Dissertations & Theses (Open Access)

Date of Award

Spring 5-2019

Degree Name

Doctor of Philosophy (PhD)

Advisor(s)

Shreela V. Sharma, Phd, Rd, Ld

Abstract

Introduction:

Clostridioides difficile is a motile, gram-negative, toxin-producing bacteria responsible for the majority of the nosocomial as well as community-acquired infection in the US and worldwide. A high rate of morbidity, mortality, recurrences, healthcare cost coupled with frequent epidemic outbreaks with drug-resistant strains has classified C. difficile as a pathogen with an urgent threat to human health. Current guidelines regarding disinfectant use are either limited or absent or are not effective in reducing the environmental burden of C. difficile. The aims for this study was first to determine the in vitro efficacy of select hospital disinfectants in eradicating the spores of C. difficile both in planktonic form as well as in biofilm and secondly, to determine the effect of community disinfectants on the eradication and propagation of C. difficile.

Methods

Following standardized protocol, low, medium and high category spores were produced and exposed to the original concentration of disinfectants at both label determined and standardized time in presence or absence of organic matter. Minimum disinfectant concentration and minimum organic matter concentration for with bactericidal effect were also determined. The efficacy of hospital disinfectants on the strictly anaerobic mono organism, mixed multi organism and strictly aerobic multi organism 72 hours and 120 hours C. difficile biofilm was quantified using both microbiological techniques as well as microscopically. The effect of community household disinfectants on C. difficile spores was determined using time-kill experiments as well as time-kill curves. Germination assay, Ca-DPA assay, spore cortex fragmentation assay, the effect on the dormant vs. activated spore, qPCR and microscopy was done to characterize the role of community disinfectants on C. difficile propagation.

Results

The effect of 7 hospital disinfectant and 7 community disinfectant were tested against16 C. difficile spores of 6 different ribotypes. All tested hospital disinfectants were effective in eradicating almost 100% C. difficile spores at manufacturer suggested contact time in absence of organic matter and at all spore concentration. Using the general linear model, sporicidal activity of the disinfectants was affected by a shorter contact time (0.52±0.10 log decrease; p<0.0001), and presence of organic matter (low organic substance: 0.77±0.11 log decrease; p<0.0001; high organic substance: 2.02±0.12 log decrease; p<0.0001). C. difficile vegetative cells and spores were recovered from biofilms regardless of type or duration of biofilm formation. No disinfectant was able to completely eliminate C. difficile from the biofilms. Overall, Clorox, OPA, and Virex were most effective at killing C. difficile spores regardless of biofilm age, ribotype, or wash conditions (p= 0.001, each). Clorox and OPA were also effective at killing total vegetative cell growth (P=0.001, each) but Virex was found to be ineffective against the total vegetative cell growth (p=0.77). Clorox and Virex were most effective in reducing biomass followed by Nixall, OPA, and Vital oxide. None of the community disinfectants except Clorox was found to be effective against C. difficile spores. Increased spore germination was observed for Lysol hydrogen peroxide and Lysol. Effect on active vs dormant spores, germination assay, Ca-DPA assay, spore cortex fragmentation assay, qPCR and microscopy ass indicate the potential for Lysol hydrogen peroxide to act as a germinant for C. difficile spores.

Conclusion:

None of the disinfectants could consistently reduce C. difficile spore count under all conditions. Furthermore, this is the first study to look at the efficacy of hospital disinfectants on C. difficile spores encased in a biofilm. This study reported for the very first time about the germinant potential of certain household disinfectants. Thus, the findings for this study may help to inform the infection control guidelines and aid to fulfill the CDCs goal of reducing C. difficile infection by 30% by 2020.

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