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1.  Evaluation of a hand-held far-ultraviolet radiation device for decontamination of Clostridium difficile and other healthcare-associated pathogens 
BMC Infectious Diseases  2012;12:120.
Background
Environmental surfaces play an important role in transmission of healthcare-associated pathogens. There is a need for new disinfection methods that are effective against Clostridium difficile spores, but also safe and rapid. The Sterilray™ Disinfection Wand device is a hand-held room decontamination technology that utilizes far-ultraviolet radiation (185-230 nm) to kill pathogens.
Methods
We examined the efficacy of disinfection using the Sterilray device in the laboratory, in rooms of hospitalized patients, and on surfaces outside of patient rooms (i.e. keyboards and portable medical equipment). Cultures for C. difficile, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE) were collected from commonly-touched surfaces before and after use of the Sterilray device.
Results
On inoculated surfaces in the laboratory, application of the Sterilray device at a radiant dose of 100 mJ/cm2 for ~ 5 seconds consistently reduced recovery of C. difficile spores by 4.4 CFU log10, MRSA by 5.4 log10CFU and of VRE by 6.9 log10CFU. A >3 log10 reduction of MRSA and VRE was achieved in ~2 seconds at a lower radiant dose, but killing of C. difficile spores was significantly reduced. On keyboards and portable medical equipment that were inoculated with C. difficile spores, application of the Sterilray device at a radiant dose of 100 mJ/cm2 for ~ 5 seconds reduced contamination by 3.2 log10CFU. However, the presence of organic material reduced the lethal effect of the far-UV radiation. In hospital rooms that were not pre-cleaned, disinfection with the Sterilray device significantly reduced the frequency of positive C. difficile and MRSA cultures (P =0.007).
Conclusions
The Sterilray™ Disinfection Wand is a novel environmental disinfection technology that rapidly kills C. difficile spores and other healthcare-associated pathogens on surfaces. However, the presence of organic matter reduces the efficacy of far-UV radiation, possibly explaining the more modest results observed on surfaces in hospital rooms that were not pre-cleaned.
doi:10.1186/1471-2334-12-120
PMCID: PMC3419611  PMID: 22591268
2.  Evaluation of an automated ultraviolet radiation device for decontamination of Clostridium difficile and other healthcare-associated pathogens in hospital rooms 
BMC Infectious Diseases  2010;10:197.
Background
Environmental surfaces play an important role in transmission of healthcare-associated pathogens. There is a need for new disinfection methods that are effective against Clostridium difficile spores, but also safe, rapid, and automated.
Methods
The Tru-D™ Rapid Room Disinfection device is a mobile, fully-automated room decontamination technology that utilizes ultraviolet-C irradiation to kill pathogens. We examined the efficacy of environmental disinfection using the Tru-D device in the laboratory and in rooms of hospitalized patients. Cultures for C. difficile, methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin-resistant Enterococcus (VRE) were collected from commonly touched surfaces before and after use of Tru-D.
Results
On inoculated surfaces, application of Tru-D at a reflected dose of 22,000 μWs/cm2 for ~45 minutes consistently reduced recovery of C. difficile spores and MRSA by >2-3 log10 colony forming units (CFU)/cm2 and of VRE by >3-4 log10 CFU/cm2. Similar killing of MRSA and VRE was achieved in ~20 minutes at a reflected dose of 12,000 μWs/cm2, but killing of C. difficile spores was reduced. Disinfection of hospital rooms with Tru-D reduced the frequency of positive MRSA and VRE cultures by 93% and of C. difficile cultures by 80%. After routine hospital cleaning of the rooms of MRSA carriers, 18% of sites under the edges of bedside tables (i.e., a frequently touched site not easily amenable to manual application of disinfectant) were contaminated with MRSA, versus 0% after Tru-D (P < 0.001). The system required <5 minutes to set up and did not require continuous monitoring.
