Two hundred nineteen Clostridium difficile isolates from 22 serogroups were screened for changes in the genes coding for toxin B (tcdB) and toxin A (tcdA). Parts of the toxin genes were amplified, and the PCR fragments were checked for length polymorphisms and cut with several restriction enzymes to monitor restriction fragment length polymorphisms (RFLPs). For 47 strains (21%), differences in the toxin genes were found compared to the toxin genes of reference strain VPI 10463. Polymorphisms were usually observed in both toxin genes. RFLPs were more commonly found in the tcdB gene, in which a single restriction enzyme could give up to five different patterns. Restriction sites seemed to be less heterogeneous in the tcdA gene, in which for most enzymes only two different RFLPs were recognized. However, deletions were observed in tcdA, and four new types of shortenedtcdA genes are described. According to the changes in their toxin genes, variant strains could be divided into 10 groups (toxinotypes I to X). A toxinotype was characterized by similar patterns of changes in the toxin genes and in other regions of the pathogenicity locus and also similar pulsed-field gel electrophoresis patterns. Variant toxinotypes were found in 9 of the 22 serogroups studied, and some toxinotypes were clearly associated with specific serogroups. Toxinotype VIII is characteristic for all strains of serogroup F. Other serogroups in which variant toxinotypes were commonly found are A1, A15, E, and X. Testing of variability inC. difficile toxin genes not only might be useful as a molecular typing system but also could have implications in diagnostics and pathogenesis.

A routine protocol for diagnosing Clostridium difficile-associated diarrhoea (CDAD) based on both faecal-cytotoxin detection and toxigenic culture was adopted by the microbiology laboratory of the St Luc-UCL University Hospital in Brussels in 1997. A toxigenic culture is a faecal culture followed, in the case of positivity, by a direct immunoassay on colonies to detect toxin A production. The results obtained over the past 7 years in the hospital are reviewed here. A total of 10 552 diarrhoeal stools from 7042 patients were analysed, of which 9494 were negative for all tests. A total of 1058 samples (10 %) from 794 patients were culture-positive, of which 460 (4 . 4 %) were positive for a faecal cytotoxin. The remaining 598 cultures were tested for toxin A on colonies; 355 of them were positive, which is 3 . 4 % of the total, and the remaining 243 (2 . 3 %) were negative. The positivity of the faecalcytotoxin assay was statistically linked to the number of colonies observed on the culture plate. In conclusion, over a 7 year period, toxigenic culture allowed the diagnosis of 355 cases of CDAD that would have been missed by a protocol using a faecal-cytotoxin assay alone. In terms of both patient care, prevention of environmental contamination and prevention of risk of a hospital outbreak, it is proposed that these results justify the recommendation to perform both faecal-toxin assay and culture in routine medical practice.

BackgroundThe equine faecal microbiota is very complex and remains largely unknown, while interspecies interactions have an important contribution to animal health. Clostridium difficile has been identified as an important cause of diarrhoea in horses. This study provides further information on the nature of the bacterial communities present in horses developing an episode of diarrhoea. The prevalence of C. difficile in hospitalised horses at the time of admission is also reported.ResultsBacterial diversity of the gut microbiota in diarrhoea is lower than that in non-diarrhoeic horses in terms of species richness (p-value <0.002) and in population evenness (p-value: 0.02). Statistical differences for Actinobacillus, Porphyromonas, RC9 group, Roseburia and Ruminococcaceae were revealed. Fusobacteria was found in horses with diarrhoea but not in any of the horses with non-diarrheic faeces. In contrast, Akkermansia was among the three predominant taxa in all of the horses studied. The overall prevalence of C. difficile in the total samples of hospitalised horses at admission was 3.7 % (5/134), with five different PCR-ribotypes identified, including PCR-ribotype 014. Two colonised horses displayed a decreased bacterial species richness compared to the remaining subjects studied, which shared the same Bacteroides genus. However, none of the positive animals had diarrhoea at the moment of sampling.ConclusionsThe abundance of some taxa in the faecal microbiota of diarrhoeic horses can be a result of microbiome dysbiosis, and therefore a cause of intestinal disease, or some of these taxa may act as equine enteric pathogens. Clostridium difficile colonisation seems to be transient in all of the horses studied, without overgrowth to trigger infection. A large proportion of the sequences were unclassified, showing the complexity of horses’ faecal microbiota.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0514-5) contains supplementary material, which is available to authorized users.

