Genomic delineation of zoonotic origins of Clostridium difficile

Clostridium difficile is toxin-producing antimicrobial resistant (AMR) enteropathogen historically associated with diarrhea and pseudomembranous colitis in hospitalized patients. In recent years, there have been dramatic increases in the incidence and severity of C. difficile infection (CDI), and associated morbidity and mortality, in both healthcare and community settings. C. difficile is an ancient and diverse species that displays a sympatric lifestyle, establishing itself in a range of ecological niches external to the healthcare system. These sources/reservoirs include food, water, soil, and over a dozen animal species, in particular, livestock such as pigs and cattle. In a manner analogous to human infection, excessive antimicrobial exposure, particularly to cephalosporins, is driving the expansion of C. difficile in livestock populations worldwide. Subsequent spore contamination of meat, vegetables grown in soil containing animal feces, agricultural by-products such as compost and manure, and the environment in general (households, lawns, and public spaces) is contributing to a persistent community source/reservoir of C. difficile and the insidious rise of CDI in the community. The whole-genome sequencing era continues to redefine our view of this complex pathogen. The application of high-resolution microbial genomics in a One Health framework (encompassing clinical, veterinary, and environment derived datasets) is the optimal paradigm for advancing our understanding of CDI in humans and animals. This approach has begun to yield critical insights into the genetic diversity, evolution, AMR, and zoonotic potential of C. difficile. In Europe, North America, and Australia, microevolutionary analysis of the C. difficile core genome shows strains common to humans and animals (livestock or companion animals) do not form distinct populations but share a recent evolutionary history. Moreover, for C. difficile sequence type 11 and PCR ribotypes 078 and 014, major lineages of One Health importance, this approach has substantiated inter-species clonal transmission between animals and humans. These findings indicate either a zoonosis or anthroponosis. Moreover, they challenge the existing paradigm and the long-held misconception that CDI is primarily a healthcare-associated infection. In this article, evolutionary, and zoonotic aspects of CDI are discussed, including the anthropomorphic factors that contribute to the spread of C. difficile from the farm to the community

Complete genome assemblies of three highly prevalent, toxigenic clostridioides difficile strains causing health care-associated infections in Australia

Clostridioides difficile infection (CDI) is the leading cause of life-threatening health care-related gastrointestinal illness worldwide. Phylogenetically appropriate closed reference genomes are essential for studies of C. difficile transmission and evolution. Here, we provide high-quality complete hybrid genome assemblies for the three most prevalent C. difficile strains causing CDI in Australia

Effect of natural products on the production and activity of Clostridium difficile toxins in vitro

Clostridium difficile infection is a toxin-mediated disease of the colon. C. difficile virulence is primarily attributed to the production of toxin A and toxin B; thus this study was aimed to investigate the effect of a range of natural products on the production and activity of C. difficile toxins in vitro. Twenty-two natural products were investigated against four C. difficile strains. The activity of products against toxins was determined using Vero and HT-29 cells cytotoxicity and neutral red uptake assays. The indirect effect of products on toxin-mediated cytotoxicity was determined using the same cell lines. The effect of seven products on toxin production by C. difficile was determined using ELISA. Zingerone (0.3 mg/ml) protected both cell lines from C. difficile cytopathic effects, confirmed by the neutral red uptake assay (P \u3c 0.05). Three Leptospermum honeys (4% w/v), fresh onion bulb extract (12.5% v/v) and trans-cinnamaldehyde (0.005% v/v) all reduced toxin production and activity significantly (P ≤ 0.023). Garlic clove powder (4.7 mg/ml) only reduced toxin activity (P ≤ 0.047). Overall, several natural products had activity against C. difficile toxins in vitro encouraging further investigation against C. difficile toxins in vivo

A species-wide genetic atlas of antimicrobial resistance in clostridioides difficile