Conclusions
The Tru-D Rapid Room Disinfection device is a novel, automated, and efficient environmental disinfection technology that significantly reduces C. difficile, VRE and MRSA contamination on commonly touched hospital surfaces.
doi:10.1186/1471-2334-10-197
PMCID: PMC2910020  PMID: 20615229
3.  Analysis of the sporicidal activity of chlorine dioxide disinfectant against Bacillus anthracis (Sterne strain) 
Summary
Routine surface decontamination is an essential hospital and laboratory procedure, but the list of effective, noncorrosive disinfectants that kill spores is limited. We investigated the sporicidal potential of an aqueous chlorine dioxide solution and encountered some unanticipated problems. Quantitative bacteriological culture methods were used to determine the log10 reduction of Bacillus anthracis (Sterne strain) spores following 3 min exposure to various concentrations of aqueous chlorine dioxide solutions at room temperature in sealed tubes, as well as spraying onto plastic and stainless steel surfaces in a biological safety cabinet. Serial 10-fold dilutions of the treated spores were then plated on 5% sheep blood agar plates, and the survivor colonies were enumerated. Disinfection of spore suspensions with aqueous chlorine dioxide solution in sealed microfuge tubes was highly effective, reducing the viable spore counts by 8 log10 in only 3 min. By contrast, the process of spraying or spreading the disinfectant onto surfaces resulted in only a 1 log10 kill because the chlorine dioxide gas was rapidly vaporised from the solutions. Full potency of the sprayed aqueous chlorine dioxide solution was restored by preparing the chlorine dioxide solution in 5% bleach (0.3% sodium hypochlorite). The volatility of chlorine dioxide can cause treatment failures that constitute a serious hazard for unsuspecting users. Supplementation of the chlorine dioxide solution with 5% bleach (0.3% sodium hypochlorite) restored full potency and increased stability for one week.
doi:10.1016/j.jhin.2009.09.017
PMCID: PMC2814968  PMID: 20061062
Bacillus anthracis; Chlorine dioxide; Disinfectant; Sodium hypochlorite; Spores
4.  Triggering Germination Represents a Novel Strategy to Enhance Killing of Clostridium difficile Spores 
PLoS ONE  2010;5(8):e12285.
Background
Clostridium difficile is an anaerobic, spore-forming bacterium that is the most common cause of healthcare-associated diarrhea in developed countries. Control of C. difficile is challenging because the spores are resistant to killing by alcohol-based hand hygiene products, antimicrobial soaps, and most disinfectants. Although initiation of germination has been shown to increase susceptibility of spores of other bacterial species to radiation and heat, it was not known if triggering of germination could be a useful strategy to increase susceptibility of C. difficile spores to radiation or other stressors.
Principal Findings
Here, we demonstrated that exposure of dormant C. difficile spores to a germination solution containing amino acids, minerals, and taurocholic acid resulted in initiation of germination in room air. Germination of spores in room air resulted in significantly enhanced killing by ultraviolet-C (UV-C) radiation and heat. On surfaces in hospital rooms, application of germination solution resulted in enhanced eradication of spores by UV-C administered by an automated room decontamination device. Initiation of germination under anaerobic, but not aerobic, conditions resulted in increased susceptibility to killing by ethanol, suggesting that exposure to oxygen might prevent spores from progressing fully to outgrowth. Stimulation of germination also resulted in reduced survival of spores on surfaces in room air, possibly due to increased susceptibility to stressors such as oxygen and desiccation.
Conclusions
Taken together, these data demonstrate that stimulation of germination could represent a novel method to enhance killing of spores by UV-C, and suggest the possible application of this strategy as a means to enhance killing by other agents.
doi:10.1371/journal.pone.0012285
PMCID: PMC2924393  PMID: 20808871
5.  Performance of exhaust-protective (class I) biological 'safety' cabinets. 
Journal of Clinical Pathology  1979;32(6):576-583.
Nineteen open-fronted (class I) safety cabinets were subjected to spore containment and airflow tests, which suggested that a flow of 0.75 m/s was the minimum required for safe operation; further tests on three of the cabinets were repeated at different air speeds and confirmed this. The airflow is required to overcome the effect of laboratory or external air currents. Contamination of surfaces (including the operator's hands) by aerosols liberated inside the cabinets was investigated and found to depend more on turbulence within the cabinet (as from a restricted front opening) than on the number of air changes. The findings suggest that the product protection provided by a well-designed class I cabinet might equal that of a class II (laminar flow) unit.
