Induced Sporicidal Activity of Chlorhexidine against Clostridium difficile Spores under Altered Physical and Chemical Conditions

Background

Chlorhexidine 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.

Conclusions

Physical 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.     

Inactivation of spores by nonthermal plasmas

Bacterial and fungal spore contamination in different industries has a greater economic impact. Because of the remarkable resistance of spores to most physical and chemical microbicidal agents, their inactivation need special attention during sterilization processes. Heat and chemical sporicides are not always well suited for different sterilization/decontamination applications and carries inherent risks. In recent years, novel nonthermal agents including nonthermal plasmas are emerging as effective sporicides against a broad spectrum of bacterial and fungal spores. The present review discusses various aspects related to the inactivation of spores using nonthermal plasmas. Different types of both low pressure plasmas (e.g., capacitively coupled plasma and microwave plasma) and atmospheric pressure plasmas (e.g., dielectric barrier discharges, corona discharges, arc discharges, radio-frequency-driven plasma jet) have been successfully applied to destroy spores of economic significance. Plasma agents contributing to sporicidal activity and their mode of action in inactivation are discussed. In addition, information on factors that affect the sporicidal action of nonthermal plasmas is included.     

Evaluation of the efficacy of electrochemically activated solutions against nosocomial pathogens and bacterial endospores

Aims: Electrochemically activated solutions (ECAS) are generated from halide salt solutions via specially designed electrolytic cells. The active solutions are known to possess high biocidal activity against a wide range of target microbial species, however, literature revealing the kill‐kinetics of these solutions is limited. The aim of the study was to identify the kill‐rate and extent of population kill for a range of target species (including endospores) using ECAS generated at the anode (anolyte). Methods and Results: Standard suspensions of methicillin‐resistant Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus atrophaeus spores and Clostridium difficile spores were treated with anolyte in a quantitative suspension assay. For vegetative cells, all concentrations of anolyte tested reduced the viable population to below the detection limit within 10 s. At a concentration of 99%, anolyte produced a log₁₀ reduction factor of greater than five in viable B. atrophaeus endospores within 90 s and reduced numbers of C. difficile endospores to below the experimental detection limit within 20 s at concentrations of 5% or greater. Conclusions: Anolyte was highly effective in killing test‐bacteria and spores. The bactericidal efficacy was retained against vegetative cells at dilutions as low as 1% and against C. difficile spores as low as 5%. Significance and Impact of Study: The results of this study demonstrate that ECAS are effective at lower concentrations and act more rapidly than previously reported. Potent bactericidal and sporicidal activity coupled with point‐of‐use generation, low production‐costs and environmental compatibility suggest that acidic ECAS has the potential to be a useful addition to the current armoury of disinfectants.     

Bacterial spore structures and their protective role in biocide resistance

The structure and chemical composition of bacterial spores differ considerably from those of vegetative cells. These differences largely account for the unique resistance properties of the spore to environmental stresses, including disinfectants and sterilants, resulting in the emergence of spore-forming bacteria such as Clostridium difficile as major hospital pathogens. Although there has been considerable work investigating the mechanisms of action of many sporicidal biocides against Bacillus subtilis spores, there is far less information available for other species and particularly for various Clostridia. This paucity of information represents a major gap in our knowledge given the importance of Clostridia as human pathogens. This review considers the main spore structures, highlighting their relevance to spore resistance properties and detailing their chemical composition, with a particular emphasis on the differences between various spore formers. Such information will be vital for the rational design and development of novel sporicidal chemistries with enhanced activity in the future.     

Cloning of Ruminococcus albus endo-β-1,4-glucanase and xylanase genes

Buffered solutions of chlorine-releasing agents, sodium hypochlorite (NaOCl), sodium dichloroisocyanurate (NaDCC), and chloramine-T, showed similar activity against vegetative cells of Bacillus subtilis but there was considerable variation in activity against spores, NaOCl showing higher activity than NaDCC, which in turn was more active than chloramine-T. The effect of coat and cortex extraction on sporicidal activity was determined. It was concluded that although spore coats play a role, they do not totally account for chlorine resistance and that the cortex is also involved, probably through its function in maintaining a low water level in the enclosed core. Observed differences in the sporicidal action of NaOCl, NaDCC, and chloramine-T may be related to their differing ability to produce core and cortex degradation.     

