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

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 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. 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.

Ontogenesis of the Gut Microbiota Composition in Healthy, Full-Term, Vaginally Born and Breast-Fed Infants over the First 3 Years of Life: A Quantitative Bird's-Eye View.

Early-life intestinal microbiota development is crucial for host's long-term health and is influenced by many factors including gestational age, birth and feeding modes, birth environment, ethnic/geographical background, etc. However, 'quantitative' data on the actual population levels of gut bacterial communities when these influences are controlled for is relatively rare. Herein, we demonstrate a quantitative perspective of microbiota development in natural and healthy milieus, i.e., in healthy, full-term, vaginally born and breast-fed infants (n = 19) born at same clinic. Fecal microbiota at age 1 and 7 days, 1, 3, and 6 months and 3 years is quantified using highly sensitive reverse-transcription-quantitative-PCR assays targeting bacterial rRNA molecules. At day 1, we detect one or more bacteria in all (100%) of the babies, wherein the microbiota is composed mainly of enterobacteria (35%), Bacteroides fragilis group (23%), enterococci (18%), staphylococci (13%), and bifidobacteria (9%). Altogether, facultative anaerobes predominate during first few weeks whereafter obligate anaerobes including bifidobacteria, B. fragilis group, Clostridium coccoides group, and Clostridium leptum subgroup gradually start prevailing. At 3 years, the composition is represented almost entirely (99%) by obligate anaerobes including C. leptum subgroup (34%), bifidobacteria (22%), B. fragilis group (21%), C. coccoides group (17%), Atopobium cluster (4%), and Prevotella (1%). The overall obligate/facultative proportion is 32/68, 37/63, 54/46, 70/30, 64/36, and 99/1% at 1 and 7 days, 1, 3, and 6 months and 3 years, respectively. However, interestingly, considerable individual-specific variations in the obligate/facultative ratios as well as in the proportions of Firmicutes, Bacteroides, Actinobacteria, and Proteobacteria communities are seen among these babies. This disparity even within this highly homogenous cohort manifests the magnitude of diverse patterns of gut microbiota configuration and hence underpins the importance of considering not only the gestational age, birth, and feeding modes, and ethnic/geographical background but also other potential outstanding factors when investigating the elements shaping the early microbiota development. In summary, the data demonstrate a quantitative bird's-eye view of the ontogenesis of early-life gut microbiota in typically natural and healthy milieus and should be informative and facilitative for future studies exploring various aspects of the human gut microbiota.

Evolution of gut Bifidobacterium population in healthy Japanese infants over the first three years of life: a quantitative assessment.

Bifidobacteria are important members of human gut microbiota; however, quantitative data on their early-life dynamics is limited. Here, using a sensitive reverse transcription-qPCR approach, we demonstrate the carriage of eight signature infant-associated Bifidobacterium species (B. longum, B. breve, B. bifidum, B. catenulatum group, B. infantis, B. adolescentis, B. angulatum and B. dentium) in 76 healthy full-term vaginally-born infants from first day to three years of life. About 21% babies carry bifidobacteria at first day of life (6.2 ± 1.9 log10 cells/g feces); and this carriage increases to 64% (8.0 ± 2.2), 79% (8.5 ± 2.1), 97% (9.3 ± 1.8), 99% (9.6 ± 1.6), and 100% (9.7 ± 0.9) at age 7 days, 1, 3 and 6 months, and 3 years, respectively. B. longum, B. breve, B. catenulatum group and B. bifidum are among the earliest and abundant bifidobacterial clades. Interestingly, infants starting formula-feed as early as first week of life have higher bifidobacterial carriage compared to exclusively breast-fed counterparts. Bifidobacteria demonstrate an antagonistic correlation with enterobacteria and enterococci. Further analyses also reveal a relatively lower/ delayed bifidobacterial carriage in cesarean-born babies. The study presents a quantitative perspective of the early-life gut Bifidobacterium colonization and shows how factors such as birth and feeding modes could influence this acquisition even in healthy infants.

Sensitive Quantitative Analysis of the Meconium Bacterial Microbiota in Healthy Term Infants Born Vaginally or by Cesarean Section.

