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Nagpal et al. BMC Microbiology (2015) 15:219 DOI 10.1186/s12866-015-0561-y
RESEARCH ARTICLE Open Access
Sensitive quantification of Clostridiumperfringens in human feces by quantitativereal-time PCR targeting alpha-toxin andenterotoxin genes
Ravinder Nagpal1*, Kiyohito Ogata2, Hirokazu Tsuji2, Kazunori Matsuda3, Takuya Takahashi2, Koji Nomoto2,Yoshio Suzuki4, Kazunari Kawashima5, Satoru Nagata6 and Yuichiro Yamashiro1
Background: Clostridium perfringens is a widespread pathogen, but the precise quantification of this subdominantgut microbe remains difficult due to its low fecal count (particularly in asymptomatic subjects) and also due to thepresence of abundant polymerase-inhibitory substances in human feces. Also, information on the intestinal carriageof toxigenic C. perfringens strains in healthy subjects is sparse. Therefore, we developed a sensitive quantitativereal-time PCR assays for quantification of C. perfringens in human feces by targeting its -toxin and enterotoxingenes. To validate the assays, we finally observed the occurrence of -toxigenic and enterotoxigenic C. perfringensin 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 wereretrieved from literature. The assays were validated for specificity and sensitivity in pre-inoculated fecal samples, andwere finally applied to quantify C. perfringens in stool samples from apparently healthy infants (n 124) and youngadults (n 221).
Results: The qPCR assays were highly specific and sensitive, with a minimum detection limit of 103 bacterial cells/gfeces. 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 -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 ofenterotoxigenic 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 andadults, respectively.
Conclusions: These data indicate that some healthy infants and young adults carry -toxigenic and enterotoxigenicC. perfringens at significant levels, and may be predisposed to related diseases. Thus, high fecal carriage of toxigenicC. perfringens in healthy children warrants further investigation on its potential sources and clinical significance inthese subjects. In summary, we present a novel qPCR assay for sensitive and accurate quantification of -toxigenicand enterotoxigenic C. perfringens in human feces, which should facilitate prospective studies of the gut microbiota.
Keywords: Alpha-toxin, Clostridium perfringens, Enterotoxin, Intestinal microbiota, Gut pathogens, Phospholipase C,Quantitative PCR
* Correspondence: email@example.comEqual contributors1Probiotics Research Laboratory, Juntendo University Graduate School ofMedicine, Tokyo, JapanFull list of author information is available at the end of the article
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BackgroundClostridium perfringens is one of the most widely dis-persed opportunistic pathogens  and is well known toproduce a number of toxins to cause several forms ofhistotoxic and enteric diseases in humans and animals. Based on the production of four major toxins i.e.,alpha, beta, epsilon and iota, it is categorized into fivetoxin-types viz. A, B, C, D and E . While it is ambigu-ous why C. perfringens produces so many diverse toxins,it is well known that it uses chromosomally-encoded -toxin (which has phospholipase C (plc) and sphingomyeli-nase activities with hemolytic, necrotic and lethal abilities)as a chief virulent factor and key mediator for mostof C. perfringens-associated diseases . Alpha-toxinis produced by all C. perfringens toxin-types but typeA produces higher amounts than the other types .In addition, few C. perfringens strains also harbor cpe(C. perfringens enterotoxin, a clinically important en-terotoxin) gene which is responsible for most of theC. perfringens food-poisoning outbreaks and diarrhealcases . Under favorable conditions, the organismcan cause gas gangrene, food-poisoning and gastro-intestinal illnesses including antibiotic-associated diar-rhea, sporadic diarrhea, and nosocomial diarrhealdiseases in humans . However, in spite of theirubiquitous environmental distribution and implicationin various food-poisoning outbreaks worldwide [10, 11],very little information is available on the relative occur-rence of -toxigenic and enterotoxigenic strains of C. per-fringens in general populations, particularly in Japanwhere C. perfringens food poisoning has been ranked asthird or fourth most common food poisoning .Generally, a high C. perfringens count (>106 cfu/g
feces) is considered as an indicator of C. perfringensfood-poisoning . C. perfringens in fecal sampleshas traditionally been detected by conventional micro-biological culture methods, biochemical analyses, tissueculture cytotoxicity assays, mouse bioassays or immuno-sorbent assays , but these methods are laboriousand time-consuming. Culture of C. perfringens is notrecommended for diagnosis, mainly because of relativeubiquity of the bacterium in human feces. Further, sincenot all isolates are toxigenic, specific toxigenic strainsthat coexist with a larger population of non-toxigenicstrains may be overlooked. Biochemical tests are also in-capable of distinguishing different types of C. perfrin-gens. As a consequence, traditional clinical diagnosis ofenteric pathogens by conventional methods lack thecompetence for large-scale investigations and the ad-equate precision and sensitivity required for low-levelsub-clinical detection. Several immunosorbent assayshave also been developed, of which some are also com-mercially available for detection of clostridial toxins in-cluding C. perfringens toxins [20, 22, 23]; however, these
assays are not as sensitive as traditional methods andoften produce false-negative results . Further,these toxin-specific immunosorbent assays may overlookthe samples of those subjects who are carrying toxigenicbacterium but in whom the toxin is not produced .Furthermore, the toxin may not be detected, for in-stance, if it is a modified toxin or neutralized by hostfactors . Therefore, to avoid such discrepancies, thepotential of PCR-based methods with specific gene tar-gets has been explored. Rapid and specific detection hasbeen achieved by molecular approaches based on PCR,and it has been used as a reliable tool to detect C. per-fringens within complex microbial backgrounds such ascomplex food products and fecal samples . How-ever, since complex samples such as human feces con-tain numerous polymerase-inhibitory substances thataffect the sensitivity of PCR reactions [33, 34], majorityof the available methods require pure cultures or pre-incubation steps in order to enumerate lower counts(< 105 cfu/g) of toxigenic strains of C. perfringens,particularly in the feces of asymptomatic subjects.In this context, we developed a rapid and sensitive
qPCR assay to detect and quantify C. perfringens directlyin human fecal samples by using a novel primer pair tar-geting the chromosomally-located plc (-toxin) gene.The assay was coupled with a commercially availablePCR buffer Ampdirect Plus (Shimadzu, Japan) as ameasure to allow more efficient PCR amplification byneutralizing the effect of inhibitory substances present inbiological samples . The method was validated forits specificity and sensitivity against various targeted(C. perfringens) and non-targeted (non-C. perfringensand non-clostridial species) bacterial strains, and alsofor detection efficiency and reproducibility in artifi-cially inoculated fecal samples. The final step in theanalytical validation of the assay was its application todetect and quantify C. perfringens in fecal samplesfrom 345 apparently healthy Japanese subjects (infants, n124; adults, n 221). In addition to plc gene, the sampleswere also analyzed for 16S rRNA and chromosomal cpegenes. Herein, we provide a specific, sensitive and rapidassay for quantitative enumeration of toxigenic C. perfrin-gens in human feces.
MethodsPrimer designThe plc gene was targeted for the design of primer. Thesequence of the gene was procured from GenBank, Na-tional Center for Biotechnology Information (NCBI)(http://www.ncbi.nlm.nih.gov/genbank/). Multiple align-ment of the gene sequences was performed with theCLUSTAL_X program (http://clustalx.software.infor-mer.com)  by using plc gene sequences from morethan 25 strains of C. perfringens including the three
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reference strains, ATCC 13124T (GenBank accessionnumber: NC_008261; Gene ID: 4201274), Str. 13 (Gen-Bank accession number