Histidine acts as a co‐germinant with glycine and taurocholate for Clostridium difficile spores

Aims: It is well established that the bile salt sodium taurocholate acts as a germinant for Clostridium difficile spores and the amino acid glycine acts as a co‐germinant. The aim of this study was to determine whether any other amino acids act as co‐germinants. Methods and Results: Clostridium difficile spore suspensions were exposed to different germinant solutions comprising taurocholate, glycine and an additional amino acid for 1 h before heating shocking (to kill germinating cells) or chilling on ice. Samples were then re‐germinated and cultured to recover remaining viable cells. Only five amino acids out of the 19 common amino acids tested (valine, aspartic acid, arginine, histidine and serine) demonstrated co‐germination activity with taurocholate and glycine. Of these, only histidine produced high levels of germination (97·9–99·9%) consistently in four strains of Cl. difficile spores. Some variation in the level of germination produced was observed between different PCR ribotypes, and the optimum concentration of amino acids with taurocholate for the germination of Cl. difficile NCTC 11204 spores was 10–100 mmol l^?1. Conclusions: Histidine was found to be a co‐germinant for Cl. difficile spores when combined with glycine and taurocholate. Significance and Impact of the Study: The findings of this study enhance current knowledge regarding agents required for germination of Cl. difficile spores which may be utilized in the development of novel applications to prevent the spread of Cl. difficile infection.     

Development of a Protocol for Predicting Bacterial Resistance to Microbicides

Regulations dealing with microbicides in Europe and the United States are evolving and now require data on the risk of the development of resistance in organisms targeted by microbicidal products. There is no standard protocol to assess the risk of the development of resistance to microbicidal formulations. This study aimed to validate the use of changes in microbicide and antibiotic susceptibility as initial markers for predicting microbicide resistance and cross-resistance to antibiotics. Three industrial isolates (Pseudomonas aeruginosa, Burkholderia cepacia, and Klebsiella pneumoniae) and two Salmonella enterica serovar Typhimurium strains (SL1344 and 14028S) were exposed to a shampoo, a mouthwash, eye makeup remover, and the microbicides contained within these formulations (chlorhexidine digluconate [CHG] and benzalkonium chloride [BZC]) under realistic, in-use conditions. Baseline and postexposure data were compared. No significant increases in the MIC or the minimum bactericidal concentration (MBC) were observed for any strain after exposure to the three formulations. Increases as high as 100-fold in the MICs and MBCs of CHG and BZC for SL1344 and 14028S were observed but were unstable. Changes in antibiotic susceptibility were not clinically significant. The use of MICs and MBCs combined with antibiotic susceptibility profiling and stability testing generated reproducible data that allowed for an initial prediction of the development of resistance to microbicides. These approaches measure characteristics that are directly relevant to the concern over resistance and cross-resistance development following the use of microbicides. These are low-cost, high-throughput techniques, allowing manufacturers to provide to regulatory bodies, promptly and efficiently, data supporting an early assessment of the risk of resistance development.     

Diarylacylhydrazones: Clostridium-selective antibacterials with activity against stationary-phase cells

Current antibiotics for treating Clostridium difficile infections (CDI), that is, metronidazole, vancomycin and more recently fidaxomicin, are mostly effective but treatment failure and disease relapse remain as significant clinical problems. The shortcomings of these agents are attributed to their low selectivity for C. difficile over normal gut microflora and their ineffectiveness against C. difficile spores. This Letter reports that certain diarylacylhydrazones identified during a high-throughput screening/counter-screening campaign show selective activity against two Clostridium species (C. difficile and Clostridium perfringens) over common gut commensals. Representative examples are shown to possess activity similar to vancomycin against clinical C. difficile strains and to kill stationary-phase C. difficile cells, which are responsible for spore production. Structure–activity relationships with additional synthesised analogues suggested a protonophoric mechanism may play a role in the observed activity/selectivity and this was supported by the well-known protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) showing selective anti-Clostridium effects and activity similar to diarylacylhydrazones against stationary-phase C. difficile cells. Two diarylacylhydrazones were shown to be non-toxic towards human FaDu and Hep G2 cells indicating that further studies with the class are warranted towards new drugs for CDI.     

