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Clostridium difficile is considered the leading cause of antibiotic-associated disease worldwide. In the past decade, a large number of studies have focused on identifying the main sources of contamination in order to elucidate the complete life cycle of the infection. Hospitals, animals and retail foods have been considered as potential vectors. However, the prevalence of C. difficile in these types of samples was found to be rather low, suggesting that other contamination routes must exist. This study explores the presence of C. difficile in the natural environment and the seasonal dynamics of the bacterium. C. difficile was isolated from a total of 45 samples out of 112 collected (40.2%) on 56 sampling points. A total of 17 points were positive only during the winter sampling (30.4%), 10 were positive only during the summer sampling (17.9%) and 9 sampling points (16.1%) were positive in both summer sampling and winter sampling. Spore counts in soil samples ranged between 50 and 250 cfu/g for 24.4% of the positive samples, with the highest concentrations detected in samples collected in the forest during winter campaign (200-250 cfu/g). A total of 17 different PCR ribotypes were identified, and 15 of them had the genes coding for toxins A and B. Most of those ribotypes had not previously been found or had been isolated only sporadically (<1% of samples) from hospitals in Belgium. Regarding antimicrobial susceptibility, most of the resistant strains were found during the summer campaign. These findings bear out that C. difficile is present in the natural environment, where the bacterium undergoes seasonal variations., (© 2019 Blackwell Verlag GmbH.)

In recent years, several studies have described the presence of Clostridium difficile in healthy and diarrhoeic farm and domestic animals. In pigs and cattle, the isolation of some PCR-ribotypes associated with human infection, especially PCR-ribotypes 014 and 078, has led us to hypothesize about the zoonotic transmission of C. difficile infections. If these animals are reservoirs of C. difficile, farmers in close contact with their animals are particularly at risk of acquiring and spreading the bacterium. This study investigates the presence of C. difficile in closely associated populations, beef cattle and farmers, as well as in the animal feed, manure and dust in five different farms in Belgium. C. difficile was isolated from calves and cattle with a prevalence varying between 5.5% and 11.3%. Furthermore, all of the isolates were toxigenic. An important age and breed effect was observed in the colonization of C. difficile. For age, there was a higher probability of colonization in calves of less than 6 months in age than in cattle over 11 months of age. For the type of breed a higher prevalence of the bacterium was detected in the Limousin breed than in the Belgian Bleu breed. By contrast, none of the human and animal feed samples tested positive for C. difficile. The results obtained indicate a persistent animal reservoir of C. difficile, but an indirect dissemination to humans, probably via the environment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Extra-colonic infections, and especially bacteremia, are infrequent manifestations of Clostridium difficile infection. C. difficile bacteremia is generally health-care associated and polymicrobial. We report the case of a patient on hunger strike that presented a C. difficile colitis and mono-microbial bacteremia on its admission to the hospital. Multilocus variable number tandem repeat analysis of stool and blood isolates indicated clonality. The strain was characterized as a ribotype 002, an emerging ribotype previously associated with high fatality rate. The patient received treatment by intra-venous amoxicillin-clavulanate and oral vancomycin but eventually died on the seventh day of admission with concomitant pneumonia and pulmonary embolism., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Increasing age, several co-morbidities, environmental contamination, antibiotic exposure and other intestinal perturbations appear to be the greatest risk factors for C. difficile infection (CDI). Therefore, elderly care home residents are considered particularly vulnerable to the infection. The main objective of this study was to evaluate and follow the prevalence of C. difficile in 23 elderly care home residents weekly during a 4-month period. A C. difficile microbiological detection scheme was performed along with an overall microbial biodiversity study of the faeces content by 16S rRNA gene analysis., Results: Seven out of 23 (30.4 %) residents were (at least one week) positive for C. difficile. C. difficile was detected in 14 out of 30 diarrhoeal samples (43.7 %). The most common PCR-ribotype identified was 027. MLVA showed that there was a clonal dissemination of C. difficile strains within the nursing home residents. 16S-profiling analyses revealed that each resident has his own bacterial imprint, which was stable during the entire study. Significant changes were observed in C. difficile positive individuals in the relative abundance of a few bacterial populations, including Lachnospiraceae and Verrucomicrobiaceae. A decrease of Akkermansia in positive subjects to the bacterium was repeatedly found., Conclusions: A high C. difficile colonisation in nursing home residents was found, with a predominance of the hypervirulent PCR-ribotype 027. Positive C. difficile status is not associated with microbiota richness or biodiversity reduction in this study. The link between Akkermansia, gut inflammation and C. difficile colonisation merits further investigations.

