Detection and quantification of four species of the genus Clostridium in infant feces

To determine the composition of Clostridium in the feces of infants approximately 30 days old, we have developed a detection and quantification method of Clostridium paraputrificum, Clostridium perfringens, Clostridium tertium, and Clostridium difficile by species-specific primers. C. perfringens and C. difficile were detected in four fecal samples from 22 infants (18.2%), whereas C. paraputrificum was detected in three samples (16.7%). C. tertium was detected in two samples (9.1%). Moreover, the occurrences of the four species in bottle-and mix-fed infants were relatively higher than in breast-fed infants (P< 0.05). Subsequently, positive samples detected by nested PCR (polymerase chain reaction) were subjected to realtime PCR. The results showed that the numbers of C. paraputrificum, C. perfringens, C. tertium, and C. difficile ranged from about 1x10(5) to 3x10(7) cells/g wet feces.     

Presence and molecular characterization of Clostridium difficile and Clostridium perfringens in intestinal compartments of healthy horses

ABSTRACT: BACKGROUND: Clostridium difficile and Clostridium perfringens are commonly associated with colitis in equids, but healthy carriers exist. Scarce information is available on the prevalence of Clostridium spp. in gastrointestinal compartments other than faeces in healthy horses, and it is unknown whether faecal samples are representative of proximal compartments. The objectives were to investigate the prevalence of C. difficile and C. perfringens in different intestinal compartments of healthy adult horses and to determine whether faecal samples are representative of colonization in proximal sites and overall carrier status. RESULTS: Toxigenic C. difficile was isolated from 14/135 (10.3%) samples from 8/15 (53.3%) horses. Between zero and three sites were positive per horse, and multiple sites were positive in four horses. Isolates were recovered from duodenum, jejunum, ileum, right dorsal colon, small colon and rectum. When multiple compartments were positive in a single horse, two different C. difficile ribotypes were always present. Clostridium perfringens Type A (CPE, beta2 toxin gene negative) was recovered from the left ventral colon of one horse (0.74%, 1/135 samples). Agreement between faeces and overall C. difficile carrier status was good. CONCLUSIONS: Clostridium difficile can be found in different compartments of the gastrointestinal tract of healthy horses, and multiple strains can be present in an individual horse. The prevalence of C. perfringens in healthy adult hoses was low, consistent with previous reports. Faecal samples were representative for presence of C. difficile in proximal compartments in 5/8 horses (63%) but were not representative for the specific strain.     

Isolation of toxigenic strains of Clostridium difficile and enterotoxin producing Bacteroides fragilis from fecal specimens of patients suspected of antibiotic associated diarrhoea

Fifty faecal samples from patients suspected of AAD (antibiotic associated diarrhoea) were studied for Clostridium difficile and enterotoxin producing Bacteroides fragilis (ETBF). Using TCD (Becton-Dickinson) and C. difficile Toxin A test (Oxoid) in 34% of specimens the presence of toxin A was detected. From all specimens 25 C. difficile strains were isolated. All isolated strains produced toxin B in vitro which was shown in Mc Coy cytotoxicity test. Eighteen strains only were toxin A positive in vitro. From all isolated C. difficile strains 28% were tox A (-) tox B (+). By means of PCR presence of toxin A and toxin B genes was tested directly in faecal samples and in strains. From the same 50 faecal samples 17 B. fragilis strains were isolated. Four of them produced the enterotoxin (fragilisin) which was detected on the HT 29/C1 cell line. Genes of fragilisin were found in strains and directly in faecal samples. Toxin producing C. difficile and B. fragilis (ETBF) together were found in 3 samples. From one faecal sample only ETBF was cultured.     

