Difficile Spores Research Articles

Outcome of relapsing Clostridium difficile infections do not correlate with virulence-, spore- and vegetative cell-associated phenotypes

One of the main clinical challenges of Clostridium difficile infections (CDI) is the high rate of relapse episodes. The main determinants involved in relapse of CDI include the presence of antibiotic-resistant C. difficile spores in the colonic environment and a permanent state of dysbiosis of the microbiota caused by antibiotic therapy. A possible scenario is that phenotypes related to the persistence of C. difficile spores might contribute to relapsing infections. In this study, 8 C. difficile isolates recovered from 4 cases with relapsing infection, and 9 isolates recovered from single infection cases were analyzed for PCR ribotyping and the presence of tcdA, tcdB and cdtAB genes. Factors associated to spore persistence, sporulation, spore adherence and biofilm formation and sporulation during biofilm formation were characterized. We also evaluated motility and cytotoxicity. However, we observed no significant difference in the analyzed phenotypes among the different clinical outcomes, most likely due to the high variability observed among strains within clinical backgrounds in each phenotype and the small sample size. It is noteworthy that C. difficile spores adhered to similar extents to undifferentiated and differentiated Caco-2 cells. By contrast, spores of all clinical isolates tested had increased germination efficiency in presence of taurocholate, while decreased sporulation rate during biofilm development in the presence of glucose. In conclusion, these results show that, at least in this cohort of patients, the described phenotypes are not detrimental in the clinical outcome of the disease.

Importance of mechanical action in a terminal disinfection process for decontamination of Clostridium difficile spores on hospital inert contact surfaces

Although the relevance of surface disinfection is increasingly being accepted, there are still a number of issues which remain controversial. In hospital environments in Quebec (Canada), a three-step technique of cleaning and disinfection is used following a case of Clostridium difficile contamination. This technique brings together three factors: chemical, mechanical and microbiological. The present study investigates the impact of the mechanical action during each of these three-steps on C. difficile spore populations. Thus, cleaning and sporicidal products were replaced by water. The reduction rate of C. difficile spores was evaluated on four contaminated types of surfaces commonly found in the hospital environment. They included: ceramic, polyvinyl chloride (PVC), melamine and 2-methylprop-2-enoate methyl (PMAM) mattress cover. According to the type of the inert surface, variation in the reduction rate of spores was observed. The mattress cover was the most difficult surface to clean, followed by melamine. In contrast, the ceramic and PVC were the easiest surface to clean. For all the four surfaces, the average rate of spore loss associated with the various steps indicates that the mechanical action in the three-step process is important in reducing the risk associated with the presence of C. difficile spores on the hospital contact surfaces.

Development of Photodynamic Antimicrobial Chemotherapy (PACT) for Clostridium difficile.

Background Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudo membranous colitis in the developed world. The aim of this study was to explore whether Photodynamic Antimicrobial Chemotherapy (PACT) could be used as a novel approach to treating C. difficile infections.MethodsPACT utilises the ability of light-activated photosensitisers (PS) to produce reactive oxygen species (ROS) such as free radical species and singlet oxygen, which are lethal to cells. We screened thirteen PS against C. difficile planktonic cells, biofilm and germinating spores in vitro, and cytotoxicity of effective compounds was tested on the colorectal adenocarcinoma cell-line HT-29.ResultsThree PS were able to kill 99.9% of bacteria in both aerobic and anaerobic conditions, both in the planktonic state and in a biofilm, after exposure to red laser light (0.2 J/cm2) without harming model colon cells. The applicability of PACT to eradicate C. difficile germinative spores indirectly was also shown, by first inducing germination with the bile salt taurocholate, followed by PACT.ConclusionThis innovative and simple approach offers the prospect of a new antimicrobial therapy using light to treat C. difficile infection of the colon.

Clostridium difficile Diarrhea in the Elderly: Current Issues and Management Options.

