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

• Published in: BMC microbiology Vol. 15; no. 1; p. 219
• Main Authors: Nagpal, Ravinder, Ogata, Kiyohito, Tsuji, Hirokazu, Matsuda, Kazunori, Takahashi, Takuya, Nomoto, Koji, Suzuki, Yoshio, Kawashima, Kazunari, Nagata, Satoru, Yamashiro, Yuichiro
• Format: Journal Article
• Language: English
• Published: London BioMed Central 19.10.2015; BioMed Central Ltd
• Subjects: Adolescent; Adult; Bacterial Load - methods; Bacterial Toxins - genetics; Biological Microscopy; Biomedical and Life Sciences; Calcium-Binding Proteins - genetics; Carrier State - microbiology; Clostridium Infections - microbiology; Clostridium perfringens - isolation & purification; Cohort Studies; Enterotoxins - genetics; Feces; Feces - microbiology; Female; Food contamination & poisoning; Genes; Genetic aspects; Genetic research; genomics and proteomics; Healthy Volunteers; Humans; Infant; Infants; Life Sciences; Male; Microbial genetics; Microbiology; Microbiota (Symbiotic organisms); Middle Aged; Mycology; Parasitology; Pathogens; Real-Time Polymerase Chain Reaction - methods; Research Article; Sensitivity and Specificity; Toxins; Type C Phospholipases - genetics; Virology; Young Adult; Young adults; Japan; Enterotoxin; Gut pathogens; Alpha-toxin; Intestinal microbiota; Quantitative PCR; Phospholipase C
• ISSN: 1471-2180; 1471-2180
• DOI: 10.1186/s12866-015-0561-y

Background Clostridium 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. Methods The 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). Results The qPCR assays were highly specific and sensitive, with a minimum detection limit of 10 3 bacterial cells/g feces. Alpha-toxigenic C. perfringens was detected in 36 % infants and 33 % adults, with counts ranging widely (10 3 -10 7 bacterial cells/g). Intriguingly, the mean count of α-toxigenic C. perfringens was significantly higher in infants (6.0 ± 1.5 log 10 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 log 10 bacterial cells/g in infants and adults, respectively. Conclusions These 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.