E. C. Keessen

Clostridium difficile infection in the community: a zoonotic disease?

Clostridium difficile infections (CDIs) are traditionally seen in elderly and hospitalized patients who have used antibiotic therapy. In the community, CDIs requiring a visit to a general practitioner are increasingly occurring among young and relatively healthy individuals without known predisposing factors. C. difficile is also found as a commensal or pathogen in the intestinal tracts of most mammals, and various birds and reptiles. In the environment, including soil and water, C. difficile may be ubiquitous; however, this is based on limited evidence. Food products such as (processed) meat, fish and vegetables can also contain C. difficile, but studies conducted in Europe report lower prevalence rates than in North America. Absolute counts of toxigenic C. difficile in the environment and food are low, however the exact infectious dose is unknown. To date, direct transmission of C. difficile from animals, food or the environment to humans has not been proven, although similar PCR ribotypes are found. We therefore believe that the overall epidemiology of human CDI is not driven by amplification in animals or other sources. As no outbreaks of CDI have been reported among humans in the community, host factors that increase vulnerability to CDI might be of more importance than increased exposure to C. difficile. Conversely, emerging C. difficile ribotype 078 is found in high numbers in piglets, calves, and their immediate environment. Although there is no direct evidence proving transmission to humans, circumstantial evidence points towards a zoonotic potential of this type. In future emerging PCR ribotypes, zoonotic potential needs to be considered.

Relatedness of Human and Animal Clostridium difficile PCR Ribotype 078 Isolates Determined on the Basis of Multilocus Variable-Number Tandem-Repeat Analysis and Tetracycline Resistance

ABSTRACT Totals of 102 and 56 Clostridium difficile type 078 strains of human and porcine origins, respectively, from four European countries were investigated by an optimized multilocus variable-number tandem-repeat analysis (MLVA) and for tetracycline susceptibility. Eighty-five percent of all isolates were genetically related, irrespective of human or porcine origin. Human strains were significantly more resistant to tetracycline than porcine strains. All tetracycline-resistant strains contained the Tn916-like transposon harboring the tet(M) gene. We conclude that strains from human and porcine origins are genetically related, irrespective of the country of origin. This may reflect a lack of diversity and/or common source.

Whole genome sequencing reveals potential spread of Clostridium difficile between humans and farm animals in the Netherlands, 2002 to 2011

Farm animals are a potential reservoir for human Clostridium difficile infection (CDI), particularly PCR ribotype 078 which is frequently found in animals and humans. Here, whole genome single-nucleotide polymorphism (SNP) analysis was used to study the evolutionary relatedness of C. difficile 078 isolated from humans and animals on Dutch pig farms. All sequenced genomes were surveyed for potential antimicrobial resistance determinants and linked to an antimicrobial resistance phenotype. We sequenced the whole genome of 65 C. difficile 078 isolates collected between 2002 and 2011 from pigs (n = 19), asymptomatic farmers (n = 15) and hospitalised patients (n = 31) in the Netherlands. The collection included 12 pairs of human and pig isolates from 2011 collected at 12 different pig farms. A mutation rate of 1.1 SNPs per genome per year was determined for C. difficile 078. Importantly, we demonstrate that farmers and pigs were colonised with identical (no SNP differences) and nearly identical (less than two SNP differences) C. difficile clones. Identical tetracycline and streptomycin resistance determinants were present in human and animal C. difficile 078 isolates. Our observation that farmers and pigs share identical C. difficile strains suggests transmission between these populations, although we cannot exclude the possibility of transmission from a common environmental source.

Clostridium difficile infection in humans and animals, differences and similarities.

Clostridium difficile is well known as the most common cause of nosocomial infections in human patients. In recent years a change in epidemiology towards an increase in incidence and severity of disease, not only inside the hospital, but also in the community, is reported. C. difficile is increasingly recognized in veterinary medicine as well and is now considered the most important cause of neonatal diarrhea in swine in North America. Research on the presence of C. difficile in production and companion animals revealed a huge overlap with strains implicated in human C. difficile infection (CDI). This has lead to the concern that interspecies transmission of this bacterium occurs. In this review C. difficile infections in humans and animals are compared. The pathogenesis, clinical signs, diagnosis and prevalence of CDI are described and similarities and differences of CDI between humans and animals are discussed.