Clostridium difficile infection (CDI) is the most common cause of infectious diarrhea in healthcare settings. Along with antimicrobial exposure, advanced age has been shown to be a significant risk factor for the development and recurrence of, and mortality from, CDI. The substantial burden of CDI in the elderly may be related to frequent healthcare exposure, the necessity for more medications, altered intestinal microbiota, and complicated comorbidities. A diagnosis of CDI is based on evidence of toxin, or the C. difficile organism itself, in a stool sample in the presence of clinical signs and symptoms. Only symptomatic patients should be tested for CDI, and routine surveillance or repeat testing on asymptomatic patients as a test of cure is discouraged. Antibiotic discontinuation alone can improve or resolve CDI in some patients, and concomitant use of antibiotics is associated with decreased response to CDI treatment. Metronidazole, vancomycin, and fidaxomicin are the therapeutic agents currently available for CDI, with the selection of these agents being based on disease severity, history of recurrence, and cost. The recurrence rate after initial treatment is 20-30%. The first recurrence can be treated with the same therapeutic agent and, for subsequent recurrences, vancomycin in a tapered and/or pulsed regimen is recommended. Fecal microbiota transplantation has shown remarkable effectiveness for recurrent anti-refractory CDI, although caution is advised in treating immunocompromised hosts and those with toxic megacolon. C. difficile can be transmitted directly and indirectly via contact with patients or their environment; therefore, isolation precautions should be initiated at the first suspicion of CDI. C. difficile spores can survive for a long time on environmental surfaces, and the patient's room and all equipment used in the room should be disinfected. In order to manage CDI in the elderly, timely diagnosis, appropriate treatment based on severity of illness, and effective infection control are essential.

Regulation of Clostridium difficile spore germination by the CspA pseudoprotease domain.

Clostridium difficile is a spore-forming obligate anaerobe that is a leading cause of healthcare-associated infections. C.difficile infections begin when its metabolically dormant spores germinate in the gut of susceptible individuals. Binding of bile salt germinants to the Csp family pseudoprotease CspC triggers a proteolytic signaling cascade consisting of the Csp family protease CspB and the cortex hydrolase SleC. Conserved across many of the Clostridia, Csp proteases are subtilisin-like serine proteases that activate pro-SleC by cleaving off its inhibitory pro-peptide. Active SleC degrades the protective cortex layer, allowing spores to resume metabolism and growth. This signaling pathway, however, is differentially regulated in C.difficile, since CspC functions both as a germinant receptor and regulator of CspB activity. CspB is also produced as a fusion to a catalytically inactive CspA domain that subsequently undergoes interdomain processing during spore formation. In this study, we investigated the role of the CspA pseudoprotease domain in regulating C.difficile spore germination. Mutational analyses revealed that the CspA domain controls CspC germinant receptor levels in mature spores and is required for optimal spore germination, particularly when CspA is fused to the CspB protease. During spore formation, the YabG protease separates these domains, although YabG itself is dispensable for germination. Bioinformatic analyses of Csp family members suggest that the CspC-regulated signaling pathway characterized in C.difficile is conserved in related Peptostreptococcaceae family members but not in the Clostridiaceae or Lachnospiraceae. Our results indicate that pseudoproteases play critical roles in regulating C.difficile spore germination and highlight that diverse mechanisms control spore germination in the Clostridia.

Unlocking the Sporicidal Potential of Ethanol: Induced Sporicidal Activity of Ethanol against Clostridium difficile and Bacillus Spores under Altered Physical and Chemical Conditions.

BackgroundDue to their efficacy and convenience, alcohol-based hand sanitizers have been widely adopted as the primary method of hand hygiene in healthcare settings. However, alcohols lack activity against bacterial spores produced by pathogens such as Clostridium difficile and Bacillus anthracis. We hypothesized that sporicidal activity could be induced in alcohols through alteration of physical or chemical conditions that have been shown to degrade or allow penetration of spore coats.Principal FindingsAcidification, alkalinization, and heating of ethanol induced rapid sporicidal activity against C. difficile, and to a lesser extent Bacillus thuringiensis and Bacillus subtilis. The sporicidal activity of acidified ethanol was enhanced by increasing ionic strength and mild elevations in temperature. On skin, sporicidal ethanol formulations were as effective as soap and water hand washing in reducing levels of C. difficile spores.ConclusionsThese findings demonstrate that novel ethanol-based sporicidal hand hygiene formulations can be developed through alteration of physical and chemical conditions.