Antimicrobial resistance (AMR) plays an important role in the pathogenesis and spread of Clostridioides difficile infection (CDI), the leading healthcare-related gastrointestinal infection in the world. An association between AMR and CDI outbreaks is well documented, however, data is limited to a few ‘epidemic’ strains in specific geographical regions. Here, through detailed analysis of 10330 publicly-available C. difficile genomes from strains isolated worldwide (spanning 270 multilocus sequence types (STs) across all known evolu-tionary clades), this study provides the first species-wide snapshot of AMR genomic epidemiology in C. difficile. Of the 10330 C. difficile genomes, 4532 (43.9%) in 89 STs across clades 1–5 carried at least one genotypic AMR determinant, with 901 genomes (8.7%) carrying AMR determinants for three or more antimicrobial classes (multidrug-resistant, MDR). No AMR genotype was identified in any strains belonging to the cryptic clades. C. difficile from Australia/New Zealand had the lowest AMR prevalence compared to strains from Asia, Europe and North America (P \u3c 0.0001). Based on the phylogenetic clade, AMR prevalence was higher in clades 2 (84.3%), 4 (81.5%) and 5 (64.8%) compared to other clades (collectively 26.9%) (P \u3c 0.0001). MDR prevalence was highest in clade 4 (61.6%) which was over three times higher than in clade 2, the clade with the second-highest MDR prevalence (18.3%). There was a strong association between specific AMR determinants and three major epidemic C. difficile STs: ST1 (clade 2) with fluoroquinolone resistance (mainly T82I substitution in GyrA) (P \u3c 0.0001), ST11 (clade 5) with tetracycline resistance (various tet-family genes) (P \u3c 0.0001) and ST37 (clade 4) with macrolide-lincosamide-streptogramin B (MLSB ) resistance (mainly ermB) (P \u3c 0.0001) and MDR (P \u3c 0.0001). A novel and previously overlooked tetM-positive transposon designated Tn6944 was identified, predominantly among clade 2 strains. This study provides a comprehensive review of AMR in the global C. difficile population which may aid in the early detection of drug-resistant C. difficile strains, and prevention of their dissemination worldwide

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

Molecular characterization of, and antimicrobial resistance in, clostridioides difficile from Thailand, 2017–2018

Antimicrobial resistance (AMR) plays an important role in the pathogenesis and spread of Clostridioides difficile infection (CDI). Many antimicrobials, such as fluoroquinolones, have been associated with outbreaks of CDI globally. This study characterized AMR among clinical C. difficile strains in Thailand, where antimicrobial use remains inadequately regulated. Stool samples were screened for tcdB and positives were cultured. C. difficile isolates were characterized by toxin profiling and PCR ribotyping. Antimicrobial susceptibility testing was performed by agar incorporation, and whole-genome sequencing and AMR genotyping were performed on a subset of strains. There were 321 C. difficile strains isolated from 326 stool samples. The most common toxigenic ribotype (RT) was RT 017 (18%), followed by RTs 014 (12%) and 020 (7%). Resistance to clindamycin, erythromycin, moxifloxacin, and rifaximin was common, especially among RT 017 strains. AMR genotyping revealed a strong correlation between resistance genotype and phenotype for moxifloxacin and rifaximin. The presence of erm-class genes was associated with high-level clindamycin and erythromycin resistance. Point substitutions in the penicillin-binding proteins were not sufficient to confer meropenem resistance, but a Y721S substitution in PBP3 was associated with a 4.37-fold increase in meropenem minimal inhibitory concentration. No resistance to metronidazole, vancomycin, or fidaxomicin was observed

Genomic analysis of clostridioides difficile recovered from horses in Western Australia

Clostridioides difficile poses an ongoing threat as a cause of gastrointestinal disease in humans and animals. Traditionally considered a human healthcare-related disease, increases in community-associated C. difficile infection (CDI) and growing evidence of inter-species transmission suggest a wider perspective is required for CDI control. In horses, C. difficile is a major cause of diarrhoea and life-threatening colitis. This study aimed to better understand the epidemiology of CDI in Australian horses and provide insights into the relationships between horse, human and environmental strains. A total of 752 faecal samples from 387 Western Australian horses were collected. C. difficile was isolated from 104 (30.9%) horses without gastrointestinal signs and 19 (37.8%) with gastrointestinal signs. Of these, 68 (55.3%) harboured one or more toxigenic strains, including C. difficile PCR ribotypes (RTs) 012 (n = 14), 014/020 (n = 10) and 087 (n = 7), all prominent in human infection. Whole-genome analysis of 45 strains identified a phylogenetic cluster of 10 closely related C. difficile RT 012 strains of equine, human and environmental origin (0–62 SNP differences; average 23), indicating recent shared ancestry. Evidence of possible clonal inter-species transmission or common-source exposure was identified for a subgroup of three horse and one human isolates, highlighting the need for a One Health approach to C. difficile surveillance

