition of IBS subjects has been widely studied with DNA-based techniques but less research has been focused on the intestinal pathogens in this disorder. Here, we optimized a highly sensitive panel of 12 quantitative real-time PCR (qPCR) assays to shed light on the putative presence of intestinal pathogens in IBS sufferers. The panel was used to screen fecal samples from 96 IBS subjects and 23 healthy controls.

Results: Fifteen IBS samples (17%) tested positive for Staphylococcus aureus with a thermonuclease (nuc) gene-targeting qPCR assay, whereas none of the healthy controls were positive for S. aureus (p <0.05). The S. aureus -positive IBS samples were confirmed by sequencing of the PCR amplicons. Clostridium perfringens was detected from IBS and control groups with a similar frequency (13% and 17%, respectively) with α-toxin (plc) gene -targeting qPCR assay while none of the samples tested positive for the Cl. perfringens enterotoxin-encoding gene (cpe).

Conclusions: The qPCR panel consisting of 12 assays for an extensive set of pathogenic microorganisms provides an efficient alternative to the conventional detection of gastrointestinal pathogens and could accelerate the initiation of targeted antibiotic therapy reducing the risk of post-infectious IBS (PI-IBS). S. aureus has not been previously reported to be associated with the onset of IBS. Although we discovered significant differences in the prevalence of S. aureus between the study groups, its importance in giving rise to IBS symptoms requires further studies.

No MeSH data available.


Related in: MedlinePlus

Real-time PCR results of the assays for Staphylococcus aureus nuc gene (A) and Clostridium perfringens plc gene (B). The values are target genomes per gram of fecal sample (log10 values). The detection limit is set to 104 bacterial genomes. The abbreviations: IBS-C and IBS-D&M stand for constipation-predominant irritable bowel syndrome and diarrhea-predominant or mixed-type irritable bowel syndrome, respectively.
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Figure 1: Real-time PCR results of the assays for Staphylococcus aureus nuc gene (A) and Clostridium perfringens plc gene (B). The values are target genomes per gram of fecal sample (log10 values). The detection limit is set to 104 bacterial genomes. The abbreviations: IBS-C and IBS-D&M stand for constipation-predominant irritable bowel syndrome and diarrhea-predominant or mixed-type irritable bowel syndrome, respectively.

Mentions: The qPCR analysis of S. aureus revealed that 15 IBS samples (prevalence of 17%) amplified within the linear range of the standard curve giving a positive result with the quantities ranging from 2.5 × 104 to 4.0 × 107 genomic equivalents per gram of feces (Figure 1A). The positive signals were initially verified by melting curve analysis (Tm = 83.5°C) and agarose gel electrophoresis. On the contrary, no positive S. aureus signals were obtained with the samples from healthy controls. This result was statistically significant at the risk level of p < 0.05 (Pearson's Chi-square test). The S. aureus-positive IBS samples were relatively evenly distributed among the two subcategories as the frequencies of positive samples in IBS-D&M and IBS-C subgroups were 16% and 13%, respectively. No clear relation to age or gender was observed among the S. aureus positive samples.


Real-time PCR analysis of enteric pathogens from fecal samples of irritable bowel syndrome subjects.

Rinttilä T, Lyra A, Krogius-Kurikka L, Palva A - Gut Pathog (2011)

Real-time PCR results of the assays for Staphylococcus aureus nuc gene (A) and Clostridium perfringens plc gene (B). The values are target genomes per gram of fecal sample (log10 values). The detection limit is set to 104 bacterial genomes. The abbreviations: IBS-C and IBS-D&M stand for constipation-predominant irritable bowel syndrome and diarrhea-predominant or mixed-type irritable bowel syndrome, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3111350&req=5

Figure 1: Real-time PCR results of the assays for Staphylococcus aureus nuc gene (A) and Clostridium perfringens plc gene (B). The values are target genomes per gram of fecal sample (log10 values). The detection limit is set to 104 bacterial genomes. The abbreviations: IBS-C and IBS-D&M stand for constipation-predominant irritable bowel syndrome and diarrhea-predominant or mixed-type irritable bowel syndrome, respectively.
Mentions: The qPCR analysis of S. aureus revealed that 15 IBS samples (prevalence of 17%) amplified within the linear range of the standard curve giving a positive result with the quantities ranging from 2.5 × 104 to 4.0 × 107 genomic equivalents per gram of feces (Figure 1A). The positive signals were initially verified by melting curve analysis (Tm = 83.5°C) and agarose gel electrophoresis. On the contrary, no positive S. aureus signals were obtained with the samples from healthy controls. This result was statistically significant at the risk level of p < 0.05 (Pearson's Chi-square test). The S. aureus-positive IBS samples were relatively evenly distributed among the two subcategories as the frequencies of positive samples in IBS-D&M and IBS-C subgroups were 16% and 13%, respectively. No clear relation to age or gender was observed among the S. aureus positive samples.

Bottom Line: The S. aureus -positive IBS samples were confirmed by sequencing of the PCR amplicons.S. aureus has not been previously reported to be associated with the onset of IBS.Although we discovered significant differences in the prevalence of S. aureus between the study groups, its importance in giving rise to IBS symptoms requires further studies.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Biosciences, Faculty of Veterinary Medicine, University of Helsinki, PO Box 66, FIN-00014, Helsinki, Finland. airi.palva@helsinki.fi.

ABSTRACT

Background: Growing amount of scientific evidence suggests that microbes are involved in the pathophysiology of irritable bowel syndrome (IBS). The predominant fecal microbiota composition of IBS subjects has been widely studied with DNA-based techniques but less research has been focused on the intestinal pathogens in this disorder. Here, we optimized a highly sensitive panel of 12 quantitative real-time PCR (qPCR) assays to shed light on the putative presence of intestinal pathogens in IBS sufferers. The panel was used to screen fecal samples from 96 IBS subjects and 23 healthy controls.

Results: Fifteen IBS samples (17%) tested positive for Staphylococcus aureus with a thermonuclease (nuc) gene-targeting qPCR assay, whereas none of the healthy controls were positive for S. aureus (p <0.05). The S. aureus -positive IBS samples were confirmed by sequencing of the PCR amplicons. Clostridium perfringens was detected from IBS and control groups with a similar frequency (13% and 17%, respectively) with α-toxin (plc) gene -targeting qPCR assay while none of the samples tested positive for the Cl. perfringens enterotoxin-encoding gene (cpe).

Conclusions: The qPCR panel consisting of 12 assays for an extensive set of pathogenic microorganisms provides an efficient alternative to the conventional detection of gastrointestinal pathogens and could accelerate the initiation of targeted antibiotic therapy reducing the risk of post-infectious IBS (PI-IBS). S. aureus has not been previously reported to be associated with the onset of IBS. Although we discovered significant differences in the prevalence of S. aureus between the study groups, its importance in giving rise to IBS symptoms requires further studies.

No MeSH data available.


Related in: MedlinePlus