Evaluation of Four Different Diagnostic Tests To Detect Clostridium difficile in Piglets

ABSTRACT Clostridium difficile is emerging as pathogen in both humans and animals. In 2000 it was described as one of the causes of neonatal enteritis in piglets, and it is now the most common cause of neonatal diarrhea in the United States. In Europe, C. difficile infection (CDI) in both neonatal piglets and adult sows has also been reported. Diagnosis of this infection is based on detection of the bacterium C. difficile or its toxins A and B. Most detection methods, however, are only validated for diagnosing human infections. In this study three commercially available enzyme immunoassays (EIAs) and a commercial real-time-PCR (Becton, Dickinson, and Company) were evaluated by testing 172 pig fecal specimens (139 diarrheic and 33 nondiarrheic piglets). The results of each test were compared to those of cytotoxicity assays (CTAs) and toxigenic culture as the “gold standards.” Compared to CTAs, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were, respectively, as follows: for real-time PCR, 91.6, 37.1, 57.6, and 82.5%; for Premier Toxins A&B (Meridian), 83.1, 31.5, 53.1, and 66.7%; for ImmunoCard Toxins A&B kit (ICTAB; Meridian), 86.6, 56.8, 66.9, and 80.7%; and for VIDAS (bioMérieux), 54.8, 92.6, 85.0, and 72.8%. Compared to toxigenic culture, the sensitivity, specificity, PPV, and NPV were, respectively, as follows: for real-time PCR, 93.0, 34.7, 50.0, and 87.5%; for Premier Toxins A&B, 80.3, 27.7, 43.8, and 66.7%; and for ICTAB, 80.0, 46.2, 52.8, and 75.4%; and for VIDAS, 56.4, 89.8, 77.5, and 76.7%. We conclude that all tests had an unacceptably low performance as a single test for the detection of C. difficile in pig herds and that a two-step algorithm is necessary, similar to that in cases of human CDI. Of all of the assays, the real-time PCR had the highest NPV compared to both reference methods and is therefore the most appropriate test to screen for the absence of C. difficile in pigs as a first step in the algorithm. The second step would be a confirmation of the positive results by toxigenic culture.

Aerial dissemination of Clostridium difficile on a pig farm and its environment

Clostridium difficile is increasingly recognized as an important enteropathogen in both humans and animals. The finding of C. difficile in air samples in hospitals suggests a role for aerial dissemination in the transmission of human C. difficile infection. The present study was designed to investigate the occurrence of airborne C. difficile in, and nearby a pig farm with a high prevalence of C. difficile. Airborne colony counts in the farrowing pens peaked on the moments shortly after or during personnel activity in the pens (P=0.043 (farrowing pens 1, 2), P=0.034 (farrowing pen 2)). A decrease in airborne C. difficile colony counts was observed parallel to aging of the piglets. Airborne C. difficile was detected up to 20 m distant from the farm. This study showed widespread aerial dissemination of C. difficile on a pig farm that was positively associated with personnel activity.

The relation between farm specific factors and prevalence of Clostridium difficile in slaughter pigs

Foodborne ingestion through pork products of Clostridium difficile has been suggested a possible route of transmission of C difficile from pigs to humans. To determine whether C. difficile bacteria are present in the intestines of slaughter pigs, rectum contents of 677 slaughter pigs from 52 farms were collected at the slaughterhouse. Data on farm specific factors were collected and the association of these factors with the presence of C. difficile in pig herds from 39 farms was assessed. The prevalence of C. difficile and the ribotypical diversity that were found in this study were much higher than previously reported in literature, with an overall C. difficile prevalence of 8.6% (58/677). Sixteen distinct C. difficile ribotypes were identified, predominantly type 078 (31.0%, 18/58). This type is also commonly found in humans with C. difficile infection (CDI). Both on individual pig level and on herd level, no significant difference between the prevalence of C. difficile in pigs derived from conventional or organic farming types was detected. Farm system, size, and presence of other animal species on the farm did not result in significant different prevalences of C. difficile.

Clostridium difficile infection associated with pig farms.

