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

BMC Microbiology

Research Article

Satoru Nagata¹  Yoshio Suzuki²  Kazunari Kawashima³  Ravinder Nagpal⁴  Yuichiro Yamashiro⁴  Koji Nomoto⁵  Hirokazu Tsuji⁵  Kiyohito Ogata⁵  Takuya Takahashi⁵  Kazunori Matsuda⁶ 

[1] Department of Pediatrics, School of Medicine, Tokyo Women’s Medical University, Tokyo, Japan;Department of Sports Science, Juntendo University School of Health and Sports Sciences, Chiba, Japan;Gonohashi Obstetrics and Gynecology Hospital, Tokyo, Japan;Probiotics Research Laboratory, Juntendo University Graduate School of Medicine, Tokyo, Japan;Yakult Central Institute, Tokyo, Japan;Yakult Honsha European Research Center for Microbiology, Ghent-Zwijnaarde, Belgium;

关键词: Alpha-toxin;    Clostridium perfringens;    Enterotoxin;    Intestinal microbiota;    Gut pathogens;    Phospholipase C;    Quantitative PCR;   

DOI  :  10.1186/s12866-015-0561-y

 received in 2015-05-14, accepted in 2015-10-08,  发布年份 2015

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© Nagpal et al. 2015