The small acid-soluble proteins of Clostridioides difficile are important for UV resistance and serve as a check point for sporulation

Clostridioides difficile is a nosocomial pathogen which causes severe diarrhea and colonic inflammation. C. difficile causes disease in susceptible patients when endospores germinate into the toxin-producing vegetative form. The action of these toxins results in diarrhea and the spread of spores into the hospital and healthcare environments. Thus, the destruction of spores is imperative to prevent disease transmission between patients. However, spores are resilient and survive extreme temperatures, chemical exposure, and UV treatment. This makes their elimination from the environment difficult and perpetuates their spread between patients. In the model spore-forming organism, Bacillus subtilis, the small acid-soluble proteins (SASPs) contribute to these resistances. The SASPs are a family of small proteins found in all endospore-forming organisms, C. difficile included. Although these proteins have high sequence similarity between organisms, the role(s) of the proteins differ. Here, we investigated the role of the main α/β SASPs, SspA and SspB, and two annotated putative SASPs, CDR20291_1130 and CDR20291_3080, in protecting C. difficile spores from environmental insults. We found that SspA is necessary for conferring spore UV resistance, SspB minorly contributes, and the annotated putative SASPs do not contribute to UV resistance. In addition, the SASPs minorly contribute to the resistance of nitrous acid. Surprisingly, the combined deletion of sspA and sspB prevented spore formation. Overall, our data indicate that UV resistance of C. difficile spores is dependent on SspA and that SspA and SspB regulate/serve as a checkpoint for spore formation, a previously unreported function of SASPs.     

Mechanism of Sporicidal Activity for the Synergistic Combination of Peracetic Acid and Hydrogen Peroxide

There is still great interest in controlling bacterial endospores. The use of chemical disinfectants and, notably, oxidizing agents to sterilize medical devices is increasing. With this in mind, hydrogen peroxide (H₂O₂) and peracetic acid (PAA) have been used in combination, but until now there has been no explanation for the observed increase in sporicidal activity. This study provides information on the mechanism of synergistic interaction of PAA and H₂O₂ against bacterial spores. We performed investigations of the efficacies of different combinations, including pretreatments with the two oxidizers, against wild-type spores and a range of spore mutants deficient in the spore coat or small acid-soluble spore proteins. The concentrations of the two biocides were also measured in the reaction vessels, enabling the assessment of any shift from H₂O₂ to PAA formation. This study confirmed the synergistic activity of the combination of H₂O₂ and PAA. However, we observed that the sporicidal activity of the combination is largely due to PAA and not H₂O₂. Furthermore, we observed that the synergistic combination was based on H₂O₂ compromising the spore coat, which was the main spore resistance factor, likely allowing better penetration of PAA and resulting in the increased sporicidal activity.     

An effective iodide formulation for killing Bacillus and Geobacillus spores over a wide temperature range

AIMS: To develop a sporicidal reagent which shows potent activity against bacterial spores not only at ambient temperatures but also at low temperatures. METHODS AND RESULTS: Suspension tests on spores of Bacillus and Geobacillus were conducted with the reagent based on a previously reported agent (N. Kida, Y. Mochizuki and F. Taguchi, Microbiology and Immunology 2003; 47: 279-283). The modified reagent (tentatively designated as the KMT reagent) was composed of 50 mmol l(-1) EDTA-2Na, 50 mmol l(-1) ferric chloride hexahydrate (FeCl(3).6H(2)O), 50 mmol l(-1) potassium iodide (KI) and 50% ethanol in 0.85% NaCl solution at pH 0.3. The KMT reagent showed significant sporicidal activity against three species of Bacillus and Geobacillus spores over a wide range of temperature. The KMT reagent had many practical advantages, i.e. activity was much less affected by organic substances than was sodium hypochlorite, it did not generate any harmful gas and it was stable for a long period at ambient temperatures. The mechanism(s) of sporicidal activity of the KMT reagent was considered to be based on active iodine species penetrating the spores with enhanced permeability of the spore cortex by a synergistic effect of acid, ethanol and generated active oxygen. CONCLUSIONS: The data suggest that the KMT reagent shows potent sporicidal activity over a wide range temperatures and possesses many advantages for practical applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate development of a highly applicable sporicidal reagent against Bacillus and Geobacillus spores.     

Assesssment of sporicidal efficacy

On the basis of their activity against bacterial spores, chemical agents can be divided into two groups: Group A contains chemicals that are bactericidal and sporistatic but not sporicidal, e.g. phenols, cresols, parabens, quaternary ammonium compounds and biguanides; Group B contains those, such as glutaraldehyde, chloride-releasing agents, ethylene oxide and hydrogen peroxide, that show both bactericidal and sporicidal activity, although much higher concentrations may be needed to achieve the latter effect.Sporistatic activity can be examined by various methods, e.g. (i) determining minimum inhibitory concentrations, (ii) detecting by phase-contrast microscopy the concentrations needed to inhibit germination or outgrowth, (iii) measuring spectrophotometrically effects on germination or outgrowth. Information thereby obtained is of considerable importance in food microbiology. Sporicidal efficacy can be tested against spores in liquid medium or suspended on appropriate carriers. Factors influencing activity such as concentration, pH, temperature and the presence of organic matter need to be assessed. The principles of evaluating sporicidal activity are essentially the same as for determining bactericidal activity. The main difference is that a treated spore has to pass through complex stages (germination and outgrowth) before a vegetative cell, and eventually a colony, is produced, if a conventional counting method is employed. It is essential that adequate quenching of residual activity is achieved and that recovery conditions are such as to permit viable but damaged spores the opportunity to revive. A recently developed procedure utilises bioluminescence as a means of determining sporicidal efficacy.     