Images
PMCID: PMC1145742  PMID: 469014
6.  Decontamination of Bacillus subtilis var. niger spores on selected surfaces by chlorine dioxide gas*  
Objective: Chlorine dioxide (CD) gas has been used as a fumigant in the disinfection of biosafety laboratories. In this study, some experiments were conducted to assess the inactivation of spores inoculated on six materials [stainless steel (SS), painted steel (PS), polyvinyl chlorid (PVC), polyurethane (PU), glass (GS), and cotton cloth (CC)] by CD gas. The main aims of the study were to determine the sporicidal efficacy of CD gas and the effect of prehumidification before decontamination on sporicidal efficacy. Methods: Material coupons (1.2 cm diameter of SS, PS, and PU; 1.0 cm×1.0 cm for PVC, GS, and CC) were contaminated with 10 μl of Bacillus subtilis var. niger (ATCC 9372) spore suspension in mixed organic burden and then dried in a biosafety cabinet for 12 h. The spores were recovered by soaking the coupons in 5 ml of extraction liquid for 1 h and then vortexing the liquid for 1 min. Results: The log reductions in spore numbers on inoculated test materials exposed to CD gas [0.080% (volume ratio, v/v) for 3 h] were in the range of from 1.80 to 6.64. Statistically significant differences were found in decontamination efficacies on test material coupons of SS, PS, PU, and CC between with and without a 1-h prehumidification treatment. With the extraction method, there were no statistically significant differences in the recovery ratios between the porous and non-porous materials. Conclusions: The results reported from this study could provide information for developing decontamination technology based on CD gas for targeting surface microbial contamination.
doi:10.1631/jzus.B1100289
PMCID: PMC3323940  PMID: 22467366
Chlorine dioxide gas; Bacillus subtilis var. niger spores; Decontamination
7.  Use of Purified Clostridium difficile Spores To Facilitate Evaluation of Health Care Disinfection Regimens▿ †  
Applied and Environmental Microbiology  2010;76(20):6895-6900.
Clostridium difficile is a major cause of antibiotic-associated diarrheal disease in many parts of the world. In recent years, distinct genetic variants of C. difficile that cause severe disease and persist within health care settings have emerged. Highly resistant and infectious C. difficile spores are proposed to be the main vectors of environmental persistence and host transmission, so methods to accurately monitor spores and their inactivation are urgently needed. Here we describe simple quantitative methods, based on purified C. difficile spores and a murine transmission model, for evaluating health care disinfection regimens. We demonstrate that disinfectants that contain strong oxidizing active ingredients, such as hydrogen peroxide, are very effective in inactivating pure spores and blocking spore-mediated transmission. Complete inactivation of 106 pure C. difficile spores on indicator strips, a six-log reduction, and a standard measure of stringent disinfection regimens require at least 5 min of exposure to hydrogen peroxide vapor (HPV; 400 ppm). In contrast, a 1-min treatment with HPV was required to disinfect an environment that was heavily contaminated with C. difficile spores (17 to 29 spores/cm2) and block host transmission. Thus, pure C. difficile spores facilitate practical methods for evaluating the efficacy of C. difficile spore disinfection regimens and bringing scientific acumen to C. difficile infection control.
doi:10.1128/AEM.00718-10
PMCID: PMC2953018  PMID: 20802075
8.  Control of infection in general practice: a survey and recommendations. 
BMJ : British Medical Journal  1988;297(6640):34-36.
Twenty general practices in four areas in Britain were surveyed to establish their needs for and practices of sterilising and disinfecting equipment. Of the 327 items of equipment and instruments examined in the survey, 190 were satisfactorily decontaminated, 100 were treated in a way judged to result in doubtful decontamination, and in 37 cases treatment was considered unsatisfactory. Decontamination apparatuses (autoclaves, hot air ovens, and hot water disinfectors) were generally in good working order, but the use of chemical disinfectants was often inappropriate. Recommendations were made on appropriate methods of decontamination for various items in common use in general practice. By virtue of the large numbers of patients treated by general practitioners there is a substantial possibility of transmitting infection; having appropriate methods for decontaminating instruments and equipment is therefore imperative.
PMCID: PMC1834143  PMID: 3408909
9.  Containment of Microbial Aerosols in a Microbiological Safety Cabinet 
Applied Microbiology  1968;16(8):1225-1229.
A microbiological safety cabinet was evaluated to determine conditions under which microorganisms might escape. Tests were conducted under three cabinet-closure conditions, various airflow velocities, and different laboratory operations, with 105, 1.1 × 105, and 106 microorganisms per cubic foot of cabinet space released per min for 5 min. The data revealed that (i) escape of a human infectious dose is possible when the cabinet is used with the glove panel off; (ii) the number of organisms that escaped from the cabinet increased with a decrease in air velocity; and (iii) an increase in the number of laboratory operations resulted in an increase in the number of organisms that escaped. Thus, when the glove panel was off, the cabinet was only safe for operations that released a small number of microorganisms into the cabinet, whereas the cabinet was safe for operations of significantly greater hazard when used with the glove panel on but with the gloves unattached.
