Abstract Title

The In Vitro Adherence and Virulence Factors of Clostridium Difficile Ribotypes 027 and Non-027 is Not Predicative of Virulence in the Murine or Hamster CDAD Model

Presenter Name

John C. Vitucci

RAD Assignment Number

1609

Presenter/Author(s) Information

John C. Vitucci, UNT Health Science CenterFollow

Abstract

Background: C. difficile ribotype 027 (RT027) is the North American epidemic strain. Studies suggest an enhanced virulence phenotype for RT027 such as increased toxin production, but the impact on disease severity on in vivo models is not well understood. This study describes the in vitro characterization of important virulence characteristics for several RT027 and non-RT027 C. difficile clinical isolates, and how these factors are not predictive of disease severity in the hamster C. difficile associated disease (HCDAD) model.

Methods: Six RT027 and six non-RT027 clinical isolates were evaluated in vitro for total spore counts and Toxin A/B titers in 72H broth cultures. Spore counts were generated from heat/ethanol shock culture samples and plated onto CB + taurocholate + antibiotics, and toxin A/B titers were determined from spent broth with an ELISA assay. The Murine C. difficile model involved antibiotics administered for 5 days through drinking water. The mice were then given 48 hours to clear the antibiotic from their system before the administration of 10 mg/kg clindamycin, followed 24H later by administration of spores from either an 027 or non-027 isolate. Survival was monitored for 10 days and fecal samples were taken each day to be processed for CFU/spore counts. The HCDAD studies involved infecting male Golden Syrian hamsters with varying titers of RT027 and non-RT027 spore isolates, followed by subcutaneous administration of 10 mg/kg clindamycin 24H post-infection. All groups were left untreated and survival was monitored for 7 days after infection, samples were collected every day for CFU/spore counts and Toxin A/B titers.

Results: The RT027 and the non-RT027 strains generated similar mean CFU/mL in 72H broth cultures, while the mean spore counts were 83 spores/one million cells for the RT027 strains and 123 spores/one million cells for the non-RT027 strains. While, the 72H broth-associated mean toxin A/B titers were 2.8-fold higher for RT027 strains when compared to the 72H titers of non-RT027 strains. In the HCDAD studies the non-027 infected hamsters survived with inoculation counts of up to 20,000 spores, while hamsters infected with the RT027 isolates survived inoculation with counts below 300 spores. The mean cecal fluid toxin A/B titers for RT027 infected hamsters were 2.3 to 9-fold higher than the titers for non-RT027 infected hamsters. In the mouse model, 90% of the animals infected with the non-027 isolate survived no matter the antibiotic dosing. In contrast, 13-26% morbidity was associated with mice infected with the RT027 isolate after being given antibiotics in multiple doses or in a single does through over time through supplemented water.

Conclusions: The results highlight that C. difficile RT027 isolates, when compared to non-RT027 clinical isolates, have enhanced virulence in vivo that does not correspond to a strain’s predicted virulence from in vitro characterization.

Research Area

Microbiology/Infectious Disease

Presentation Type

Oral

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The In Vitro Adherence and Virulence Factors of Clostridium Difficile Ribotypes 027 and Non-027 is Not Predicative of Virulence in the Murine or Hamster CDAD Model

Background: C. difficile ribotype 027 (RT027) is the North American epidemic strain. Studies suggest an enhanced virulence phenotype for RT027 such as increased toxin production, but the impact on disease severity on in vivo models is not well understood. This study describes the in vitro characterization of important virulence characteristics for several RT027 and non-RT027 C. difficile clinical isolates, and how these factors are not predictive of disease severity in the hamster C. difficile associated disease (HCDAD) model.

Methods: Six RT027 and six non-RT027 clinical isolates were evaluated in vitro for total spore counts and Toxin A/B titers in 72H broth cultures. Spore counts were generated from heat/ethanol shock culture samples and plated onto CB + taurocholate + antibiotics, and toxin A/B titers were determined from spent broth with an ELISA assay. The Murine C. difficile model involved antibiotics administered for 5 days through drinking water. The mice were then given 48 hours to clear the antibiotic from their system before the administration of 10 mg/kg clindamycin, followed 24H later by administration of spores from either an 027 or non-027 isolate. Survival was monitored for 10 days and fecal samples were taken each day to be processed for CFU/spore counts. The HCDAD studies involved infecting male Golden Syrian hamsters with varying titers of RT027 and non-RT027 spore isolates, followed by subcutaneous administration of 10 mg/kg clindamycin 24H post-infection. All groups were left untreated and survival was monitored for 7 days after infection, samples were collected every day for CFU/spore counts and Toxin A/B titers.

Results: The RT027 and the non-RT027 strains generated similar mean CFU/mL in 72H broth cultures, while the mean spore counts were 83 spores/one million cells for the RT027 strains and 123 spores/one million cells for the non-RT027 strains. While, the 72H broth-associated mean toxin A/B titers were 2.8-fold higher for RT027 strains when compared to the 72H titers of non-RT027 strains. In the HCDAD studies the non-027 infected hamsters survived with inoculation counts of up to 20,000 spores, while hamsters infected with the RT027 isolates survived inoculation with counts below 300 spores. The mean cecal fluid toxin A/B titers for RT027 infected hamsters were 2.3 to 9-fold higher than the titers for non-RT027 infected hamsters. In the mouse model, 90% of the animals infected with the non-027 isolate survived no matter the antibiotic dosing. In contrast, 13-26% morbidity was associated with mice infected with the RT027 isolate after being given antibiotics in multiple doses or in a single does through over time through supplemented water.

Conclusions: The results highlight that C. difficile RT027 isolates, when compared to non-RT027 clinical isolates, have enhanced virulence in vivo that does not correspond to a strain’s predicted virulence from in vitro characterization.