Summary. Most toxigenic strains of Clostridium di#iciie produce two toxins : an enterotoxin (toxin A) and a cytotoxin (toxin B). Only one strain (strain 8864) has been reported to produce toxin B but no toxin A. Serogroup F strains (44) of C. dzficile, often isolated from asymptomatic infants, have been examined for toxin production. These strains, which were from distinct geographical and clinical sources, did not produce any detectable toxin A in citro when examined in three distinct immunoassays. Nevertheless, all the strains produced a cytotoxin. Immunological differences between the cytotoxin of the serogroup F strains and that produced by C. dz3ciie strain VPI 10463 (serogroup G) were demonstrated with monoclonal antibodies specific for either the toxin B produced by C . dificiie strain VPI 10463 or C. sordellii lethal toxin (LT). Polymerase chain reaction amplification with primers derived from C. dtficile strain VPI 10463 toxin A and B genes showed that serogroup F strains seem to possess a toxin B gene homologous with that of strain VPI 10463 and at least fragments of the toxin A gene. When axenic mice were inoculated with serogroup F strains, the animals survived; they did not develop diarrhoea and no toxin A could be detected in their faeces. However, cytotoxin was detected. Furthermore, these mice were protected against subsequent challenge with the otherwise lethally toxigenic C . dzflcile strain VPI 10463. The serogroup F strains appeared to be homogeneous and distinct from other C. diflcile strains with regard to toxin production.

Eighty-six. A gallery of nine conventional and enzymatic tests was developed for the rapid identification of the most common species isolated during this survey. Pyrrolidonyl aminopeptidase, ␥-glutamyl aminopeptidase, ␣-mannosidase, and ␣-glucosidase were found to be highly discriminating and could be used to develop an identification scheme.Nocardia species are isolated with an increased frequency from clinical specimens, especially in specimens from immunocompromised patients (19). The taxonomy of the genus has dramatically been revised during the last decade, and at least 30 valid species have been reported, besides a number of unnamed genomospecies (18). Not all of them have been found in humans, and Nocardia asteroides, previously most frequently isolated from clinical specimens, has proved to be heterogeneous and has been divided into several species (18). More recently, additional species of human origin have been described (6, 8, 9, 11, 12, 17, 28).The routine identification of Nocardia strains at the species level is difficult in the laboratory. This, and the nomenclature changes, may explain that species distribution in clinical isolates has been poorly documented up to now, and even recent surveys still report the "N. asteroides complex" as the most frequent Nocardia species isolated in humans (7,10,16,19). Identification studies have not been systematically carried out since the several recent taxonomic changes.The aim of this study was to assess the species distribution of a large number of Nocardia isolates and to propose simple and rapid identification tests that may be helpful to identify the species most commonly encountered in clinical material. MATERIALS AND METHODSBacterial strains. Eighty-six Nocardia strains isolated from clinical specimens in Belgium were collected for the study. Most strains were isolated during the past decade, but a few were received before 1990. They were isolated by several laboratories in different parts of the country. All the Nocardia isolates were included in the study to avoid any bias in the selection of the strains. Only one strain per patient was considered. The clinical origins of the isolates were as follows: 36 strains were isolated from the respiratory tract, 18 from pyogenic lesions and wounds, 8 from blood, 4 from brain abscesses, and 1 from cerebral fluid. Nineteen were of unknown origin.The type strains of the most relevant species were included for phenotypic comparison as well as reference strains of some less common species. Nocardia species and strains are listed in Table 1.Sequencing of the 16S rRNA gene. The full-length 16S rRNA gene sequences (Ϯ1,400 nucleotides) of all strains were determined as described previously (24), and sequences were compared to those of the type strains deposited in the GenBank database.Cellular fatty acids were analyzed by gas-liquid chromatography as outlined elsewhere (22).Phenotypic characterization. Strains were cultured on tryptic soy blood agar plates at 35°C. They were examined for partial acid-fastness, presenc...