Bactericidal activity of telavancin,vancomycin and metronidazole against Clostridium difficile

We compared the time-kill activities of telavancin, vancomycin and metronidazole at concentrations of 2 times, 4 times, and 8 times their respective MICs against five Clostridium difficile strains including REA type BI, using inocula that contained predominantly either vegetative cells or spores. Telavancin MICs (0.125–0.25 μg/ml) were 2–8-fold lower than those of vancomycin. Telavancin was bacteriostatic, reducing the inoculum by 1–2.5 log₁₀ after 24 h. No major differences occurred with the different inoculum forms or the REA types. Although telavancin MICs were 2–8 times lower than vancomycin, both demonstrated similar bacteriostatic activity against C. difficile while metronidazole was bactericidal.     

Interaction of Bacillus subtilis spores with sodium hypochlorite, sodium dichloroisocyanurate and chloramine-T

S.F. BLOOMFIELD AND M. ARTHUR. 1992. Solutions of chlorine-releasing agents (CRAs) show varying activity against Bacillus subtilis spores; sodium hypochlorite (NaOCl) shows higher activity than sodium dichloroisocyanurate (NaDCC) which is more active than chloramine-T. Investigations with coat- and cortex-extracted spores indicate that resistance to CRAs depends not only on the spore coat but also the cortex. Whereas extraction of alkali-soluble coat protein increased sensitivity to NaOCl and NaDCC, degradation of coat and cortex material was required to achieve significant activity with chloramine-T. NaOCl (in the presence and absence of NaOH) and NaDCC (in the presence of NaOH only) produced degradation of spore coat and cortes material which may be related to their rapid sporicidal action at low concentrations under these conditions. By contrast, chloramine-T produced no degradation of cortex peptidoglycan and was only effective against normal and alkali-treated spores at high concentrations, requiring extraction of peptidoglycan with urea/dithiothreitol/sodium lauryl sulphate (UDS) or UDS/lysozyme to achieve significant activity at low concentrations. Results suggest that the sporicidal action of CRAs is associated with spore coat and cortex degradation causing rehydration of the protoplast allowing diffusion to the site of action on the underlying protoplast.     

Activities of a broad-spectrum antimicrobial peptide analogue SAMP-A4-C8 and its combat against pneumonia in Staphylococcus aureus-infected mice

Antimicrobial peptides and their analogues have become substitutes for antibiotics in recent years. The antimicrobial peptide analogue SAMP-A4-C8 (n-octanoic-VRLLRRRI) with high antimicrobial activity was found in our lab. We speculate that it may kill pathogens by some lethal mechanism of action. In the present investigation, the microbicidal activities of SAMP-A4-C8 and its mechanism of action were investigated. The results demonstrated that SAMP-A4-C8 had lethal activities against Staphylococcus aureus and Candida albicans by cell disruption. Based on its microbicidal activities, we believe that it is worth further research for its potential as drug candidate. The results showed that SAMP-A4-C8, with low propensity to induce the resistance of S. aureus and C. albicans, could kill the persister cells of S. aureus and C. albicans, exhibited biofilm forming inhibition activity and preformed biofilm eradication ability against S. aureus and C. albicans, and displayed therapeutic potential on pneumonia in S. aureus-infected mice by reducing lung inflammation. The present study provided a promising drug candidate in the war against multidrug resistance.     

Interaction of Bacillus subtilis spores with sodium hypochlorite, sodium dichloroisocyanurate and chloramine-T.

Solutions of chlorine-releasing agents (CRAs) show varying activity against Bacillus subtilis spores; sodium hypochlorite (NaOCl) shows higher activity than sodium dichloroisocyanurate (NaDCC) which is more active than chloramine-T. Investigations with coat- and cortex-extracted spores indicate that resistance to CRAs depends not only on the spore coat but also the cortex. Whereas extraction of alkali-soluble coat protein increased sensitivity to NaOCl and NaDCC, degradation of coat and cortex material was required to achieve significant activity with chloramine-T. NaOCl (in the presence and absence of NaOH) and NaDCC (in the presence of NaOH only) produced degradation of spore coat and cortex material which may be related to their rapid sporicidal action at low concentrations under these conditions. By contrast, chloramine-T produced no degradation of cortex peptidoglycan and was only effective against normal and alkali-treated spores at high concentrations, requiring extraction of peptidoglycan with urea/dithiothreitol/sodium lauryl sulphate (UDS) or UDS/lysozyme to achieve significant activity at low concentrations. Results suggest that the sporicidal action of CRAs is associated with spore coat and cortex degradation causing rehydration of the protoplast allowing diffusion to the site of action on the underlying protoplast.     