To the Editor: Clostridium difficile of PCR ribotype 078 causes enteric disease in humans and pigs (1,2); a recent pan-European study revealed that this type was the third most frequently found type of C. difficile (1). The finding of identical C. difficile PCR ribotype 078 isolates in piglets with diarrhea and in humans with C. difficile infection (CDI) led to the suggestion that interspecies transmission might occur (3,4). Because C. difficile can be detected in the immediate environment of pig farms, we investigated intestinal colonization with C. difficile in pigs and in pig farmers, their relatives, and their employees in the Netherlands. Persons living on 32 pig farms were enrolled as part of a longitudinal intervention study of several zoonotic agents. Pig farmers were partly recruited through the Dutch Farmer’s Association or by veterinarians, who informed potential participants about the aims of the study. Inclusion criteria for participants were that they should work and/or live on the farm; the farms were either closed farms or multipliers (farms at which piglets are bred and then sold to other farms, where they are raised until ready for slaughter). The number of persons willing to submit a fecal sample per farm ranged from 1 to 10 (mean 4, median 5). Veterinarians who normally provided veterinary services to each farm collected fresh fecal samples from the floors of 10 animal wards per farm. No a priori knowledge of C. difficile colonization status of the pigs on the farms was available. Fecal samples from humans and from animal wards were cultured for the presence of toxinogenic C. difficile by using previously described methods (1,3,4). Of the 128 persons who enrolled in the study, 48 had daily contact with pigs, 22 had weekly contact with pigs, and 36 had contact with pigs varying from monthly to less than yearly; no contact information was available for 22 participants. A total of 12 (25%) of 48 persons who had daily contact with pigs had fecal samples positive for C. difficile colonization; for persons who had weekly contact with pigs, 3 (14%) of 22 had positive samples. Daily to weekly contact with pigs versus monthly to less than yearly contact was significantly associated with an intestinal presence of C. difficile (p = 0.003). C. difficile was also found in fecal samples from 3 persons for whom no contact information was available. The C. difficile carriage rate among those with daily to weekly contact with pigs (15/70, 21%) was higher than the carriage rate of <5% reported for nonhospitalized adults with CDI (5). A total of 18 C. difficile–positive human samples were detected at 16 of 32 pig farms investigated. At 2 of these farms, only 1 person submitted a sample, but at the other 14 farms, the number of participants ranged from 2 to 9 (mean 4, median 3). C. difficile was found in pig manure at all farms; 10%–80% of the wards were positive per farm. Corresponding C. difficile PCR ribotypes were cultured from samples from pigs and humans; type 078 was found in humans and pigs on 15 farms and type 045 in a farmer and his pigs on 1 farm. Multilocus variable number tandem repeat analysis (MLVA) and antimicrobial drug susceptibility testing (E-test) were performed on human isolates from 15 farms and 1 porcine isolate per farm. One human isolate could not be typed because the isolate was lost during laboratory activities. MLVA results showed that, at 2 farms, the human and porcine isolates were not genetically related, whereas at the other 13 farms, human and porcine isolates were genetically related, including 100% identical MLVA results for type 078 human and porcine isolates at 3 farms. Isolates were considered genetically related when the summed tandem repeat differences were <10 (3,4,6). Antimicrobial drug susceptibility testing demonstrated similar susceptibility levels among isolates. For human and porcine isolates from 9 of 15 farms, MIC variability of <1 μg/L was found for imipenem, cotrimoxazole, erythromycin, clindamycin, tetracycline, and moxifloxacin. For the remaining 6 farms, drug susceptibility patterns for human isolates differed from pig isolates for 1 drug only: for 1 farm, MICs of erythromycin were 256 μg/L for human isolates and 0.38 μg/L for pig isolates; for 3 farms, MICs of erythromycin were 256 μg/L for pig isolates and 0.25 μg/L for human isolates; and for 2 farms, MICs of imipenem were 32 μg/L for pig isolates and 1.5 or 2 μg/L for human isolates. In summary, the high C. difficile carriage rate among persons who had direct contact with pigs and the fact that these C. difficile isolates were genotypically and phenotypically similar to the pig isolates from the same farms indicates that transmission occurs either by direct contact or through the environment. Prospective studies are needed to determine the relationship between C. difficile carriage and development of CDI in this population.