Clostridium difficile in retail chicken meat parts and liver in the Eastern Region of Turkey

In recent studies, the presence of Clostridium difficile has been shown in foods and animals that may be a source of infection in susceptible humans. In the present study, it was aimed to investigate the prevalence of toxigenic Clostridium difficile spores and vegetative cells in chicken meat parts and liver. For this purpose, 310 samples (75 leg quarters, 75 breast, 60 wings, 60 drumsticks and 40 chicken livers) were collected from two cities in eastern region of Turkey for 6 months and analyzed for the presence of C. difficile. C. difficile was isolated from 25 (8.06 %) of 310 samples. The prevalence of C. difficile was 12, 8, 6.6, 5 and 7.5 % for the leg quarter, breast, wing, drumstick and liver, respectively. The results showed that the prevalence of C. difficile in chicken samples was higher in winter months (December, January and February). Multiplex PCR results showed that 5 of 25 C. difficile isolates had toxin B (tcdB) gene, and 8 isolates had toxin A (tcdA) gene. None of the isolates was having binary toxin (cdtA or cdtB) genes. The findings of the present study show that toxigenic C. difficile spores can be found in both chicken meat parts and in livers although their numbers is low.

Microbiology for Radiologists: How to Minimize Infection Transmission in the Radiology Department.

The implementation of standardized infection control and prevention practices is increasingly relevant as modern radiology practice evolves into its more clinical role. Current Centers for Disease Control and Prevention, National Institutes of Health, and World Health Organization guidelines for the proper use of personal protective equipment, decontamination of reusable medical equipment, and appropriate management of bloodborne pathogen exposures will be reviewed. Standard precautions apply to all patients at all times and are the mainstay of infection control. Proper hand hygiene includes washing hands with soap and water when exposed to certain infectious particles, such as Clostridium difficile spores, which are not inactivated by alcohol-based hand rubs. The appropriate use of personal protective equipment in accordance with recommendations from the Centers for Disease Control and Prevention includes wearing a surgical mask during lumbar puncture. Because radiologists may perform lumbar punctures for patients with prion disease, it is important to appreciate that incineration is the most effective method of inactivating prion proteins. However, there is currently no consensus recommendation on the decontamination of prion-contaminated reusable items associated with lumbar puncture, and institutional policies should be consulted for directed management. In the event of a needlestick injury, radiology staff must be able to quickly provide appropriate initial management and seek medical attention, including laboratory testing for bloodborne pathogens.

Pathogen transfer and high variability in pathogen removal by detergent wipes

The rise in health care-associated infections has placed a greater emphasis on cleaning and disinfection practices. The majority of policies advocate using detergent-based products for routine cleaning, with detergent wipes increasingly being used; however, there is no information about their ability to remove and subsequently transfer pathogens in practice. Seven detergent wipes were tested for their ability to remove and transfer Staphylococcus aureus, Acinetobacter baumannii, and Clostridium difficile spores using the 3-stage wipe protocol. The ability of the detergent wipes to remove S aureus, A baumannii, and C difficile spores from a stainless steel surface ranged from 1.50 log10 (range, 0.24-3.25), 3.51 log10 (range, 3.01-3.81), and 0.96 log10 (range, 0.26-1.44), respectively, following a 10-second wiping time. All wipes repeatedly transferred significant amounts of bacteria/spores over 3 consecutive surfaces, although the percentage of total microorganisms transferred from the wipes after wiping was low for a number of products. Detergent-based wipe products have 2 major drawbacks: their variability in removing microbial bioburden from inanimate surfaces and a propensity to transfer pathogens between surfaces. The use of additional complementary measures such as combined detergent/disinfectant-based products and/or antimicrobial surfaces need to be considered for appropriate infection control and prevention.

Treatment with Nontoxigenic

Recurrent Clostridium difficile infection (CDI) appears to stem from the germination of toxigenic C. difficile spores in the intestinal tract in the

The yype I IFN pathway in C. difficile infection: impact on morbidity and mortality in mice (MPF4P.737)

