Johan Van Broeck

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.

Presence of Clostridium difficile in pigs and cattle intestinal contents and carcass contamination at the slaughterhouse in Belgium.

The objective of this study was to evaluate the presence of Clostridium difficile in intestinal and carcass samples collected from pigs and cattle at a single slaughterhouse. C. difficile was isolated in 1% and 9.9% of the pig and cattle intestinal contents and in 7.9% and 7% of cattle and pig carcass samples respectively. A total of 19 different PCR-ribotypes were identified, among them types 078 and 014. Seven of 19 ribotypes correlated with the PCR-ribotypes involved in human C. difficile infections in Belgium. This study confirms that animals are carriers of C. difficile at slaughter and ribotypes are identical than those in humans, and that carcass contamination occurs inside the slaughterhouse.

Identification of a Novel Brevibacterium Species Isolated from Humans and Description of Brevibacterium sanguinis sp. nov.

ABSTRACT 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.

Inhibition by lactate dehydrogenase-elevating virus of in vivo interleukin 4 production during immunization with keyhole limpet haemocyanin.

Interleukin 4 (IL-4) and gamma interferon (IFN-gamma) gene expression in lymph node cells from mice immunized with keyhole limpet haemocyanin in the presence of complete Freunds adjuvant was analysed by polymerase chain reaction. This immunization clearly induced the expression of IL-4 message, whereas IFN-gamma message was only slightly increased. A concomitant infection with lactate dehydrogenase-elevating virus drastically decreased IL-4 expression whereas IFN-gamma message was not modified. Analysis of the kinetics of cytokine production indicated that the virus did not induce a mere change in the timing of lymphokine production, but a persistent inhibition of IL-4.

The LMW surface-layer proteins of Clostridium difficile PCR ribotypes 027 and 001 share common immunogenic properties.

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.

Carriage and acquisition rates of Clostridium difficile in hospitalized horses, including molecular characterization, multilocus sequence typing and antimicrobial susceptibility of bacterial isolates

Clostridium difficile has been identified as a significant agent of diarrhoea and enterocolitis in both foals and adult horses. Hospitalization, antibiotic therapy or changes in diet may contribute to the development of C. difficile infection. Horses admitted to a care unit are therefore at greater risk of being colonized. The aim of this study was to investigate the carriage of C. difficile in hospitalized horses and the possible influence of some risk factors in colonization. During a seven-month period, faecal samples and data relating the clinical history of horses admitted to a veterinary teaching hospital were collected. C. difficile isolates were characterized through toxin profiles, cytotoxicity activity, PCR-ribotyping, antimicrobial resistance and multilocus sequence typing (MLST). Ten isolates were obtained with a total of seven different PCR-ribotypes, including PCR-ribotype 014. Five of them were identified as toxinogenic. A high resistance to gentamicin, clindamycin and ceftiofur was found. MLST revealed four different sequencing types (ST), which included ST11, ST26, ST2 and ST15, and phylogenetic analysis showed that most of the isolates clustered in the same lineage. Clinical history suggests that horses frequently harbour toxigenic and non-toxigenic C. difficile and that in most cases they are colonized regardless of the reason for hospitalization; the development of diarrhoea is more unusual.

Clostridium difficile in beef cattle farms, farmers and their environment: assessing the spread of the bacterium

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.

Laboratory identification of anaerobic bacteria isolated on Clostridium difficile selective medium.

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.

Surveillance of Clostridium difficile infections in a long-term care psychogeriatric facility: outbreak analysis and policy improvement

BackgroundFollowing 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).MethodsFor 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.ResultsThe 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.ConclusionsAside 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).