Clostridioides Difficile Spores Research Articles

The effect of pulsed xenon ultraviolet light disinfection on healthcare-associated Clostridioides difficile rates in a tertiary care hospital

Ultraviolet (UV) light has destructive activity against pathogenic bacteria including Clostridioides difficile spores. Portable pulsed-xenon UV disinfecting devices were implemented for terminal room cleaning in 6 units of our academic hospital with high C. difficile infection (CDI) rates. CDI rates were measured in a 9-month period before and a 9-month period after device implementation. Despite documented administration of UV disinfection for 87% of terminal room cleaning, no impact on CDI rates was detected.

Activity of Hospital Disinfectants against Vegetative Cells and Spores of Clostridioides difficile Embedded in Biofilms.

Clostridioides difficile spores can survive in the environment in either mono- or mixed-species biofilms. However, no previous studies have investigated chemical disinfection of C. difficile spores embedded in biofilms. Thus, the purpose of this study was to assess the in vitro effectiveness of hospital disinfectants against C. difficile spores embedded within biofilms. Five unique C. difficile strains embedded in three different biofilm types grown for 72 or 120 h were exposed to seven different hospital disinfectants. C. difficile abundance [as log(number of CFU/milliliter)] was calculated after manufacturer-determined contact times along with biofilm biomass and microscopy. The primary analysis compared differences between C. difficile vegetative cell and spore counts as well as amounts of biomass after exposure to disinfectants. C. difficile vegetative cells and spores were recovered from biofilms regardless of the type of biofilm growth or biofilm growth time. No disinfectant was able to completely eliminate C. difficile from the biofilms. Overall, Clorox, ortho-phthalaldehyde (OPA), and Virex were most effective at killing C. difficile spores regardless of biofilm age, ribotype, or wash conditions (whether biofilms are washed or unwashed) (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 vegetative cell growth in biofilms (P = 0.77). Clorox and Virex were most effective in reducing biomass, followed by Nixall, OPA, and Vital Oxide. No disinfectant was able to completely eliminate C. difficile embedded within biofilms although differences among disinfectants were noted. Future research will be required to determine methods to eradicate this persister reservoir.

Efficacy of an ultraviolet-A lighting system for continuous decontamination of health care–associated pathogens on surfaces

We found that ultraviolet-A (UV-A) light exposure resulted in a modest reduction in recovery of methicillin-resistant Staphylococcus aureus (MRSA), Candida auris, bacteriophage MS2, and bacteriophage Phi X174, but not Clostridioides difficile spores, on steel disk carriers. Four hours of UV-A exposure from a ceiling light fixture resulted in a significant reduction in pathogenic microorganisms recovered from in-use medical equipment. These findings suggest that UV-A could be useful as a means to provide continuous low-level decontamination of surfaces in health care facilities.

Development of a rapid-viability PCR method for detection of Clostridioides difficile spores from environmental samples

Clostridioides difficile is a common pathogen that is well known to survive for extended periods of time on environmental healthcare surfaces from fecal contamination. During epidemiological investigations of healthcare-associated infections, it is important to be able to detect whether or not there are viable spores of C. difficile on surfaces. Current methods to detect C. difficile can take up to 7 days for culture and in the case of detection by PCR, viability of the spores cannot be ascertained. Prevention of C. difficile infection in healthcare settings includes adequate cleaning and disinfection of environmental surfaces which increases the likelihood of detecting dead organisms from an environmental sample during an investigation. In this study, we were able to adapt a rapid-viability PCR (RV-PCR) method, first developed for detection of viable Bacillus anthracis spores, for the detection of viable C. difficile spores. RV-PCR uses the change in cycle threshold after incubation to confirm the presence of live organisms. Using this modified method we were able to detect viable C. difficile after 22 h of anaerobic incubation in Cycloserine Cefoxitin Fructose Broth (CCFB). This method also used bead beating combined with the Maxwell 16 Casework kit for DNA extraction and purification and a real-time duplex PCR assay for toxin B and cdd3 genes to confirm the identity of the C. difficile spores. Spiked environmental sponge-wipes with and without added organic load were tested to determine the limit of detection (LOD). The LOD from spiked environmental sponge-wipe samples was 104 spores/mL but after incubation initial spore levels of 101 spores/mL were detected. Use of this method would greatly decrease the amount of time required to detect viable C. difficile spores; incubation of samples is only required for germination (22 h or less) instead of colony formation, which can take up to 7 days. In addition, PCR can then quickly confirm or deny the identity of the organism at the same time it would confirm viability. The presence of viable C. difficile spores could be detected at very low levels within 28 h total compared to the 2 to 10-day process that would be needed for culture, identification and toxin detection.

The CspC pseudoprotease regulates germination of Clostridioides difficile spores in response to multiple environmental signals.

The gastrointestinal pathogen, Clostridioides difficile, initiates infection when its metabolically dormant spore form germinates in the mammalian gut. While most spore-forming bacteria use transmembrane germinant receptors to sense nutrient germinants, C. difficile is thought to use the soluble pseudoprotease, CspC, to detect bile acid germinants. To gain insight into CspC’s unique mechanism of action, we solved its crystal structure. Guided by this structure, we identified CspC mutations that confer either hypo- or hyper-sensitivity to bile acid germinant. Surprisingly, hyper-sensitive CspC variants exhibited bile acid-independent germination as well as increased sensitivity to amino acid and/or calcium co-germinants. Since mutations in specific residues altered CspC’s responsiveness to these different signals, CspC plays a critical role in regulating C. difficile spore germination in response to multiple environmental signals. Taken together, these studies implicate CspC as being intimately involved in the detection of distinct classes of co-germinants in addition to bile acids and thus raises the possibility that CspC functions as a signaling node rather than a ligand-binding receptor.