Novel Strategies for Enhanced Removal of Persistent Bacillus anthracis Surrogates and Clostridium difficile Spores from Skin

Background

Removing spores of Clostridium difficile and Bacillus anthracis from skin is challenging because they are resistant to commonly used antimicrobials and soap and water washing provides only modest efficacy. We hypothesized that hygiene interventions incorporating a sporicidal electrochemically generated hypochlorous acid solution (Vashe^®) would reduce the burden of spores on skin.

Methods

Hands of volunteers were inoculated with non-toxigenic C. difficile spores or B. anthracis spore surrogates to assess the effectiveness of Vashe solution for reducing spores on skin. Reduction in spores was compared for Vashe hygiene interventions versus soap and water (control). To determine the effectiveness of Vashe solution for removal of C. difficile spores from the skin of patients with C. difficile infection (CDI), reductions in levels of spores on skin were compared for soap and water versus Vashe bed baths.

Results

Spore removal from hands was enhanced with Vashe soak (>2.5 log₁₀ reduction) versus soap and water wash or soak (~2.0 log₁₀ reduction; P <0.05) and Vashe wipes versus alcohol wipes (P <0.01). A combined approach of soap and water wash followed by soaking in Vashe removed >3.5 log₁₀ spores from hands (P <0.01 compared to washing or soaking alone). Bed baths using soap and water (N =26 patients) did not reduce the percentage of positive skin cultures for CDI patients (64% before versus 57% after bathing; P =0.5), whereas bathing with Vashe solution (N =21 patients) significantly reduced skin contamination (54% before versus 8% after bathing; P =0.0001). Vashe was well-tolerated with no evidence of adverse effects on skin.

Conclusions

Vashe was safe and effective for reducing the burden of B. anthracis surrogates and C. difficile spores on hands. Bed baths with Vashe were effective for reducing C. difficile on skin. These findings suggest a novel strategy to reduce the burden of spores on skin.     

Control of the growth of Alicyclobacillus acidoterrestris in industrialized orange juice using rosemary essential oil and nisin

The use of rosemary essential oil (RO) and its combination with nisin (RO+N) in preventing the multiplication of Alicyclobacillus acidoterrestris in orange juice was evaluated. The minimum inhibitory and bactericidal concentrations (MIC and MBC) for RO were both 125 μg ml⁻¹ while RO+N displayed a synergistic effect. The use of RO and RO+N at concentrations of 1, 4 and 8× MIC in orange juice for 96 h was evaluated in terms of their sporicidal effectiveness. With regard to the action against A. acidoterrestris spores, RO at 8× MIC was sporostatic, whereas RO+N at 1× MIC was sporicidal. Morphological changes in the structure of the micro-organism after treatment were also observed by microscopy. Furthermore, flow cytometric analysis showed that most cells were damaged or killed after treatment. In general, the antioxidant activity after addition of RO+N decreased with time. The results demonstrate that using the combination of RO and nisin can prevent the A. acidoterrestris growth in orange juice.     

CotL,a new morphogenetic spore coat protein of Clostridium difficile

The strict anaerobe Clostridium difficile is the most common cause of antibiotic-associated diarrhoea. The oxygen-resistant C. difficile spores play a central role in the infectious cycle, contributing to transmission, infection and recurrence. The spore surface layers, the coat and exosporium, enable the spores to resist physical and chemical stress. However, little is known about the mechanisms of their assembly. In this study, we characterized a new spore protein, CotL, which is required for the assembly of the spore coat. The cotL gene was expressed in the mother cell compartment under the dual control of the RNA polymerase sigma factors, σ^E and σ^K. CotL was localized in the spore coat, and the spores of the cotL mutant had a major morphologic defect at the level of the coat/exosporium layers. Therefore, the mutant spores contained a reduced amount of several coat/exosporium proteins and a defect in their localization in sporulating cells. Finally, cotL mutant spores were more sensitive to lysozyme and were impaired in germination, a phenotype likely to be associated with the structurally altered coat. Collectively, these results strongly suggest that CotL is a morphogenetic protein essential for the assembly of the spore coat in C. difficile.