Genome Analysis of Clostridium difficile PCR Ribotype 014 Lineage in Australian Pigs and Humans Reveals a Diverse Genetic Repertoire and Signatures of Long-Range Interspecies Transmission.

Clostridium difficile PCR ribotype (RT) 014 is well-established in both human and porcine populations in Australia, raising the possibility that C. difficile infection (CDI) may have a zoonotic or foodborne etiology. Here, whole genome sequencing and high-resolution core genome phylogenetics were performed on a contemporaneous collection of 40 Australian RT014 isolates of human and porcine origin. Phylogenies based on MLST (7 loci, STs 2, 13, and 49) and core orthologous genes (1260 loci) showed clustering of human and porcine strains indicative of very recent shared ancestry. Core genome single nucleotide variant (SNV) analysis found 42% of human strains showed a clonal relationship (separated by ≤2 SNVs in their core genome) with one or more porcine strains, consistent with recent inter-host transmission. Clones were spread over a vast geographic area with 50% of the human cases occurring without recent healthcare exposure. These findings suggest a persistent community reservoir with long-range dissemination, potentially due to agricultural recycling of piggery effluent. We also provide the first pan-genome analysis for this lineage, characterizing its resistome, prophage content, and in silico virulence potential. The RT014 is defined by a large "open" pan-genome (7587 genes) comprising a core genome of 2296 genes (30.3% of the total gene repertoire) and an accessory genome of 5291 genes. Antimicrobial resistance genotypes and phenotypes varied across host populations and ST lineages and were characterized by resistance to tetracycline [tetM, tetA(P), tetB(P) and tetW], clindamycin/erythromycin (ermB), and aminoglycosides (aph3-III-Sat4A-ant6-Ia). Resistance was mediated by clinically important mobile genetic elements, most notably Tn6194 (harboring ermB) and a novel variant of Tn5397 (harboring tetM). Numerous clinically important prophages (Siphoviridae and Myoviridae) were identified as well as an uncommon accessory gene regulator locus (agr3). Conservation in the pathogenicity locus and S-layer correlated with ST affiliation, further extending the concept of clonal C. difficile lineages. This study provides novel insights on the genetic variability and strain relatedness of C. difficile RT014, a lineage of emerging One Health importance. Ongoing molecular and genomic surveillance of strains in humans, animals, food, and the environment is imperative to identify opportunities to reduce the overall CDI burden.

Persistence of Clostridium difficile RT 237 infection in a Western Australian piggery.

Clostridium difficile is commonly associated with healthcare-related infections in humans, and is an emerging pathogen in food animal species. There is potential for transmission of C. difficile from animals or animal products to humans. This study aimed to determine if C. difficile RT 237 had persisted in a Western Australian piggery or if there had been a temporal change in C. difficile diversity. C. difficile carriage in litters with and without diarrhea was investigated, as was the acquisition of C. difficile over time using cohort surveys. Rectal swabs were obtained from piglets aged 1-10 days to determine prevalence of C. difficile carriage and samples were obtained from 20 piglets on days 1, 7, 13, 20, and 42 of life to determine duration of shedding. Isolation of C. difficile from feces was achieved by selective enrichment culture. All isolates were characterized by standard molecular typing. Antimicrobial susceptibility testing was performed on selected isolates (n = 29). Diarrheic piglets were more likely to shed C. difficile than the non-diseased (p = 0.0124, χ2). In the cohort study, C. difficile was isolated from 40% samples on day 1, 50% on day 7, 20% on day 13, and 0% on days 20 and 42. All isolates were RT 237 and no antimicrobial resistance was detected. The decline of shedding of C. difficile to zero has public health implications because slaughter age pigs have a low likelihood of spreading C. difficile to consumers via pig meat.

Nationwide surveillance study of Clostridium difficile in Australian neonatal pigs shows high prevalence and heterogeneity of PCR ribotypes.

Clostridium difficile is an important enteric pathogen of humans and the cause of diarrhea and enteritis in neonatal pigs. Outside Australia, prevalence in piglets can be up to 73%, with a single PCR ribotype (RT), 078, predominating. We investigated the prevalence and genotype of C. difficile in Australian pig herds. Rectal swabs (n = 229) were collected from piglets aged <7 days from 21 farms across Australia. Selective culture for C. difficile was performed and isolates characterized by PCR for toxin genes and PCR ribotyping. C. difficile was isolated from 52% of samples by direct culture on chromogenic agar and 67% by enrichment culture (P = 0.001). No association between C. difficile recovery or genotype and diarrheic status of either farm or piglets was found. The majority (87%; 130/154) of isolates were toxigenic. Typing revealed 23 different RTs, several of which are known to cause disease in humans, including RT014, which was isolated most commonly (23%; 36/154). RT078 was not detected. This study shows that colonization of Australian neonatal piglets with C. difficile is widespread in the herds sampled.