Abstract Infections with C. difficile are increasingly prevalent in individuals in health care settings and are becoming a concern in food animals. C. difficile is a gram positive, anaerobic bacteria known for its ability to form spores that are exceedingly difficult to kill. Disruption of the normal gut microbiota allows C. difficile spores to germinate, divide and produce toxins that compromise the intestinal barrier resulting in extreme inflammation. Currently, little is known about the innate immune response in Clostridium difficile infection (CDI). We hypothesized that type I IFNs may play a crucial role in innate protection from CDI. We found that IFNAR-deficient mice succumbed to CDI more rapidly compared to wild type mice. In addition, wild type mice treated with type I IFN-inducing dsRNA mimetic, polyIC, had delayed onset of symptoms compared to mice treated with saline. Since type I IFNs can block damaging inflammasome activity, this potential mechanism was explored. Wild type and IFNAR-deficient mice were treated with DMSO or the inflammasome-blocking agent, Glibenclamide. Glibenclamide-treated IFNAR-deficient mice faired better than all groups. This finding suggests that type I IFN signaling may protect from CDI in part by blocking the inflammasome. Considering that recombinant type I IFN is already used clinically, investigation of this pathway may present new treatment options for this emerging infection.

Novel handwashes are superior to soap and water in removal of Clostridium difficile spores from the hands

We examined the efficacy of 5 experimental handwash formulations in removing nontoxigenic Clostridium difficile spores from the hands of health care workers. Handwashing with sand resulted in an additional 0.5-log reduction in spore recovery compared with the current standard of soap and water.

Spore Cortex Hydrolysis Precedes Dipicolinic Acid Release during Clostridium difficile Spore Germination.

Bacterial spore germination is a process whereby a dormant spore returns to active, vegetative growth, and this process has largely been studied in the model organism Bacillus subtilis. In B. subtilis, the initiation of germinant receptor-mediated spore germination is divided into two genetically separable stages. Stage I is characterized by the release of dipicolinic acid (DPA) from the spore core. Stage II is characterized by cortex degradation, and stage II is activated by the DPA released during stage I. Thus, DPA release precedes cortex hydrolysis during B. subtilis spore germination. Here, we investigated the timing of DPA release and cortex hydrolysis during Clostridium difficile spore germination and found that cortex hydrolysis precedes DPA release. Inactivation of either the bile acid germinant receptor, cspC, or the cortex hydrolase, sleC, prevented both cortex hydrolysis and DPA release. Because both cortex hydrolysis and DPA release during C. difficile spore germination are dependent on the presence of the germinant receptor and the cortex hydrolase, the release of DPA from the core may rely on the osmotic swelling of the core upon cortex hydrolysis. These results have implications for the hypothesized glycine receptor and suggest that the initiation of germinant receptor-mediated C. difficile spore germination proceeds through a novel germination pathway. Clostridium difficile infects antibiotic-treated hosts and spreads between hosts as a dormant spore. In a host, spores germinate to the vegetative form that produces the toxins necessary for disease. C. difficile spore germination is stimulated by certain bile acids and glycine. We recently identified the bile acid germinant receptor as the germination-specific, protease-like CspC. CspC is likely cortex localized, where it can transmit the bile acid signal to the cortex hydrolase, SleC. Due to the differences in location of CspC compared to the Bacillus subtilis germinant receptors, we hypothesized that there are fundamental differences in the germination processes between the model organism and C. difficile. We found that C. difficile spore germination proceeds through a novel pathway.

Microfiber cloths reduce the transfer of Clostridium difficile spores to environmental surfaces compared with cotton cloths

Environmental surfaces in health care facilities contaminated with Clostridium difficile spores can be a reservoir that contribute to transmission of hospital-acquired infections. Microfiber cleaning cloths may improve the effectiveness of surface cleaning. The objective of this study was to assess the removal and transfer of C difficile spores on surfaces cleaned by microfiber compared with cotton cloths. C difficile spores (approximately 4.2 log(10)/site) were applied to ceramic surfaces. Microfiber or cotton cloths were used to wipe the surfaces that were sprayed with either buffer or a nonsporicidal cleaning agent. To ensure reproducible pressure and surface contact time, a drill apparatus was used. The pressure was 1.5-1.77N, and the total number of rotations was 10. Viable counts were used to assess the efficiency of microfiber and cotton cloths in removing and transferring spores. Of 4.4 log(10)C difficile spores inoculated on a ceramic surface, microfiber and cotton cloths removed 2.4 and 1.7 log(10), respectively. Microfiber cloths containing 4.2 log(10)C difficile spores transferred 1.7 log(10)C difficile spores when used to wipe a ceramic surface compared with cotton cloths that transferred 2.4 log(10). Similarly microfiber wipes transferred fewer spores on consecutive surfaces wiped compared with cotton cloths (0.8 log(10) vs 1.80 log(10)). The use of microfiber cloths may reduce the risk of C difficile spore transfer during surface cleaning.

