Recipient Organization
UNIVERSITY OF ARIZONA
888 N EUCLID AVE
TUCSON,AZ 85719-4824
Performing Department
Animal & Comparative Biomedical Sciences
Non Technical Summary
Clostridium difficile is an important pathogen of both human and veterinary populations. In humans, it is the leading healthcare-associated infection in the United States. Clostridium difficile (CD) is a gram-positive, anaerobic, spore-forming bacillus that colonizes the gastrointestinal tract and causes diarrhea which, in some cases, can progress to pseudomembranous colitis, toxic megacolon and death. Risk factors in both human and veterinary populations include age, microbiota status, and prior antibiotic treatment. Disease- causing strains of CD produce 1-3 toxins that enter intestinal epithelial cells and glucosylate or ribosylate Rho family GTPases, leading to cell death. Though it is increasingly appreciated that factors other than the toxins play a role in disease, the identity of such factors, and their functions, remain to be defined.Since the year 2000, new "epidemic-" and outbreak-associated" strains of CD have emerged that cause more severe disease with higher mortality. These strains have been found in many North American communities. CD is also established in food animals, and is a significant problem in the agriculture industry, with non-human neonates (piglets, calves and foals) being particularly affected. These animals are all susceptible to CD infection (CDI) within 1-14 days of birth, and disease manifestation resembles that in humans. Worryingly, the greatest numbers of CD isolates are recovered from suckling piglets inside the farrowing barn. Further, particular molecular types of CD found in pigs have been recovered from human patients. On some farms, up to 75% of all piglets carry CD, and many of these CD strains are resistant to multiple antibiotics. Specifically, since the immune system is un- or under-developed at this time-frame, classic approaches such as vaccination are not used for disease prevention in agriculturally-relevant neonates. Ironically, only antibiotic treatment is considered for suckling piglets [erythromycin, tylosin or tetracycline]; these will further delay the development and establishment of a healthy gastrointestinal microbiota. Neonates are lost in high numbers if symptomatic, and those that survive infection are asymptomatically colonized, and shed CD to further contaminate the environment, and potentially, food sources. Recent findings are suggestive of zoonotic spread, with food-borne transmission of CD spores as a hypothesized route for human infection. Epidemic-associated CD spores, such as those of ribotype 078, may thus be introduced into hospitals by patients infected in the community.In Arizona, the last published CD infection (CDI) surveillance in humans was done 18 years ago. In the past 5 years, and in collaboration with colleagues at multiple Tucson-area hospitals, we have re- initiated robust surveillance efforts; these are currently ongoing. Sampling of the veterinary population in Arizona has also been extremely limited and sporadic, and there have been no standardized surveillance efforts. Therefore, and commencing in early this year (2017), we began tracking and collecting CD isolates from farm-based piglets in Southern Arizona. From this recent (and ongoing) surveillance, we conclude that "hypervirulent" veterinary strains (Ribotype 078) are present in significant levels in piglets the region. Importantly, we have also found the same Ribotype 078 strains in our recent human surveillance; their recovery from human patients is highly suggestive of CD's zoonotic potential. Thus, our current collection of these recent CD isolates of both human and veterinary origin presents a unique opportunity for in-depth, clinically-relevant, molecular analyses.In this proposal, we will therefore perform comparative analyses of CD strains of recent veterinary origin with the goal of understanding the molecular determinants that confer disease and species-specificity.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
In this project, we will perform comparative analyses of Clostridium difficile (C. difficile; CD) strains of recent veterinary origin with the goal of understanding the molecular determinants that confer disease and species-specificity. This proposal draws on our various strengths and resources. Most importantly, we have a collection of veterinary C. difficile isolates collected in Arizona. We also have extensive expertise in cutting-edge omics approaches, as well as in vitro and in vivo host- pathogen interaction studies.The overarching goal of this project, therefore, is to evaluate the in vitro and in vivo attributes of specific C. difficile (CD) strains of veterinary origin. The Specific Objectives will be to determine if our veterinary clinical samples:• Encode all known CD toxin genes (toxin A, toxin B, and binary toxin)• Express or produce measurable and functional amounts of these toxins• Adhere to human and porcine intestinal epithelial cells• Express antibiotic resistance• Contain unique genetic signatures• Are virulent in animal models of severe C. difficile disease
Project Methods
I. In vitro studies to assess Clostridium difficile (CD) toxin gene presence, toxin production, CD adherence, CD antibiotic susceptibility and genomic signatures:Stool samples will be plated on selective media (TCCFA) to isolate CD. Pure culture of CD in brain-heart infusion will grow for 72 hours, after which supernatant will be collected. Toxin production will be measured using the supernatant on a commercially available toxin A/B ELISA kit as we have previously performed. Detection of CD toxin genes tcdA, tcdB, cdtA, and cdtB will be done using polymerase chain reaction amplification of the genes, also as we have previously performed. Crude template will be prepared from pure colonies using a standard colony lyse solution. Amplicons will be separated on 1% agarose. Adherence will be measured as previously described; both human (C2BBE) and neonatal porcine intestinal epithelial (IEC) cell lines will be used. Antibiotic susceptibility testing [erythromycin, tetracycline, ciprofloxacin, vancomycin (human and veterinary antimicrobials)] will be determined through growth media supplemented with antibiotics in serial dilution as we have previously performed, and then confirmed through PCR amplification of suspected resistance genes. Strains that are found to be most virulent from the studies above will be used for genomics. Whole-cell DNA will be extracted from these strains, and sequenced using an Illumina platform at the University of Arizona's Core Genomics facility.II. In vivo studies to assess CD virulence in animal models for severe CDI:CD strains will be tested in the Syrian golden hamster model of C. difficile infection as we have previously performed and published. In brief, animals will be sensitized to CD infection with a single oral administration of clindamycin (30mg/kg; similar to humans). 3-5 days post-antibiotic, animals will be challenged with 100 spores of veterinary CD. This is a lethal infection model, and death usually occurs within 48-96 hours post-challenge. CDI symptoms will be monitored (diarrhea, inappetence, weight loss), and CD burden quantitated in fecal material, and in cecal contents upon necropsy. We will also perform neonatal piglet infections with at least two recent veterinary-origin (Ribotype 078) CD strains. Studies will involve CD challenges of ~5-day-old piglets obtained from a high health-status herd housed south of Tucson; we currently work with this farm as part of our regular veterinary CD surveillance. CD challenge will include 103-5 spore administrations with two well-described veterinary-associated strains (Ribotype 078). Disease symptoms will be monitored and time-to- death or recovery documented similar to parameters measured in Syrian hamsters. Piglets will be housed at the UA Central Animal Facility according to Dr. Vedantam's current and approved IACUC Protocol.