Laboratory detection of Clostridium difficile in piglets in Australia.

Clostridium difficile is a well-known enteric pathogen of humans and the causative agent of high-morbidity enteritis in piglets aged 1 to 7 days. C. difficile prevalence in Australian piglets is as high as 70%. The current diagnostic assays have been validated only for human infections, and there are no published studies assessing their performance in Australian piglets. We evaluated the suitability of five assays for detecting C. difficile in 157 specimens of piglet feces. The assays included a loop-mediated isothermal amplification (LMIA)-PCR for tcdA (illumigene C. difficile; Meridian), a real-time PCR for tcdB (GeneOhm Cdiff; Becton Dickinson), two-component enzyme immunoassays (EIA) for C. difficile glutamate dehydrogenase (GDH) (EIA-GDH) and TcdA/TcdB (EIA-TcdA/TcdB) (C. diff Quik Chek; Alere), and direct culture (DC) (C. difficile chromID agar; bioMérieux). The assays for detection of the organism were compared against enrichment culture (EC), and assays for detection of toxins/toxin genes were compared against EC followed by PCR for toxin genes (toxigenic EC [TEC]). The recovery of C. difficile by EC was 39.5% (n = 62/157), and TEC revealed that 58.1% (n = 36/62) of isolates were positive for at least one toxin gene (tcdA/tcdB). Compared with those for EC/TEC, the sensitivities, specificities, positive predictive values, and negative predictive values were, respectively, as follows: DC, 91.9, 100.0, 100.0, and 95.0%; EIA-GDH, 41.9, 92.6, 78.8, and 71.0%; EIA-TcdA/TcdB, 5.6, 99.2, 66.7, and 77.9%; real-time PCR, 42.9, 96.7, 78.9, and 85.4% and LMIA-PCR, 25.0, 95.9, 64.3, and 81.1%. The performance of the molecular methods was poor, suggesting that the current commercially available assays for diagnosis of C. difficile in humans are not suitable for use in piglets. C. difficile recovery by the DC provides a cost-effective alternative.

Comparison of ChromID C. difficile agar and cycloserine-cefoxitin-fructose agar for the recovery of Clostridium difficile.

AIM: The rapidly changing epidemiology of Clostridium difficile infection highlights the need for improved and continuing surveillance involving stool culturing to enable molecular tracking. Culture of C. difficile can be difficult and time consuming. In this report ChromID C. difficile agar (CDIF) was compared to cycloserine-cefoxitin-fructose-egg-yolk agar which contained 0.1% sodium taurocholate (TCCFA) as a germinant. RESULTS: All ribotypes of C. difficile tested (n=90) grew well on CDIF within 24 h and most gave characteristic small irregular black colonies with a raised umbonate profile. Counts from standard suspensions of C. difficile at 24 h (p<0.005) and 48 h (p=0.01) were significantly higher on CDIF than on TCCFA. Similar results were achieved after alcohol shock. When temperature shock was used to differentiate vegetative cells and spores, the total number of culturable and vegetative cells on CDIF was significantly higher than on TCCFA (culturable cells, p=0.003 at 24 h and p=0.002 at 48 h; vegetative cells, p=0.0003 at 24 h and p=0.0002 at 48 h). CONCLUSIONS: These data suggest that CDIF is a better medium for the recovery of vegetative C. difficile than TCCFA and equal to TCCFA for spore recovery.

Novel molecular type of Clostridium difficile in neonatal pigs, Western Australia.

Clostridium difficile causes neonatal enteritis in piglets; strains of PCR ribotype 078 are most commonly identified. We investigated C. difficile prevalence in piglets in Australia and isolated a novel strain with a unique pathogenicity locus. In a mouse infection model, this strain produced more weight loss than did a ribotype 078 strain.

Clostridium difficile infection in humans and piglets: a 'One Health' opportunity.

Clostridium difficile causes infectious diarrhoea in humans and animals. It has been found in both diarrhoeal and non-diarrhoeal pigs, horses and cattle, suggesting a potential reservoir for human insection, and in 20-40 % of meat products in Canada and the USA, suggesting the possibility, albeit not proven, of food-borne transmission. Although it is not yet completely clear, 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. 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 human infection 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, a fact that sits well with the notion that animals are a reservoir for human infection. Thus, there are three problems that require resolution: a human health issue, an animal health issue and the factor common to both these problems, environmental contamination. To successfully deal with these recent changes in the epidemiology of CDI will require a 'one health' approach involving human health physicians, veterinarians and environmental scientists.