For decades, babies were thought to be born germ-free, but recent evidences suggest that they are already exposed to various bacteria in utero. However, the data on population levels of such pioneer gut bacteria, particularly in context to birth mode, is sparse. We herein aimed to quantify such bacteria from the meconium of 151 healthy term Japanese infants born vaginally or by C-section. Neonatal first meconium was obtained within 24-48 h of delivery; RNA was extracted and subjected to reverse-transcription-quantitative PCR using specific primers for Clostridium coccoides group, C. leptum subgroup, Bacteroides fragilis group, Atopobium cluster, Prevotella, Bifidobacterium, Lactobacillus, Enterococcus, Enterobacteriaceae, Staphylococcus, Enterococcus, Streptococcus, C. perfringens, and C. difficile. We detected several bacterial groups in both vaginally- and cesarean-born infants. B. fragilis group, Enterobacteriaceae, Enterococcus, Streptococcus, and Staphylococcus were detected in more than 50% of infants, with counts ranging from 105 to 108 cells/g sample. About 30-35% samples harbored Bifidobacterium and Lactobacillus (104-105 cells/g); whereas C. coccoides group, C. leptum subgroup and C. perfringens were detected in 10-20% infants (103-105 cells/g). Compared to vaginally-born babies, cesarean-born babies were significantly less often colonized with Lactobacillus genus (6% vs. 37%; P = 0.01) and Lactobacillus gasseri subgroup (6% vs. 31%; P = 0.04). Overall, seven Lactobacillus subgroups/species, i.e., L. gasseri subgroup, L. ruminis subgroup, L. casei subgroup, L. reuteri subgroup, L. sakei subgroup, L. plantarum subgroup, and L. brevis were detected in the samples from vaginally-born group, whereas only two members, i.e., L. gasseri subgroup and L. brevis were detected in the cesarean group. These data corroborate that several bacterial clades may already be present before birth in term infants' gut. Further, lower detection rate of lactobacilli in cesarean-born babies suggests that the primary source of lactobacilli in infant gut is mainly from maternal vaginal and-to a lesser extent-anal microbiota during vaginal delivery, and that the colonization by some important Lactobacillus species is delayed in babies delivered via cesarean-section.

Rectal mucosal dissection during transanal pull-through for Hirschsprung disease: the anorectal or the dentate line?

PURPOSE: Both the dentate line (DL) and anorectal line (ARL) are anatomic landmarks for rectal mucosal dissection during transanal pull-through for Hirschsprung disease. We compared outcome after rectal mucosal dissection commencing above the DL (DL group; n = 8) with outcome after rectal mucosal dissection commencing on the ARL (ARL group; n = 6) with normal babies (Cont group; n = 10). METHODS: Rectal mucosal dissection commenced on the ARL in the ARL group and at various levels (0-10 mm) above the DL in the DL group. Outcome was assessed prospectively for 6 months using a standard structured questionnaire. RESULTS: Subject demographics were not significantly different. Differences in frequency of motions between the ARL and Cont groups were not statistically significant after 3 months of age, but the DL group had significantly more motions than the other 2 groups after 4 months of age (P < .01). At 6 months of age, fecal staining was 17% in the ARL group, 63% in the DL group, and 0% in the Cont group. Anal shape was normal in the ARL and Cont groups, but 50% of the DL group had visible anal mucosa. CONCLUSION: Bowel function in the ARL group is similar to normal, and because the ARL is easily identifiable without the need for subjective interpretation, we recommend using the ARL as a landmark for rectal mucosal dissection during transanal pull-through for Hirschsprung disease.

Repair of hypospadias with severe chordee using a long, wide, U-shaped flap that preserves ventral penile tissues intact for second-stage urethroplasty.

PURPOSE: The aim of the study was to report a new technique for repairing hypospadias with severe chordee (HSC). METHODS: Our new technique involves making a long, wide, U-shaped incision on the ventral penis from the coronal sulcus to very distal to the meatus and dissecting to create a flap (U-flap). During dissection, the urethra is divided just proximal to the meatus. After release of chordee, the U-flap is returned to the ventral penile shaft and sutured in place. A buttonhole made distally in the U-flap is anastomosed to the cut end of the urethra to create a neomeatus. Snodgrass urethroplasty is performed 6 to 18 months later. We have treated 11 patients with HSC (mean age, 22.3 months) using this technique. RESULTS: Postoperatively, all U-flaps were viable. The neomeatus appeared to be more proximal because the penis was straighter. Urethroplasty using the central part of the U-flap was uncomplicated by scar tissue and successful in all cases. After a mean follow-up of 15.7 months, all patients have satisfactory penises without stenosis or diverticulum, although 1 had fistula. CONCLUSION: Our U-flap technique allows the ventral penis to be preserved intact without scarring for second-stage urethroplasty and as a result is well suited for treating HSC.