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A yellow-pigmented coryneform rod was isolated from the blood of a child with acute lymphoblastic leukemia who was perfused with a central venous catheter. The culture bottles were positive twice, at a 2-month interval. The isolate was identified as a Microbacterium sp. and studied along with five other similar strains. Phenotypic, chemotaxonomic, and genetic characteristics indicated that they are closely related to Microbacterium oxydans but that they belong to a distinct species, for which the name Microbacterium paraoxydans sp. nov. is proposed. The type strain of M. paraoxydans is CF36T ‫؍‬ DSM 15019 T . The G؉C content of its DNA is 69.9 mol%. CASE REPORTA 5-year-old boy was diagnosed with acute lymphoblastic leukemia in December 1994 and included in the Fralle 93 protocol. The child achieved hematologic remission 6 weeks after induction chemotherapy was initiated. He was seen as an outpatient and responded well to treatment in the following years. However, at a March 1997 consultation, the patient had a febrile episode when being perfused with the Port-a-cath system. Clinical examination showed no septic localization. The leukocyte count was 10,860/ml, with an absolute neutrophil count of 8,850/ml. One set of blood cultures was collected from the Port-a-cath, and the aerobic bottle culture yielded yellow-pigmented colonies of gram-positive coryneform rods. The boy received ceftriaxone (1 g) intravenously once, followed by cefadroxil 500 mg twice a day for 7 days. Because of a persistent fever, cefadroxil was given for another week and immunosuppressive drugs were discontinued during the same period. The patient's history was unremarkable for the next 2 months, and no blood culture was performed when he came for his monthly visits. In June 1997, the child was presented for consultation as subfebrile and complaining of fatigue. A physical examination revealed no focus of infection, and the leukocyte count was not elevated. One set of blood cultures was taken from the Port-a-cath, and the aerobic vial revealed the same gram-positive coryneform rods, subsequently identified as Microbacterium sp. Catheter-related bacteremia was diagnosed, and the central venous catheter was removed. Ampicillin (100 mg/kg/day) was given intravenously perioperatively, with the first dose given 24 h prior to surgery. The patient's clinical condition improved rapidly afterwards. The Port-a-cath was cultured on Columbia blood agar. Within 24 h at 37°C, a pure culture of yellow-pigmented colonies of a nonfermentative, gram-positive, somewhat discolored rod had grown. Unfortunately, subcultures were lost and no further examination could be performed.The strain isolated from the patient's blood cultures, labeled CF36, was a yellow-pigmented, motile coryneform with an oxidative metabolism, proteolytic activity, and cellular fatty acids of the branched type. These general features are suggestive of the genus Microbacterium, which includes both fermentative and oxidative species, the latter formerly called Aureobacterium (13).The 16S rRN...

This study investigated whether differences in fecal and serum antitoxin A antibody levels may account for the duration of Clostridium difficile-associated diarrhea (CDAD) and the occurrence of relapses. By an enzyme linked-immunosorbent assay, we tested 40 patients with CDAD including 25 patients without immunodeficiency and 15 patients receiving antineoplastic drugs. Two hundred eighty serum samples and 80 normal stool samples were investigated as controls. In nonimmunocompromised patients, serum immunoglobulin (IgG) and fecal IgA antitoxin A antibody titers were significantly higher in patients who suffered a single episode (n = 21) than in those with relapsing CDAD (n = 4) whose titers were at control levels. Of these 25 patients, eight suffered from diarrhea which lasted for more than 2 weeks. These patients had significantly lower serum-and feces-specific antibody levels than the others who presented symptoms of shorter duration. In cytostatic-treated patients, antitoxin A antibody levels were similar to controls, but relapses occurred in a single case. These data suggest an association between a defective humoral response to toxin A and a more severe form of C. difficile infection. They also indicate that other host-related factors control the severity of CDAD and remain to be elucidated.