PMCID: PMC547623  PMID: 4877500
10.  Effect of Inoculation Techniques and Relative Humidity on the Growth of Molds on the Surfaces of Yellow Layer Cakes 
Four inoculation techniques were compared for initiation of growth on cake surfaces: spot, air cabinet, spray (atomizer), and talc addition methods. Molds were isolated from commercial cakes and were identified as Aspergillus sydowii, Aspergillus ochraceus, Penicillium funiculosum, and Eurotium herbariorum. Cake surfaces were inoculated with mold spores and incubated under three equilibrium relative humidity (ERH) levels: 97, 85, and 75%. Random contamination by spores in a ventilated air cabinet was the simplest method of inoculation, but standard deviations in the inoculation rates (20% on a relative scale) were almost twice those observed with the other methods. The spot method was the most reproducible. Cake samples inoculated in the air cabinet had colony counts 10 times lower than those obtained for potato dextrose agar plates at 97% ERH, which was not the case with the spray and talc methods. Growth of molds was much slower in the samples incubated in 75% relative humidity, with all methods. Colony counts were generally similar in systems adjusted at 85 to 97% ERH but were lower for samples incubated at 75% ERH. In comparisons of the shelf life estimates obtained by the various inoculation methods, a correlation coefficient (r2) of 0.70 was obtained between the spot method and the other methods of inoculation, while talc, air cabinet, and spray shelf life data were correlated better (r2 ≈ 0.97). The spot method appeared to be the method of choice in consideration of ease of use, precision, and the ability to enable the study of the effects of the environment on mold-free shelf life as well as on the rate of growth of molds on cakes.
PMCID: PMC124692  PMID: 16349479
11.  Bile Salts and Glycine as Cogerminants for Clostridium difficile Spores▿  
Journal of Bacteriology  2008;190(7):2505-2512.
Spore formation by Clostridium difficile is a significant obstacle to overcoming hospital-acquired C. difficile-associated disease. Spores are resistant to heat, radiation, chemicals, and antibiotics, making a contaminated environment difficult to clean. To cause disease, however, spores must germinate and grow out as vegetative cells. The germination of C. difficile spores has not been examined in detail. In an effort to understand the germination of C. difficile spores, we characterized the response of C. difficile spores to bile. We found that cholate derivatives and the amino acid glycine act as cogerminants. Deoxycholate, a metabolite of cholate produced by the normal intestinal flora, also induced germination of C. difficile spores but prevented the growth of vegetative C. difficile. A model of resistance to C. difficile colonization mediated by the normal bacterial flora is proposed.
doi:10.1128/JB.01765-07
PMCID: PMC2293200  PMID: 18245298
12.  Reduction of Clostridium Difficile and vancomycin-resistant Enterococcus contamination of environmental surfaces after an intervention to improve cleaning methods 
Background
Contaminated environmental surfaces may play an important role in transmission of some healthcare-associated pathogens. In this study, we assessed the adequacy of cleaning practices in rooms of patients with Clostridium difficile-associated diarrhea (CDAD) and vancomycin-resistant Enterococcus (VRE) colonization or infection and examined whether an intervention would result in improved decontamination of surfaces.
Methods
During a 6-week period, we cultured commonly touched surfaces (i.e. bedrails, telephones, call buttons, door knobs, toilet seats, and bedside tables) in rooms of patients with CDAD and VRE colonization or infection before and after housekeeping cleaning, and again after disinfection with 10% bleach performed by the research staff. After the housekeeping staff received education and feedback, additional cultures were collected before and after housekeeping cleaning during a 10-week follow-up period.
Results
Of the 17 rooms of patients with VRE colonization or infection, 16 (94%) had one or more positive environmental cultures before cleaning versus 12 (71%) after housekeeping cleaning (p = 0.125), whereas none had positive cultures after bleach disinfection by the research staff (p < 0.001). Of the 9 rooms of patients with CDAD, 100% had positive cultures prior to cleaning versus 7 (78%) after housekeeping cleaning (p = 0.50), whereas only 1 (11%) had positive cultures after bleach disinfection by research staff (p = 0.031). After an educational intervention, rates of environmental contamination after housekeeping cleaning were significantly reduced.
