Difficile In Animals Research Articles

High Prevalence of Clostridium difficile in Home Gardens in Western Australia.

In recent years, community-associated Clostridium difficile infection (CA-CDI) has emerged as a significant health problem, accounting for ∼50% of all CDI cases. We hypothesized that the home garden environment could contribute to the dissemination of C. difficile spores in the community and investigated 23 homes in 22 suburbs of Perth, Western Australia. We identified a high prevalence of toxigenic C. difficile in this environment. In total, 97 samples consisting of soil (n = 48), compost (n = 15), manure (n = 12), and shoe sole swabs (n = 22) were collected. All samples were cultured anaerobically on C. difficile ChromID agar and enriched in brain heart infusion broth, and isolates were characterized by toxin gene PCR and PCR ribotyping. Two-thirds (67%; 95% confidence interval [CI], 57 to 76%) of home garden samples, including 79% (95% CI, 68 to 91%) of soil, 67% (95% CI, 43 to 90%) of compost, 83% (95% CI, 62% to 100%) of manure, and 32% (95% CI, 12 to 51%) of shoe sole samples, contained C. difficile Of 87 isolates, 38% (95% CI, 28 to 48%) were toxigenic, and 26 PCR ribotypes (RTs), 5 of which were novel, were identified. The toxigenic C. difficile strain RT014/020 was the most prevalent RT. Interestingly, 19 esculin hydrolysis-negative strains giving white colonies were identified on C. difficile ChromID agar, 5 of which were novel toxigenic RTs that produced only toxin A. Clearly, there is the potential for transmission of C. difficile in the community due to the contamination of home gardens. Our findings highlight the importance of a "One Health" approach to dealing with CDI.IMPORTANCE Recently, community-associated Clostridium difficile infection (CA-CDI) has emerged as a significant problem, accounting for ∼50% of all CDI cases and reported to affect a younger population without traditional risk factors. Possible sources of CA-CDI are soil, food, and water contaminated by animal feces, and recent reports show overlapping ribotypes of C. difficile in animals, humans, and the environment; however, the epidemiology of CA-CDI and related risk factors need to be better understood. Our research aimed to determine the prevalence of C. difficile in home gardens and on the shoe soles of homeowners in Perth, Western Australia. There were high rates of contamination with C. difficile in gardens, and some of the ribotypes identified had been isolated from human cases of CDI in Western Australia. This study shows that home gardens and shoes may be a source of C. difficile in CA-CDI.

Clostridium difficile in Food and Animals: A Comprehensive Review.

Zoonoses are infections or diseases that can be transmitted between animals and humans through direct contact, close proximity or the environment. Clostridium difficile is ubiquitous in the environment, and the bacterium is able to colonise the intestinal tract of both animals and humans. Since domestic and food animals frequently test positive for toxigenic C. difficile, even without showing any signs of disease, it seems plausible that C. difficile could be zoonotic. Therefore, animals could play an essential role as carriers of the bacterium. In addition, the presence of the spores in different meats, fish, fruits and vegetables suggests a risk of foodborne transmission. This review summarises the current available data on C. difficile in animals and foods, from when the bacterium was first described up to the present.

Is the prevalence of Clostridium difficile in animals underestimated?

Reported prevalence rates of Clostridium difficile infection in animals differ considerably depending on the nature of the study and the population surveyed. The methods used to recover this organism from faecal samples may account for some of the prevalence variation. The objective of this study was to assess the performance of two different methods of detecting C. difficile in animal faeces in comparison with a conventional isolation procedure (‘ethanol shock’ of faecal samples followed by culture on a single plate of solid selective medium). Samples were obtained from two populations of pigs where the expected prevalence rate of C. difficile infection was anticipated to differ, namely, ‘high prevalence’ (<7-day old piglets) and ‘low prevalence’ (2–3-month old pigs).The first alternative detection method required culturing faecal samples on 10 (instead of one) plate of solid selective medium after ethanol shock, while the second method included an intermediate enrichment step in selective broth prior to ethanol shock and subsequent plating. Both alternative methods considerably increased bacterial recovery in tested samples from both surveyed populations and highlighted the existence of a considerable proportion (⩾22%) of false negatives. The results confirm previous suggestions that the procedure used to isolate C. difficile can have a significant impact on prevalence data for this pathogen.

Improved detection of Clostridium difficile in animals by using enrichment culture followed by LightCycler real-time PCR

The performance of our previously published TaqMan real-time PCR (TMrtPCR) for the detection of Clostridium difficile directly from animal faeces was found to be inadequate due to TMrtPCR false negative results. Therefore, we developed a new internally controlled LightCycler real-time PCR (LC rtPCR) capable of detecting variant strains in diarrhoeic and subclinical animals by using two hybridisation probes instead of one hydrolysis probe used in TMrtPCR. While LC rtPCR did not provide better results than TMrtPCR, improved detection of C. difficile in samples with low number of bacteria was introduced, using a pre-enrichment step followed by LC rtPCR. Hence, 40 (100%) rectal swabs were sampled and subjected to direct LC rtPCR, culture without enrichment and enrichment culture; after 24, 48 and 72 h, and seven days of incubation, DNA was extracted and amplified with LC rtPCR. Only one (2.5%) sample was culture positive/LC rtPCR positive without the enrichment step, while 33 (82.5%) samples were culture positive after seven days of enrichment. Only one day of enrichment evidently increases the number of culture positive (15; 37.5%) and LC rtPCR positive (28; 70%) samples. Results of this study demonstrate that one day enrichment culture for C. difficile followed by LC rtPCR assay targeting multiple genes can be applied as accurate and rapid screening test, especially in samples with low number of C. difficile, as no culture positive/LC rtPCR negative samples were observed.