Induced sporicidal activity of chlorhexidine against Clostridium difficile spores under altered physical and chemical conditions.

BackgroundChlorhexidine is a broad-spectrum antimicrobial commonly used to disinfect the skin of patients to reduce the risk of healthcare-associated infections. Because chlorhexidine is not sporicidal, it is not anticipated that it would have an impact on skin contamination with Clostridium difficile, the most important cause of healthcare-associated diarrhea. However, although chlorhexidine is not sporicidal as it is used in healthcare settings, it has been reported to kill spores of Bacillus species under altered physical and chemical conditions that disrupt the spore’s protective barriers (e.g., heat, ultrasonication, alcohol, or elevated pH). Here, we tested the hypothesis that similarly altered physical and chemical conditions result in enhanced sporicidal activity of chlorhexidine against C. difficile spores.Principal Findings C. difficile spores became susceptible to heat killing at 80°C within 15 minutes in the presence of chlorhexidine, as opposed to spores suspended in water which remained viable. The extent to which the spores were reduced was directly proportional to the concentration of chlorhexidine in solution, with no viable spores recovered after 15 minutes of incubation in 0.04%–0.0004% w/v chlorhexidine solutions at 80°C. Reduction of spores exposed to 4% w/v chlorhexidine solutions at moderate temperatures (37°C and 55°C) was enhanced by the presence of 70% ethanol. However, complete elimination of spores was not achieved until 3 hours of incubation at 55°C. Elevating the pH to ≥9.5 significantly enhanced the killing of spores in either aqueous or alcoholic chlorhexidine solutions.ConclusionsPhysical and chemical conditions that alter the protective barriers of C. difficile spores convey sporicidal activity to chlorhexidine. Further studies are necessary to identify additional agents that may allow chlorhexidine to reach its target within the spore.

Protein composition of the outermost exosporium-like layer of Clostridium difficile 630 spores

Clostridium difficile spores are considered the morphotype of infection, transmission and persistence of C. difficile infections. There is a lack of information on the composition of the outermost exosporium layer of C. difficile spores. Using recently developed exosporium removal methods combined with MS/MS, we have established a gel-free approach to analyze the proteome of the exosporium of C. difficile spores of strain 630. A total of 184 proteins were found in the exosporium layer of C. difficile spores. We identified 7 characterized spore coat and/or exosporium proteins; 6 proteins likely to be involved in spore resistance; 6 proteins possibly involved in pathogenicity; 13 uncharacterized proteins; and 146 cytosolic proteins that might have been encased into the exosporium during assembly, similarly as reported for Bacillus anthracis and Bacillus cereus spores. We demonstrate through Flag-fusions that CotA and CotB are mainly located in the spore coat, while the exosporium collagen-like glycoproteins (i.e. BclA1, BclA2 and BclA3), the exosporium morphogenetic proteins CdeC and CdeM, and the uncharacterized exosporium proteins CdeA and CdeB are mainly located in the exosporium layer of C. difficile 630 spores. This study offers novel candidates of C. difficile exosporium proteins as suitable targets for detection, removal and spore-based therapies. Biological significanceThis study offers a novel strategy to identify proteins of the exosporium layer of C. difficile spores and complements previous proteomic studies on the entire C. difficile spores and spore coat since it defines the proteome of the outermost layer of C. difficile spores, the exosporium. This study suggests that C. difficile spores have several proteins involved in protection against environmental stress as well as putative virulence factors that might play a role during infection. Spore exosporium structural proteins were also identified providing the ground basis for further functional studies of these proteins. Overall this work provides new protein target for the diagnosis and/or therapeutics that may contribute to combat C. difficile infections.