Clostridium difficile strains not producing Toxin A,but producing Toxin B [toxA(-)tox B(+)]--etiologic agent of antibiotic associated diarrhoea (AAD) in Poland

Previously, toxin A-negative/toxin B-positive Clostridium difficile strains were not thought to be associated with clinically significant diseases. In our study among 159 tested C. difficile strains isolated from feacal samples from 413 patients with antibiotic associated diarrhoea (AAD) 17 strains (11%) were negative in the "Culturette Brand Toxin" CD (Becton-Dickinson) for detection toxin A and positive in the TOX A/B test, designed for detection of both toxins. The conserved regions of both toxin genes were detectable in all of isolates studied by the PCR. Nine of these C. difficile strains had a deletion in the A gene and remaining 8 strains, revealed an amplicon with the expected size of approximately 2500 bp. In this paper we described the first time the toxin A-negative/toxin B-positive C. difficile strains with deletion in toxin A gene, isolated from the faecal samples of patient with AAD in Poland.     

Quantification of Bifidobacterium spp., Escherichia coli and Clostridium difficile in faecal samples of breast-fed and formula-fed infants by real-time PCR

To determine the influence of either exclusive breast-feeding or formula feeding on both composition and quantity of the gut microbiota in infants, we have developed real-time, quantitative PCR assays for the detection of Bifidobacterium spp. and Clostridium difficile. Furthermore, we have monitored the prevalence and counts of Escherichia coli by applying a previously described real-time PCR assay. We found all 100 infants tested to be colonized by Bifidobacterium spp. The bifidobacterial counts were comparable between the 50 breast-fed and 50 formula-fed infants with median values of 10.56 log10 and 10.24 log10 CFU g(-1) wet weight faeces, respectively. C. difficile was detected in 14% of the breast-fed and 30% of the formula-fed infants. In addition, the C. difficile counts were significantly lower in breast-fed infants than in the formula-fed group (median values of 3.28 log10 and 7.43 log10 CFU g(-1), respectively; p=0.03). The prevalence of E. coli in the breast-fed and formula-fed group was 80% and 94%, respectively. Also, the E. coli counts in colonized infants was significantly lower in the breast-fed infants than in the formula-fed group (median values of 9.11 log10 and 9.57 log10 CFU g(-1), respectively; p=0.004). We conclude that the prevalence and counts of C. difficile as well as E. coli are significantly lower in the gut microbiota of breast-fed infants than in that of formula-fed infants, whereas the prevalence and counts of Bifidobacterium spp. is similar among both groups.     

Fecal lactoferrin and Clostridium spp. in stools of autistic children

Stools from autistic and healthy children were studied for fecal lactoferrin, Clostridium difficile toxins, Clostridium perfringens enterotoxin and cultured for Clostridium spp. Elevated level of FLA was demonstrated in 24.4% stools, all from boys (31.25%). No toxins were detected. Clostridium spp. was isolated with similar frequency from all samples. C. perfringens were isolated significantly often from the autistic stools, intermediate sensitive strains to penicillin 19%, to clindamycin 11.3%, and to metronidazole 7.5% were detected. Further studies on fecal microflora and inflammatory mediators, with larger groups of patients, are required in order to explain their role in neurological deficits.     

Intestinal flora of patients with suspected antibiotic associated diarrhea (AAD). I. Clostridium perfringens

Stool samples of 158 patients suspected of antibiotic-associated diarrhoea (AAD) were studied. Toxin A of C. difficile and enterotoxin of C. perfringens were detected in stool samples by immunoenzymatic assays and PCR. In 35 stool samples toxin A of C. difficile was detected and in 48 cases (30%) C. difficile strains were cultured from 21 stool samples (13%). The presence of the cpe gene of C. perfringens, enabling the production of enterotoxin, could not be detected by PCR, both in stool samples and in isolated strains, using ent 1 and ent 2 primer pairs. C. difficile and C. perfringens were isolated from the same stool samples in 4 cases. From stool samples of two patients with AAD C. perfringens strains, thermoresistant spores were cultured.     