Infection: the next big thing!

Clostridium difficile causes infectious diarrhoea in humans and animals. It has been found in pigs, horses, and cattle, suggesting a potential reservoir for human infection, and in 20-40% of meat products in Canada and the USA, suggesting the possibility of food-borne transmission. It is likely that excessive antimicrobial exposure is driving the establishment of C. difficile in animals, in a manner analogous to human infection, rather than the organism just being normal flora of the animal gastrointestinal tract. Outside Australia, PCR ribotype 078 is the most common ribotype of C. difficile found in pigs (83% in one study in the USA) and cattle (up to 100%) and this ribotype is now the third most common ribotype of C. difficile found in humans in Europe. Human and pig strains of C. difficile are genetically identical in Europe confirming that a zoonosis exists. Rates of community-acquired C. difficile infection (CDI) are increasing world-wide, and a new community strain of unidentified origin has recently emerged in Australia. Environmental contamination may also play a role. C. difficile spores survive in treated piggery effluent, the by-products of which are used to irrigate crops and pasture and manufacture compost. There is abundant evidence that food products intended for human consumption contain toxigenic strains of C. difficile but food-borne transmission remains unproven. Thus there are four problems that require resolution: a human health issue, an animal health issue and the factors common to both these problems, environmental contamination and antimicrobial misuse.

Detection of Clostridium difficile in animals: comparison of real-time PCR assays with the culture method

Clostridium difficile has emerged as a pathogen or commensal in food animals. There is overlap between isolates from animals, retail meats and humans, suggesting that animals may be a C. difficile reservoir. For direct detection of variant C. difficile strains in faecal samples of symptomatic and asymptomatic animals, we developed and validated a new TaqMan real-time PCR (TMrtPCR) assay targeting the tcdA, tcdB and cdtB genes. We compared it with the enrichment culture method and with two real-time PCR (rtPCR) assays, BrtPCR and PCRFast, targeting tcdB and tcdA/tcdB, respectively. All ten tested C. difficile toxinotypes, except one (XIa) with PCRFast and two (X, XIa) with BrtPCR, were detected with the test assays. A total of 340 (100 %) samples were cultured and amplified with TMrtPCR. Results correlated in 75.3 % samples. Forty (11.8 %) samples were culture positive/TMrtPCR negative, possibly because of the low numbers of bacteria in the samples or because of DNA extraction failure. Forty (11.8 %) samples were TMrtPCR positive/culture negative. Among 79 samples included in the rtPCR assays/culture comparison, 50.6 % were in complete concordance. The results showed that TMrtPCR performed better than BrtPCR and PCRFast, and 67 % of the culture-positive samples were TMrtPCR positive in comparison to 40 % of the samples positive in BrtPCR and 7 % of the samples positive in PCRFast, respectively. Another advantage of TMrtPCR over BrtPCR and PCRFast is its ability to detect a binary toxin gene. Therefore, the TMrtPCR results can provide the first information about the toxin type present in the sample. According to the results of our study, TMrtPCR could be a preferred screening method for the rapid detection of C. difficile in animal faecal samples, although an enrichment culture has to be performed for the specimens with negative or inconclusive rtPCR results.

Tigecycline Exhibits Inhibitory Activity against Clostridium difficile in the Colon of Mice and Does Not Promote Growth or Toxin Production

Tigecycline is a broad-spectrum glycylcycline antibiotic with potent in vitro activity against Clostridium difficile. We used a mouse model to test the hypothesis that tigecycline has a low propensity to promote colonization and toxin production by C. difficile due to inhibitory activity in the colon. Mice (5 to 8 per group) received subcutaneous injections of tigecycline (low and high doses) alone or in combination with clindamycin for 6 days. Growth of and toxin production by 3 strains of C. difficile (tigecycline MICs ≤ 0.012 μg/ml) were measured in cecal contents collected 6 h or 3 days after the final antibiotic dose. Antibiotic concentrations were measured using a bioassay, and concentrations of total anaerobes and Bacteroides spp. were measured. The effects of tigecycline on rendering mice susceptible to colonization with and reducing the burden of C. difficile were also examined. In comparison to saline controls, clindamycin promoted the growth of C. difficile (P < 0.001) in cecal contents, whereas tigecycline did not. Tigecycline did not suppress total anaerobes or Bacteroides spp. in comparison to saline controls. Concurrent administration of tigecycline prevented clindamycin-induced promotion of C. difficile in cecal contents collected 6 h or 3 days (high dose only) after the final antibiotic dose. Tigecycline did not promote the establishment of colonization in mice, yet it did not reduce concentrations of C. difficile in animals with established colonization. In summary, tigecycline did not promote the growth of or toxin production by C. difficile, probably due to inhibitory activity against C. difficile and relative sparing of indigenous anaerobic microflora.

Clostridium difficilein Food and Domestic Animals: A New Foodborne Pathogen?

Clostridium difficile infection is increasingly recognized as a cause of diarrhea in outpatients and persons with no apparent health care facility contacts. In contrast to C. difficile infection acquired in health care settings, few risk factors for development of community-associated C. difficile infection are known. Foodborne transmission of C. difficile has been hypothesized as a possible source for community-associated infections; however, the evidence to confirm or refute this hypothesis is incomplete. Recent studies have demonstrated isolation of C. difficile from foods in the United States, Canada, and Europe and from meat products intended for consumption by pets. This raises questions about foodborne transmission of this pathogen to humans through consumption of contaminated products. This review summarizes the available data on C. difficile in animals and food and discusses the potential for foodborne transmission of this pathogen.