Effect of age on Clostridium difficile infection in a murine model of infection

Clostridium difficile, a Gram-positive, spore-forming bacillus, is the leading cause of nosocomial antibiotic-associated diarrhea in the U.S. A major risk factor for developing C. difficile infection (CDI) is advanced age (>65 years), which also accounts for 93% of CDI-related deaths. It is unknown why CDI becomes more severe with age. The objective of this study was to develop a CDI mouse model to study the effects of aging on the pathogenesis of CDI. To achieve this goal we compared CDI in aged (12 months) versus young (2 months) C57BL/6J mice. Six days after C. difficile spore challenge, clinical symptoms (e.g. wet tail, hunching, dehydration) were exhibited by a greater proportion of aged mice compared to young mice (p=0.08). Aged mice experienced greater weight loss than younger animals (p=0.013) and shed greater numbers of bacteria into the feces (p=0.023). Not surprisingly, aged mice suffered a higher cumulative mortality (44%) compared to the young (20%). Histological examination of cecum and colon tissue revealed the severity of inflammation and mucosal damage was greater in aged mice than younger animals (p=0.004). Because mortality is typically associated with lower anti-toxin antibody titers, splenic B cells (CD45+CD19+) numbers were measured by flow cytometry. In comparison to young mice, the proportion of splenic B cells was significantly reduced in the aged group (p=0.006). These results demonstrate that the consequences of CDI are more severe in aged mice and associated with B cell changes. Utilizing our model will help to advance our understanding of age-related changes on host immune defenses against CDI, which is crucial for designing new prophylactic and treatment strategies to reduce morbidity and mortality in older adults.

Clostridium difficile drug pipeline: challenges in discovery and development of new agents.

In the past decade Clostridium difficile has become a bacterial pathogen of global significance. Epidemic strains have spread throughout hospitals, while community acquired infections and other sources ensure a constant inoculation of spores into hospitals. In response to the increasing medical burden, a new C. difficile antibiotic, fidaxomicin, was approved in 2011 for the treatment of C. difficile-associated diarrhea. Rudimentary fecal transplants are also being trialed as effective treatments. Despite these advances, therapies that are more effective against C. difficile spores and less damaging to the resident gastrointestinal microbiome and that reduce recurrent disease are still desperately needed. However, bringing a new treatment for C. difficile infection to market involves particular challenges. This review covers the current drug discovery pipeline, including both small molecule and biologic therapies, and highlights the challenges associated with in vitro and in vivo models of C. difficile infection for drug screening and lead optimization.

Cold-air atmospheric pressure plasma against Clostridium difficile spores: a potential alternative for the decontamination of hospital inanimate surfaces.

Clostridium difficile spores survive for months on environmental surfaces and are highly resistant to decontamination. We evaluated the effect of cold-air plasma against C. difficile spores. The single-jet had no effect while the multi-jet achieved 2-3 log10 reductions in spore counts and may augment traditional decontamination.

The recombinant Lactococcus lactis oral vaccine induces protection against C. difficile spore challenge in a mouse model

Clostridium difficile infection (CDI) causes nosocomial antibiotic-associated diarrhea and colitis in the developed world. Two potent cytotoxins, toxin A (TcdA) and toxin B (TcdB) are the virulence factors of this disease and can be a good vaccine candidate against CDI. In the present study, we genetically engineered Lactococcus lactis to express the nontoxic, recombinant fragments derived from TcdA and TcdB C-terminal receptor binding domains (Tcd-AC and Tcd-BC) as an oral vaccine candidate. The immunogenicity of the genetically engineered L. lactis oral vaccine delivery system (animal groups LAC and LBC or the combination of both, LACBC) was compared with the recombinant TcdA and TcdB C-terminal receptor binding domain proteins (animal groups PAC and PBC or the combination of both, PACBC), which were expressed and purified from E. coli. After the C. difficile challenge, the control groups received PBS or engineered L. lactis with empty vector, showed severe diarrhea symptoms and died within 2–3 days. However, both the oral vaccine and recombinant protein vaccine groups had significantly lower mortalities, body weight decreases and histopathologic lesions than the control sham-vaccine groups (p<0.05) except group LBC which only had a 31% survival rate after the challenge. The data of post infection survival showed that an average of 86% of animals survived in groups PAC and PACBC, 75% of animals survived in group LACBC, and 65% of animals survived in group LAC. All of the vaccinated animals produced higher titers of both IgG and IgA than the control groups (p<0.05), and the antibodies were able to neutralize the cytopathic effect of toxins in vitro. The results of this study indicate that there is a potential to use L. lactis as a delivery system to develop a cost effective oral vaccine against CDI.