Despite increasing interest in the bacterium, the methodology for Clostridium difficile recovery has not yet been standardized. Cycloserine-cefoxitin fructose taurocholate (CCFT) has historically been the most used medium for C. difficile isolation from human, animal, environmental, and food samples, and presumptive identification is usually based on colony morphologies. However, CCFT is not totally selective. This study describes the recovery of 24 bacteria species belonging to 10 different genera other than C. difficile, present in the environment and foods of a retirement establishment that were not inhibited in the C. difficile selective medium. These findings provide insight for further environmental and food studies as well as for the isolation of C. difficile on supplemented CCFT.

Background: The 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., Results: Bacterial 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., Conclusions: The 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.

Background: Following an exceptionally high Clostridium difficile infections (CDI) incidence (Spring 2011) in a psychogeriatric long-term care facility, a bidirectional study (2009-2012) was initiated to identify determinants (retrospectively) and to assess intervention measures taken (prospectively)., Methods: For every CDI patient (de novo cases, relapses, and recurrences), a control patient (patient in the opposite room) was selected and risk factor analysis performed. Following the epidemic peak a more stringent hygienic protocol and surveillance program were implemented, as well as uniform guidelines for metronidazole and vancomycin prescription., Results: The nutritional state (total protein/prealbumine) significantly differed between the CDI group (poorer nutritional state at admission) and the control group, and also antibiotic use (general) could be confirmed as a risk factor. A multi-disciplinary nutritional team has been established in order to improve the nutritional balance of our patients., Conclusions: Aside from stringent hygiene and antibiotic prescription stewardship, malnutrition of patients is a factor to be taken into account to contain a CDI outbreak in a long term care facility (LTCF).

The aim of this study was to investigate the S-layer proteins (SLPs) of the hypervirulent Clostridium difficile PCR ribotype 027 and compare them with those of PCR ribotype 001 and other PCR ribotypes involved in C. difficile infection and outbreaks, by molecular analysis and immunological assays. It has been demonstrated previously that PCR ribotype 027 SlpA is conserved in C. difficile strains belonging to this PCR ribotype and that it is a new variant, showing 88 % identity with SlpA of PCR ribotype 001. As the low-molecular-weight (LMW) SLPs of C. difficile are immunodominant antigens, attention was focused on this region of the genome. Sequencing of strains of different PCR ribotypes (001, 012, 014, 017, 027 and 078) showed that SlpA was conserved among strains belonging to the same PCR ribotype. Comparison of the LMW SLP region among these strains identified ten regions with sequence identity between PCR ribotypes 027 and 001, and low conservation with the other PCR ribotypes. In particular, two of these regions corresponded to areas predicted to be surface exposed. Three specific peptides, including those of the two surface-exposed regions, were recognized by human sera against PCR ribotypes 027 and 001 and by a rabbit polyclonal serum against the SLPs of PCR ribotype 027. In contrast, these peptides were not recognized by a polyclonal serum against the SLPs of PCR ribotype 012 used as a control. These results confirm the antigenic role of the LMW SLP and suggest that it may have a role in evasion of the host immune response.

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 faecal-cytotoxin 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.

Six coryneforms isolated from blood and dialysate fluid were phenotypically similar to Brevibacterium casei, but 16S rRNA gene sequencing and DNA-DNA hybridization indicate that they belong to a new species for which the name Brevibacterium sanguinis is proposed.