Typing of Clostridium perfringens by in vitro amplification of toxin genes

G. DAUBE, B. CHINA, P. SIMON, K. HVALA AND J. MAINIL. 1994. The strains of Clostridium perfringens are classified according to major toxins produced. Classically, this determination involves the seroneutralization of their lethal effect in mice. However, this method requires specific antisera and a large number of mice. In this work, a new typing method was developed based on the amplification of toxin genes by polymerase chain reaction (PCR). By combination of several pairs of primers, the toxinotype of a Cl. perfringens strain was determined by looking at the pattern of bands on an agarose gel electrophoresis. This mixture contained primers amplifying simultaneously a part of α-toxin, α-toxin, β-toxin and enterotoxin genes. In order to distinguish between toxinotype A and E, the *** l -toxin gene fragment must be amplified in a separate PCR reaction. Moreover, with the primers combination, in most cases, a PCR product corresponding to the α-toxin gene was obtained from direct enrichments of animal intestinal contents.     

Non Digestible Oligosaccharides Modulate the Gut Microbiota to Control the Development of Leukemia and Associated Cachexia in Mice

We tested the hypothesis that changing the gut microbiota using pectic oligosaccharides (POS) or inulin (INU) differently modulates the progression of leukemia and related metabolic disorders. Mice were transplanted with Bcr-Abl-transfected proB lymphocytes mimicking leukemia and received either POS or INU in their diet (5%) for 2 weeks. Combination of pyrosequencing, PCR-DGGE and qPCR analyses of the 16S rRNA gene revealed that POS decreased microbial diversity and richness of caecal microbiota whereas it increased Bifidobacterium spp., Roseburia spp. and Bacteroides spp. (affecting specifically B. dorei) to a higher extent than INU. INU supplementation increased the portal SCFA propionate and butyrate, and decreased cancer cell invasion in the liver. POS treatment did not affect hepatic cancer cell invasion, but was more efficient than INU to decrease the metabolic alterations. Indeed, POS better than INU delayed anorexia linked to cancer progression. In addition, POS treatment increased acetate in the caecal content, changed the fatty acid profile inside adipose tissue and counteracted the induction of markers controlling β-oxidation, thereby hampering fat mass loss. Non digestible carbohydrates with prebiotic properties may constitute a new nutritional strategy to modulate gut microbiota with positive consequences on cancer progression and associated cachexia.     

Prevalence of enterohaemorrhagic Escherichia coli from serotype O157 and other attaching and effacing Escherichia coli on bovine carcasses in Algeria

AIMS: Bovine meat is the principal source of human contamination of attaching and effacing Escherichia coli, including enterohaemorrhagic E. coli O157. The aim was to study the prevalence of these strains on bovine carcasses in Algeria. METHODS AND RESULTS: Two-hundred and thirty carcasses were swabbed and analysed by classical microbiological methods for total E. coli counts and for the presence of pathogenic E. coli. The E. coli counts were high, with a 75th percentile of 444.75 CFUs cm(-2). For pathogenic E. coli, more than 7% of the tested carcasses were positive for E. coli O157. Eighteen E. coli O157 strains were isolated and typed by multiplex PCR. The main isolated pathotype (78%) was eae+ stx2+ ehxA+. In addition to E. coli O157, other attaching and effacing E. coli (AEEC) were also detected from carcasses by colony hybridization after pre-enrichment and plating on sorbitol MacConkey agar using eae, stx1 and stx2 probes. Thirty carcasses (13%) on the 230 analysed harboured at least one colony positive for one of the tested probes. These positive carcasses were different from those positive for E. coli O157. Sixty-six colonies (2.9%) positive by colony hybridization were isolated. The majority (60.6%) of the positive strains harboured an enteropathogenic E. coli-like pathotype (eae+ stx-). Only three enterohaemorrhagic E. coli (EHEC)-like (eae+ stx1+) colonies were isolated from the same carcass. These strains did not belong to classical EHEC serotypes. CONCLUSIONS: In this study, the global hygiene of the slaughterhouse was low, as indicated by the high level of E. coli count. The prevalence of both E. coli O157 and other AEEC was also high, representing a real hazard for consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study of this type in Algeria, which indicates that the general hygiene of the slaughterhouse must be improved.     

Clostridium difficile in young farm animals and slaughter animals in Belgium

Faecal carriage of Clostridium difficile in healthy animals has been reported recently, especially in piglets and calves. However there is limited data about carriage in animals just prior to slaughter in Europe. The main objective of this study was to determine the presence of C. difficile in pigs and cattle at the slaughterhouse. C. difficile was isolated in 6.9% of the cattle at the slaughterhouse. None of the pig slaughter samples were positive for C. difficile after an enrichment time of 72 h. For complementary data, a short study was conducted in piglets and calves at farms. C. difficile was more prevalent in piglets (78.3%) than in calves (22.2%) on the farms. Regarding the piglet samples, 27.8% of the positive samples were detected without enrichment of stools. The PCR ribotype 078 was predominant in farm animals. Samples isolated from slaughter cattle presented the widest range in PCR-ribotype variety, and the most prevalent PCR ribotype was 118a UCL. The results of this study confirm that C. difficile is present in slaughter animals in Belgium with a large percentage of toxigenic strains also commonly found in humans.     

Optimal stimulation settings for CMAP scan registrations

Background

The CMAP (Compound Muscle Action Potential) scan is a non-invasive electrodiagnostic tool, which provides a quick and visual assessment of motor unit potentials as electrophysiological components that together constitute the CMAP. The CMAP scan records the electrical activity of the muscle (CMAP) in response to transcutaneous stimulation of the motor nerve with gradual changes in stimulus intensity. Large MUs, including those that result from collateral reinnervation, appear in the CMAP scan as so-called steps, i.e., clearly visible jumps in CMAP amplitude. The CMAP scan also provides information on nerve excitability. This study aims to evaluate the influence of the stimulation protocol used on the CMAP scan and its quantification.

Methods

The stimulus frequency (1, 2 and 3 Hz), duration (0.05, 0.1 and 0.3 ms), or number (300, 500 and 1000 stimuli) in CMAP scans of 23 subjects was systematically varied while the other two parameters were kept constant. Pain was measured by means of a visual analogue scale (VAS). Non-parametric paired tests were used to assess significant differences in excitability and step variables and VAS scores between the different stimulus parameter settings.

Results

We found no effect of stimulus frequency on CMAP scan variables or VAS scores. Stimulus duration affected excitability variables significantly, with higher stimulus intensity values for shorter stimulus durations. Step variables showed a clear trend towards increasing values with decreasing stimulus number.

Conclusions

A protocol delivering 500 stimuli at a frequency of 2 Hz with a 0.1 ms pulse duration optimized CMAP scan quantification with a minimum of subject discomfort, artefact and duration of the recording. CMAP scan variables were influenced by stimulus duration and number; hence, these need to be standardized in future studies.     

Design of an optical switch for studying conformational dynamics in individual molecules of GroEL

We describe the design of an optical switch in the chaperonin GroEL that is opened and closed by its ATP- and cochaperonin GroES-driven conformational changes. The switch, based on a fluorophore and a quencher, is engineered into the single-ring variant of the chaperone, and shows dramatic modulation of its fluorescent intensity in response to the transition of the protein between its allosteric states. It, therefore, forms a sensitive probe for the dynamics of the allosteric transitions of this machine, both in the bulk and in single molecules.