Significance of pulmonary artery size and blood flow as a predictor of outcome in congenital diaphragmatic hernia.

AIM: To determine if pulmonary artery size and blood flow have prognostic value in congenital diaphragmatic hernia (CDH). METHODS: Twenty-eight consecutive left-sided CDH patients treated according to a standard protocol with high frequency oscillatory ventilation (HFOV) + nitric oxide (NO) had right and left pulmonary artery (RPA, LPA) diameters, LPA/RPA diameter (L/R) ratios, and PA blood flows examined by echocardiography (EC) on days 0, 2, and 5 after birth and compared prospectively. RESULTS: Twenty-two patients (78.6%) survived. Of these, 15 required NO (NO-s), and seven did not (non-NO-s). All six patients that died required NO (NO-d). RPA in the NO-d group was significantly smaller than in the NO-s or non-NO-s groups on day 0 (2.90 +/- 0.41 vs. 3.40 +/- 0.49 or 4.01 +/- 0.43; P < 0.01, respectively). LPA in the NO-d group was significantly smaller than in the non-NO-s on day 0 (2.13 +/- 0.45 vs. 3.39 +/- 0.34; P < 0.01). L/R ratios in NO subjects were significantly smaller (NO-s 0.74 +/- 0.11; NO-d 0.73 +/- 0.11) than in non-NO-s subjects (0.84 +/- 0.03) on day 0 (P < 0.01). PA diameters and L/R ratios did not change significantly from day 0 to day 5 in all three groups. There was LPA flow on day 0 in all non-NO-s subjects, but none in all NO subjects. In the NO-s group, LPA flow was confirmed in 87% (13/15) on day 2 and in 100% on day 5, however, there was no LPA flow from day 0 to day 5 in any of the NO-d group. CONCLUSION: Our data indicate that PA diameters on day 0 and LPA flow are strongly prognostic in left-sided CDH and L/R ratio would appear to be a simple highly reliable indicator of the necessity for NO therapy.

Management of pulmonary hypertension in congenital diaphragmatic hernia: nitric oxide with prostaglandin-E1 versus nitric oxide alone.

AIM: Prostaglandin-E1 (PGE1) is used at most centers for treating pulmonary hypertension (PH) in congenital diaphragmatic hernia (CDH) because it has been regarded as effective. The aim of this study was to investigate the role of PGE1 for treating PH in CDH. METHODS: We reviewed 49 CDH cases with echocardiography-proven PH. PH was treated with PGE1 and nitric oxide (NO) and high frequency oscillatory ventilation (HFOV) from 1997 to 2001 (PG + NO; n = 19) and with NO and HFOV from 2002 to 2007 (NO; n = 30). RESULTS: Subject demographics, severity of PH, and presence of other anomalies were not significantly different between the two groups. In the PG + NO group, 12/19 (63.2%) survived (PG + NO-s) and 7/19 (36.8%) died (PG + NO-d). In the NO group, 21/30 (70.0%) survived (NO-s) and 9/30 (30.0%) died (NO-d). Survival rates were not significantly different. In the NO-s group, spontaneous closure of the ductus arteriosus (DA) was significantly earlier compared with the PG + NO-s group (P < 0.01; 4.0 +/- 0.9 vs. 9.5 +/- 2.2 days after birth). DA diameters were significantly larger in groups that died compared with groups that survived (P < 0.01), and PH persisted in groups that died. In the NO-s group, surgery was possible significantly earlier compared with the PG + NO-s group (P < 0.01; 3.75 +/- 0.67 vs. 6.12 +/- 0.78 days after birth). No NO-s case developed a PH crisis even though PGE1 was not used. Hospital stay was significantly shorter in the NO-s group compared with the PG + NO-s group (P < 0.05; 39.9 +/- 19 vs. 53.2 +/- 23 days). CONCLUSION: Nitric oxide alone would appear to simplify the management of CDH with PH and provide better outcome.