Conclusion
Our findings provide additional evidence that simple educational interventions directed at housekeeping staff can result in improved decontamination of environmental surfaces. Such interventions should include efforts to monitor cleaning and disinfection practices and provide feedback to the housekeeping staff.
doi:10.1186/1471-2334-7-61
PMCID: PMC1906786  PMID: 17584935
13.  Use of Hydrogen Peroxide Vapor for Deactivation of Mycobacterium tuberculosis in a Biological Safety Cabinet and a Room▿  
Journal of Clinical Microbiology  2006;45(3):810-815.
Mycobacterium tuberculosis is an important human pathogen that is routinely cultured in clinical and research laboratories. M. tuberculosis can contaminate surfaces and is highly resistant to disinfection. We investigated whether hydrogen peroxide vapor (HPV) is effective for the deactivation of M. tuberculosis on experimentally contaminated surfaces in a biological safety cabinet (BSC) and a room. Biological indicators (BIs) consisting of an ∼3-log10 inoculum of M. tuberculosis on stainless steel discs and a 6-log10 inoculum of Geobacillus stearothermophilus were exposed to HPV in BSC time course experiments and at 10 locations during room experiments. In three separate BSC experiments, M. tuberculosis BIs were transferred to growth media at 15-min intervals during a 180-min HPV exposure period. No M. tuberculosis BIs grew following 30 min of HPV exposure. In three separate room experiments, M. tuberculosis and G. stearothermophilus BIs were exposed to HPV for 90, 120, and 150 min, respectively. BIs for both microorganisms were deactivated in all 10 locations following 90 min of HPV exposure. HPV provides an alternative to traditional decontamination methods, such as formaldehyde fumigation, for laboratories and other areas contaminated with M. tuberculosis.
doi:10.1128/JCM.01797-06
PMCID: PMC1829131  PMID: 17166957
14.  Mass Airflow Cabinet for Control of Airborne Infection of Laboratory Rodents 
Applied Microbiology  1973;26(2):167-172.
A mass airflow cabinet for handling and housing of laboratory rodents has been developed and tested. The unit consists of a high-efficiency particulate air filter and uniform distribution of air at a vertical velocity of 19 cm per s. Animals are maintained without bedding in mesh-bottomed cages that rest on rollers for rotation inside the cabinet. There is an air barrier of 90 cm per s separating the cabinet air from room air. Sampling for airborne bacteria yielded an average of 0.03 colony-forming units (CFU) per ft3 of air inside the cabinet, whereas 28.8 CFU per ft3 was simultaneously detected outside the cabinet during housekeeping, a reduction of almost three logs. The efficiency of the air barrier was tested by aerosolization of T3 phage. When phage was aerosolized 5 cm outside the cabinet, no phage could be detected 5 cm inside when the fans were operating; with the fans off an average of 1.6 × 104 plaque-forming units (PFU) per ft3 was detected in six tests. Aerosolization of phage inside the cabinet yielded an average of 9 × 10 PFU per ft3 outside; an average of 4.1 × 106 PFU per ft3 were detected with the fans not in operation, a reduction of more than four logs. In-use studies on effectiveness showed that the cabinet significantly reduced the incidence of mice originally titer-free to Reo-3 virus. Hemagglutination inhibition antibodies to Reo-3 were detected in 9/22 (42%) mice housed in a conventionally ventilated animal laboratory while no seroconversion was detected in any of 22 mice housed in the mass air flow cabinet in the same laboratory.
Images
PMCID: PMC379745  PMID: 4355261
15.  Improved eradication of Clostridium difficile spores from toilets of hospitalized patients using an accelerated hydrogen peroxide as the cleaning agent 
BMC Infectious Diseases  2010;10:268.
Background
C. difficle spores in the environment of patients with C. difficile associated disease (CDAD) are difficult to eliminate. Bleach (5000 ppm) has been advocated as an effective disinfectant for the environmental surfaces of patients with CDAD. Few alternatives to bleach for non-outbreak conditions have been evaluated in controlled healthcare studies.