The efficacy of a simulated tunnel washer process on removal and destruction of Clostridioides difficile spores from health care textiles

Research on reducing Clostridioides difficile spore contamination of textiles via laundering is needed. We evaluated the sporicidal properties of 5 laundry chemicals and then determined the ability of a peracetic acid (PAA) laundry cycle to inactivate and/or remove spores from cotton swatches during a simulated tunnel washer (TW) process. In phase I, spore-inoculated swatches were immersed in alkaline detergent, sodium hypochlorite, hydrogen peroxide, or PAA for 8 minutes. In phase II, inoculated swatches were passed through a simulated 24-minute TW process employing 5 wash liquids. Spore survivors on swatches and in test chemical fluids in both studies were enumerated using standard microbiologic assay methods. In phase I, hypochlorite solutions achieved >5 log10 spore reductions on swatches and >3 log10 reductions for wash solutions. PAA achieved minimal spore reduction in the wash solution (0.26 log10). In phase II, the PAA equilibrium-containing process achieved a >5 log10 spore reduction on swatches. In wash solution tests, the cumulative spore reduction peaked at >3.08 log10 in the final module. Sodium hypochlorite as a laundry additive is sporicidal. The cumulative effects of a TW process, coupled with a PAA bleach agent at neutral pH, may render textiles essentially free of C difficile spore contamination.

Molecular epidemiology of Clostridioides difficile in domestic dogs and zoo animals

Animals such as domestic dogs and zoo animals reside in close proximity to humans and could contribute to the dissemination of Clostridioides difficile spores which are common in the community environment. The purpose of this study was to assess C. difficile colonization in domestic dogs attending a day boarding facility and zoo animals receiving systemic antibiotics.Stool samples and paw swabs were collected from dogs who attended a day boarding facility. Stool samples were also collected from zoo animals starting systemic antibiotics. Finally, environmental samples were collected from nearby public parks. Stool samples and swabs were incubated anaerobically in enrichment broth for C. difficile growth, PCR was done to confirm presence of toxin genes, and PCR ribotyping was performed for strain characterization.During the study period, 136 dog stool samples were obtained, the paws of 16 dogs were swabbed, and 250 environmental swabs from surrounding public parks were obtained. Twenty-three of 136 dog stool samples (17%) and 9 of 16 dog paws sampled (56%) grew toxigenic C. difficile. One hundred and four stool samples from 49 zoo animals were collected of which 19 (18%) grew toxigenic C. difficile. Rates of toxigenic C. difficile colonization increased significantly during antibiotic therapy (33%) and then returned to baseline during the follow-up (11%) period (p = 0.019). Fifty-five of 250 environmental swabs from public parks (22%) grew toxigenic C. difficile. Ribotypes associated with human disease including 106 and 014-020 were isolated from all sources.This study demonstrated a high rate of toxigenic C. difficile colonization in domestic dogs and zoo animals with ribotypes similar to those causing human disease. These results demonstrate the relationship between humans, animals, and the environment in the dissemination of spores.

Isolation and characterization of Clostridioides difficile spores from contaminated single-used surgical gowns

Clostridium difficile is the primary cause of antibiotic associated diarrhoea globally. In the UK there has been a decline in the prevalence of C. difficile due to implementation of surveillance and infection control procedures. At Rideout Hospital, USA, however, there is a high incidence of C. difficile infection, which has been partly attributed to poor infection control measures. Other factors include the ability of spores to adhere to fomites such as surgical gowns. It has been demonstrated that the single-use polypropylene surgical gowns used at Rideout can ‘trap’ hydrophobic epidemic spores of C. difficile within the fibres, which can then be transferred to stainless steel surfaces and hospital floor vinyl; even with use of appropriate sporicides such as sodium dichloroisocyanurate. This study sought to establish the strains of C. difficile present on the gowns and thus inside the nosocomial environment. Contaminated gowns from Rideout were cultured for 5 days anaerobically in Brain –Heart Infusion broth supplemented with 0.1 % Sodium taurocholate. Broth culture was screened for the presence of C. difficile using CCFA media, C. DIFF QUIK CHEK COMPLETE®, 16 s-23s RNA analysis and toxin PCR. Once isolated, strains were sequenced and tested for biocide susceptibility to in-use concentrations of Sodium dichloroisocyanurate. In total 23 suspected C. difficile samples were isolated from the gowns; of which 8 were confirmed. Sporicide susceptibility testing is ongoing. Once infective strains have been identified measures can be taken to enforce appropriate infection control procedures in order to limit the prevalence of spores and reduce infection rates.

Hierarchical recognition of amino acid co-germinants during Clostridioides difficile spore germination

Bile acids are an important signal for germination of Clostridioides difficile spores; however, the bile acid signal alone is not sufficient. Amino acids, such as glycine, are another signal necessary for germination by C. difficile spores. Prior studies on the amino acid signal required for germination have shown that there is a preference for the amino acid used as a signal for germination. Previously we found that d-alanine can function as a co-germinant for C. difficile spores at 37 °C but not at 25 °C. Here, we tested the ability of other amino acids to act as co-germinants with taurocholate (TA) at 37 °C and found that many amino acids previously categorized as non-co-germinants are co-germinants at 37 °C. Based on the EC50 values calculated for two different strains, we found that C. difficile spores recognize different amino acids with varying efficiencies. Using this data, we ranked the amino acids based on their effect on germination and found that in addition to d-alanine, other D-forms of amino acids are also used by C. difficile spores as co-germinants. Among the different types of amino acids, ones with branched chains such as valine, leucine, and isoleucine are the poorest co-germinants. However, glycine is still the most effective amino acid signal for both strains. Our results suggest that the yet-to-be-identified amino acid germinant receptor is highly promiscuous.