Sensitive quantification of Clostridium perfringens in human feces by quantitative real-time PCR targeting alpha-toxin and enterotoxin genes

Abstract

BackgroundClostridium perfringens is a widespread pathogen, but the precise quantification of this subdominant gut microbe remains difficult due to its low fecal count (particularly in asymptomatic subjects) and also due to the presence of abundant polymerase-inhibitory substances in human feces. Also, information on the intestinal carriage of toxigenic C. perfringens strains in healthy subjects is sparse. Therefore, we developed a sensitive quantitative real-time PCR assays for quantification of C. perfringens in human feces by targeting its α-toxin and enterotoxin genes. To validate the assays, we finally observed the occurrence of α-toxigenic and enterotoxigenic C. perfringens in the fecal microbiota of healthy Japanese infants and young adults.MethodsThe plc-specific qPCR assay was newly validated, while primers for 16S rRNA and cpe genes were retrieved from literature. The assays were validated for specificity and sensitivity in pre-inoculated fecal samples, and were finally applied to quantify C. perfringens in stool samples from apparently healthy infants (n 124) and young adults (n 221).ResultsThe qPCR assays were highly specific and sensitive, with a minimum detection limit of 103 bacterial cells/g feces. Alpha-toxigenic C. perfringens was detected in 36 % infants and 33 % adults, with counts ranging widely (103-107 bacterial cells/g). Intriguingly, the mean count of α-toxigenic C. perfringens was significantly higher in infants (6.0 ± 1.5 log10 bacterial cells/g), as compared to that in adults (4.8 ± 1.2). Moreover, the prevalence of enterotoxigenic C. perfringens was also found to be significantly higher in infants, as compared to that in adults. The mean enterotoxigenic C. perfringens count was 5.9 ± 1.9 and 4.8 ± 0.8 log10 bacterial cells/g in infants and adults, respectively.ConclusionsThese data indicate that some healthy infants and young adults carry α-toxigenic and enterotoxigenic C. perfringens at significant levels, and may be predisposed to related diseases. Thus, high fecal carriage of toxigenic C. perfringens in healthy children warrants further investigation on its potential sources and clinical significance in these subjects. In summary, we present a novel qPCR assay for sensitive and accurate quantification of α-toxigenic and enterotoxigenic C. perfringens in human feces, which should facilitate prospective studies of the gut microbiota.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0561-y) contains supplementary material, which is available to authorized users.

Chronic Psychological Stress Disrupted the Composition of the Murine Colonic Microbiota and Accelerated a Murine Model of Inflammatory Bowel Disease

“…Clostridium perfringens in colonic contents was quantified by means of quantitative PCR by using the C . perfringens phospholipase C–specific primer set described previously [ 31 ].…”

Production of multiple bacteriocins, including the novel bacteriocin gassericin M, by Lactobacillus gasseri LM19, a strain isolated from human milk

“…qPCR was used to detect the presence or absence of C. perfringens (Nagpal et al 2015) and L. gasseri LM19 bacteriocin genes (Treven et al 2013). Primers (Table 1, Sigma Genosys, Haverhill, UK) were designed using Primer 3 (v. 0.4.0) (Untergasser et al 2012) and Netprimer (Premier Biosoft, San Francisco, California, USA) and tested using genomic DNA.…”

Molecular structure and function of the carboxy‐terminus of the alpha‐toxin from Clostridium perfringens type A

“…As the most important exotoxin of C. perfringens , alpha‐toxin is a powerful necrosis factor, researchers have regarded it as the most important research object in recent years (Goossens et al, , , ; Siqueira et al, ; Takehara, ). Alpha‐toxin is a multifunctional metalloenzyme with phospholipase C (PLC) and sphingomyelinase (SMase) enzyme activities that simultaneously hydrolyse phosphatidylcholine and sphingomyelin (Goossens et al, ; Nagpal et al, ; Uzal et al, ). Depending on the activity of these two enzymes, alpha‐toxin hydrolyses the membrane phospholipid, which is the main component of the cell membrane, destroying the integrity of the cell membrane structure and leading to cell lysis.…”