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 ß-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 ß-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.

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.

Synthetic Studies of Neoclerodane Diterpenes from Salvia divinorum: Identification of a Potent and Centrally Acting µ Opioid Analgesic with Reduced Abuse Liability.

Opioids are widely used to treat millions suffering from pain, but their analgesic utility is limited due to associated side effects. Herein we report the development and evaluation of a chemical probe exhibiting analgesia and reduced opioid-induced side effects. This compound, kurkinorin (5), is a potent and selective µ-opioid receptor (MOR) agonist (EC50 = 1.2 nM, >8000 µ/κ selectivity). 5 is a biased activator of MOR-induced G-protein signaling over ß-arrestin-2 recruitment. Metadynamics simulations of 5's binding to a MOR crystal structure suggest energetically preferred binding modes that differ from crystallographic ligands. In vivo studies with 5 demonstrate centrally mediated antinociception, significantly reduced rewarding effects, tolerance, and sedation. We propose that this novel MOR agonist may represent a valuable tool in distinguishing the pathways involved in MOR-induced analgesia from its side effects.