Development of an extensive set of 16S rDNA-targeted primers for quantification of pathogenic and indigenous bacteria in faecal samples by real-time PCR

AIMS: The microbiota of the human intestinal tract constitutes a complex ecosystem. We report the design and optimization of an extensive set of 16S rDNA-targeted species- and group-specific primers for more accurate quantification of bacteria from faecal samples with real-time PCR. METHODS AND RESULTS: A linear range of quantification between 0.1-10 pg and 10 ng of specific target genome was obtained, which corresponds to detection of ca 30-4500 to 1.9 x 10(6)-6.0 x 10(6) target bacterial genomes. Functionality of the assays was confirmed by quantification of target bacterial DNA from faecal DNA preparations of healthy volunteers and irritable bowel syndrome (IBS) patients. Additionally, spiking of faecal preparations with Helicobacter pylori, Clostridium difficile or Campylobacter jejuni was used to confirm the accurate and sensitive quantification. CONCLUSIONS: Real-time PCR is a very sensitive and precise technique for an extensive quantitative evaluation of gut microbiota and is feasible for detection of human pathogens from faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: To design and optimize an extensive set of real-time PCR assays targeting a large group of predominant and pathogenic GI microbial species for further use in updating the current knowledge of the putative role of gut microbiota in health and disease.     

Clostridium difficile and enterotoxigenic Bacteroides fragilis strains isolated from patients with antibiotic associated diarrhoea

From the fecal samples of 332 patients with a clinical diagnosis of antibiotic associated diarrhoea (AAD), 131 Clostridium difficile strains were isolated. For detection of toxin A in the isolated strains the enzymatic immunoassay was used. The cytopathic effect was determined on McCoy cell line. PCR was used for the detection of non-repeating and repeating sequences of toxin A gene and non-repeating sequences of toxin B gene. One hundred and six isolated C. difficile strains were TcdA(+)TcdB(+), 10 strains TcdA(-)TcB(+) and 15 were non-toxigenic TcdA(-)TcdB(-). Out of the same fecal samples 50 Bacteroides fragilis strains were isolated. All B. fragilis strains were tested in PCR reaction for fragilysine gene detection (bft). In 9 strains (18%) this gene was detected and the strains could be assumed as enterotoxigenic Bacteroides fragilis (ETBF). In 4 fecal samples toxigenic C. difficile (TcdA(+)TcdB(+)) was found simultaneously with ETBF. One sample contained C. difficile (TcdA(-)TcdB(+)) and ETBF. Out of 4 fecal samples only ETBF was isolated. The cytotoxicity of ETBF strains was tested on HT29/C1 human colon carcinoma cell line. The cytotoxicity titer in the range of 20 and 80 was observed.     

A sporulation medium for Clostridium perfringens

A new solidified medium for inducing sporulation of Clostridium perfringens is described. The essential components of the medium are bile, bicarbonate and quinoline. The medium induced significant sporulation in all of 100 strains of Cl. perfringens isolated at random from human faecal specimens. The majority (94%) of strains sporulated profusely.     

Cultivation of fungi from fecal specimens in cases of antibiotic associated diarrhea (AAD)

The aim of performed examinations was to isolate, identify and determine a drug susceptibility of fungi cultured from faecal specimens submitted for detection of Clostridium difficile in cases of antibiotic-associated diarrhoea (AAD). One hundred samples of diarrhoeic faeces were examined using routine bacteriological methods (isolation and identification of C. difficile), serological test (detection of C. difficile toxins A/B) and mycological methods (isolation, identification and drug susceptibility testing of fungi). Out of twenty seven specimens of diarrhoeic faeces fungal strains were isolated, in 20 samples C. difficile strain and/or C. difficile toxins A/B were detected, in 23 specimens fungal strains, C. difficile strains and/or toxins A/B of this species were present. The most active in vitro agent against cultured fungal strains was nystatin. In conclusion it can be stated, that fungal strains are responsible for some cases of antibiotic-associated diarrhoea. So, mycological diagnostics of faecal samples from patients with diarrhoea after antibiotic therapy is necessary. Cases of diarrhoea with mixed bacterial and fungal aetiology (C. difficile + yeast-like fungus) were observed.     