Complete genome sequences of evolutionary clade C-III strains of Clostridioides (Clostridium) difficile isolated from the environment in Western Australia

Clostridioides (Clostridium) difficile in the environment is thought to contribute to C. difficile infection in community settings. Here, we provide complete genome assemblies for two esculin hydrolysis-negative strains of C. difficile that were isolated from soils in Western Australia; the strains produce white colonies on chromogenic media and belong to evolutionarily divergent clade C-III

Esculin hydrolysis negative and TcdA-only producing strains of Clostridium (Clostridioides) difficile from the environment in Western Australia

Background and Aims: Clostridium (Clostridiodes) difficile clade 3 ribotype (RT) 023 strains that fail to produce black colonies on bioMérieux ChromID agar have been reported, as well as variant strains of C. difficile that produce only toxin A. We have recently isolated strains of C. difficile from the environment in Western Australia (WA) with similar characteristics. The objective of this study was to characterize these strains. It was hypothesized that a putative β-glucosidase gene was lacking in these strains of C. difficile, including RT 023, leading to white colonies. Methods and Results: A total of 17 environmental isolates of C. difficile from garden soil and compost, and gardening shoe soles in Perth, WA, failed to produce black colonies on ChromID agar. MALDI-TOF MS analysis confirmed these strains as C. difficile. Four strains contained only a tcdA gene (A+B−CDT−) by PCR and were a novel RT (QX 597). All isolates were susceptible to all antimicrobials tested except one with low-level resistance to clindamycin (MIC = 8 mg/L). The four tcdA-positive strains were motile. All isolates contained neither bgl locus but only bgl K or a putative β-glucosidase gene by PCR. Whole-genome sequencing showed the 17 strains belonged to novel multi-locus sequence types 632, 848, 849, 850, 851, 852 and 853, part of the evolutionarily divergent clade C-III. Four isolates carried a full-length tcdA but not tcdB nor binary toxin genes. Conclusions: ChromID C. difficile agar is used for the specific detection of C. difficile in the samples. To date, all strains except RT 023 strains from clinical samples hydrolyse esculin. This is the first report to provide insights into the identification of esculin hydrolysis negative and TcdA-only producing (A+B−CDT−) strains of C. difficile from environmental samples. Significance and Impact of the Study: White colonies of C. difficile from environmental samples could be overlooked when using ChromID C. difficile agar, leading to false-negative results, however, whether these strains are truly pathogenic remains to be proven

Mapping of A(1 )conferring resistance to the aphid Amphorophora idaei and dw (dwarfing habit) in red raspberry (Rubus idaeus L.) using AFLP and microsatellite markers

BACKGROUND: Raspberry breeding programmes worldwide aim to produce improved cultivars to satisfy market demands and within these programmes there are many targets, including increased fruit quality, yield and season, and improved pest and disease resistance and plant habit. The large raspberry aphid, Amphorophora idaei, transmits four viruses and vector resistance is an objective in raspberry breeding. The development of molecular tools that discriminate between aphid resistance genes from different sources will allow the pyramiding of such genes and the development of raspberry varieties with superior pest resistance. We have raised a red raspberry (Rubus idaeus) F(1 )progeny from the cross 'Malling Jewel' × 'Malling Orion' (MJ × MO), which segregates for resistance to biotype 1 of the aphid Amphorophora idaei and for a second phenotypic trait, dwarf habit. These traits are controlled by single genes, denoted (A(1)) and (dw) respectively. RESULTS: The progeny of 94 seedlings was scored for the segregation of 95 AFLP and 22 SSR markers and a linkage map was constructed that covers a total genetic distance of 505 cM over seven linkage groups. The average linkage group length was 72.2 cM and there was an average of 17 markers per linkage group, of which at least two were codominant SSRs, allowing comparisons with previously published maps of raspberry. The two phenotypic traits, A(1 )and dw, mapped to linkage groups 3 and 6 respectively. CONCLUSION: The mapping of A(1 )will facilitate the discrimination of resistance genes from different sources and the pyramiding of aphid resistance genes in new raspberry cultivars; the mapping of dw will allow further investigations into the genetics of dwarfing habit in Rubus