Germination response of spores of the pathogenic bacterium Clostridium perfringens and Clostridium difficile to cultured human epithelial cells

Spores of pathogenic Clostridium perfringens and Clostridium difficile must germinate in the food vehicle and/or host's intestinal tract to cause disease. In this work, we examined the germination response of spores of C. perfringens and C. difficile upon incubation with cultured human epithelial cell lines (Caco-2, HeLa and HT-29). C. perfringens spores of various sources were able to germinate to different extents; while spores of a non-food-borne isolate germinated very well, spores of food-borne and animal isolates germinated poorly in human epithelial cells. In contrast, no detectable spore germination (i.e., loss of spore heat resistance) was observed upon incubation of C. difficile spores with epithelial cells; instead, there was a significant (p?相似文献   

Design of species-specific primers to identify 13 species of Clostridium harbored in human intestinal tracts

The genus Clostridium is dominant in human intestinal tracts and plays an important role in human health. We designed species-specific primers to identify 13 species of Clostridium (C. perfringens, C. paraputrificum, C. bifermentans, C. difficile, C. clostridiiforme, C. nexile, C. sphenoides, C. indolis, C. ramosum, C. cocleatum, C. butyricum, C. sordellii, and C. innocuum) easily and rapidly. The PCR annealing temperature was set at a uniform 60 C for application to all strains at the same time. To confirm the specificities of these primers, 85 intestinal bacteria in total, including type strains, reference strains, and isolates were used. Ten primers (including those for C. perfringens to C. cocleatum) indicated high specificities. Although there were some cross-reactions with the other three primers, the target species were distinguishable from other bacteria by the different sizes of PCR products.     

Laboratory diagnosis of Clostridium difficile associated diarrhoea and molecular characterization of clinical isolates

We evaluated a three-step algorithm for laboratory diagnosis of Clostridium difficile-associated diarrhoea (CDAD). First, stool specimens were screened using an EIA test for glutamate dehydrogenase detection. Screen-positive specimens were tested by a rapid cytotoxintoxin A/B assay and subjected to stool culture. All cultures positive for C. difficile underwent toxigenic culture. The results showed that toxigenic culture allowed us to recover 37/156 (24.4%) stool samples harbouring toxigenic C. difficile that would have been missed by using faecal cytotoxin assay alone. This determined an increase in infection prevalence of 4.2% (from 11.4% to 15.6 %). Furthermore, to characterize the clinical Clostridium difficile isolates and the distribution of PCR ribotypes circulating in the San Carlo Borromeo hospital, molecular typing using semi-automated repetitive-sequence-based PCR (rep- PCR) and PCR ribotyping, and an evaluation of the antibiotic resistance were also performed. Among them, 71 indistinguishable strains were detected by rep-PCR and 83 by PCR-ribotyping revealing C. difficile outbreaks in our hospital. A total of 6 different ribotypes were obtained by PCR ribotyping. The most frequent ribotype was 018 (88.2%) that also showed resistance to moxifloxacin. In one case, uncommon PCR ribotype 186 was also identified.     

Hybridization analysis of the class P tetracycline resistance determinant from the Clostridium perfringens R-plasmid, pCW3

The tetracycline resistance determinant from pCW3, a conjugative plasmid from Clostridium perfringens, has been identified and the structural gene localized to within a 1.4-kb region. Hybridization analysis, which utilized an internal 0.8-kb specific gene probe, showed that eight nonconjugative tetracycline resistant C. perfringens strains all carried homologous resistance determinants. No homology was detected in DNA prepared from tetracycline resistant isolates of Clostridium difficile or Clostridium sporogenes. However, the one strain of Clostridium paraputrificum that was tested did contain an homologous determinant. No homology was found to any of the recognized classes of tetracycline resistance determinants. The C. perfringens tetracycline resistance determinant represents a new hybridization group, Class P.     

Occurrence of Clostridium difficile in fecal samples of HIV-infected children in Poland

The prevalence of Clostridium difficile and its toxins (A and B) in HIV-positive children in Poland was investigated in a group of 18 children, aged 6 months to 8 1/2 years. Stool samples were tested using an antigen detection method for toxin A/B, cytotoxicity-neutralization and culture. In 3 cases (17%) C. difficile toxins were detected in both stool samples and strains recovered from culture. The three strains isolated were shown by PCR methods to contain toxins A and B genes. All children had been treated previously with antimicrobial and antiviral agents. All three C. difficile-positive children had mild diarrhea that resolved without specific therapy. Further studies involving a large number of children and molecular analyses of isolated C. difficile strains are necessary to determine the frequency and rate of carriage of C. difficile strains among HIV-positive children in Poland.     

Profile of toxigenicity of Clostridium difficile strains isolated from paediatric patients with clinical diagnosis of antibiotic associated diarrhea (AAD)

This study was performed to determine profile of toxigenicity of 18 Clostridium difficile strains isolated from paeditric patients suffering from antibiotic associated diarrhea (AAD). Toxigenicity of C. difficile strains was tested for detection toxin A and toxin B by phenotypic methods and for detection of the tcdA and tcdB genes using of PCR. Changes in the repeating regions of the tcdA genes were detected with the NK9/NKV011 primer pairs. For detection of binary toxin (CDT) cdtA and cdtB genes, cdtApos/cdtArev i cdtBpos/cdtBrev two pair primers in PCR was used. Among C. difficile strains was detected three profiles of toxigenicity: C. difficile strains possesing of tcdA and tcdB genes but not possesing cdtA and cdtB genes of binary toxin (A+B+CDT-), strains possesing tcdA and tcdB and cdtA and cdtB genes (A+B+CDT+), strains with deletion of toxin A gene (A-B+CDT-). This is the first report on the occurence of binary positive C. difficile strains isolated from paediatric patients.     

Prophage Carriage and Diversity within Clinically Relevant Strains of Clostridium difficile

Prophages are encoded in most genomes of sequenced Clostridium difficile strains. They are key components of the mobile genetic elements and, as such, are likely to influence the biology of their host strains. The majority of these phages are not amenable to propagation, and therefore the development of a molecular marker is a useful tool with which to establish the extent and diversity of C. difficile prophage carriage within clinical strains. To design markers, several candidate genes were analyzed including structural and holin genes. The holin gene is the only gene present in all sequenced phage genomes, conserved at both terminals, with a variable mid-section. This allowed us to design two sets of degenerate PCR primers specific to C. difficile myoviruses and siphoviruses. Subsequent PCR analysis of 16 clinical C. difficile ribotypes showed that 15 of them are myovirus positive, and 2 of them are also siphovirus positive. Antibiotic induction and transmission electron microscope analysis confirmed the molecular prediction of myoviruses and/or siphovirus presence. Phylogenetic analysis of the holin sequences identified three groups of C. difficile phages, two within the myoviruses and a divergent siphovirus group. The marker also produced tight groups within temperate phages that infect other taxa, including Clostridium perfringens, Clostridium botulinum, and Bacillus spp., which suggests the potential application of the holin gene to study prophage carriage in other bacteria. This study reveals the high incidence of prophage carriage in clinically relevant strains of C. difficile and correlates the molecular data to the morphological observation.     

Clostridium difficile and its cytotoxin in infants admitted to hospital with infectious gastroenteritis.

During a prospective study of infectious gastroenteritis in children under 2 years, 19 out of 390 patients (4.9%) were found to have Clostridium difficile cytotoxin in the faeces. In several there was no history of use of antibiotics. The symptoms of many infants with toxin settled spontaneously, but one child became acutely and severely ill and developed a toxic megacolon and five others required, and responded to, vancomycin. Cl difficile was cultured from the stools in 191 (49%) of the children. The highly significant increased prevalence of past use of antibiotics in 118 control patients was not associated with an increased incidence of either isolation of Cl difficile or presence of faecal cytotoxin. Cl difficile should not be overlooked as a cause of acute diarrhoea and vomiting in children under 2 years.