Methods
This study was a prospective clinical comparison during non-outbreak conditions of the efficacy of an accelerated hydrogen peroxide cleaner (0.5% AHP) to the currently used stabilized hydrogen peroxide cleaner (0.05% SHP at manufacturer recommended use-dilution) with respect to spore removal from toilets in a tertiary care facility. The toilets used by patients who had diarrhea with and without C. difficile associated disease (CDAD) were cultured for C. difficile and were monitored using an ultraviolet mark (UVM) to assess cleaning compliance on a daily basis 5 days per week. A total of 243 patients and 714 samples were analysed. The culture results were included in the analysis only if the UVM audit from the same day confirmed that the toilet had been cleaned.
Results
Our data demonstrated that the efficacy of spore killing is formulation specific and cannot be generalized. The OxivirTB® AHP formulation resulted in statistically significantly (p = 0.0023) lower levels of toxigenic C. difficile spores in toilets of patients with CDAD compared to the SHP formulation that was routinely being used (28% vs 45% culture positive). The background level of toxigenic C. difficile spores was 10% in toilets of patients with diarrhea not due to CDAD. The UVM audit indicated that despite the enhanced twice-daily cleaning protocol for CDAD patients cleaning was not achieved on approximately 30 - 40% of the days tested.
Conclusion
Our data indicate that the AHP formulation evaluated that has some sporicidal activity was significantly better than the currently used SHP formulation. This AHP formulation provides a one-step process that significantly lowers the C. difficile spore level in toilets during non-outbreak conditions without the workplace safety concerns associated with 5000 ppm bleach.
doi:10.1186/1471-2334-10-268
PMCID: PMC2949757  PMID: 20843348
16.  First Evidence of Division and Accumulation of Viable but Nonculturable Pseudomonas fluorescens Cells on Surfaces Subjected to Conditions Encountered at Meat Processing Premises▿  
Cleaning and disinfection of open surfaces in food industry premises leave some microorganisms behind; these microorganisms build up a resident flora on the surfaces. Our goal was to explore the phenomena involved in the establishment of this biofilm. Ceramic coupons were contaminated, once only, with Pseudomonas fluorescens suspended in meat exudate incubated at 10°C. The mean adhering population after 1 day was 102 CFU·cm−2 and 103 total cells·cm−2, i.e., the total number of cells stained by DAPI (4′,6′-diamidino-2-phenylindole). The coupons were subjected daily to a cleaning product, a disinfectant, and a further soiling with exudate. The result was a striking difference between the numbers of CFU, which reached 104 CFU·cm−2, and the numbers of total cells, which reached 2 × 106 cells·cm−2 in 10 days. By using hypotheses all leading to an overestimation of the number of dead cells, we showed that the quantity of nonculturable cells (DAPI-positive cells minus CFU) observed cannot be accounted for as an accumulation of dead cells. Some nonculturable cells are therefore dividing on the surface, although cell division is unable to continue to the stage of macrocolony formation on agar. The same phenomenon was observed when only a chlorinated alkaline product was used and the number of cells capable of reducing 5-cyano-2,3-ditolyl tetrazolium chloride was close to the number of total cells, confirming that most nonculturable cells are viable but nonculturable. Furthermore, the daily shock applied to the cells does not prompt them to enter a new lag phase. Since a single application of microorganisms is sufficient to produce this accumulation of cells, it appears that the phenomenon is inevitable on open surfaces in food industry premises.
doi:10.1128/AEM.02267-06
PMCID: PMC1892859  PMID: 17337551
17.  Hypervirulent Clostridium difficile PCR-Ribotypes Exhibit Resistance to Widely Used Disinfectants 
PLoS ONE  2011;6(10):e25754.
The increased prevalence of Clostridium difficile infection (CDI) has coincided with enhanced transmissibility and severity of disease, which is often linked to two distinct clonal lineages designated PCR-ribotype 027 and 017 responsible for CDI outbreaks in the USA, Europe and Asia. We assessed sporulation and susceptibility of three PCR-ribotypes; 012, 017 and 027 to four classes of disinfectants; chlorine releasing agents (CRAs), peroxygens, quaternary ammonium compounds (QAC) and biguanides. The 017 PCR-ribotype, showed the highest sporulation frequency under these test conditions. The oxidizing biocides and CRAs were the most efficacious in decontamination of C. difficile vegetative cells and spores, the efficacy of the CRAs were concentration dependent irrespective of PCR-ribotype. However, there were differences observed in the susceptibility of the PCR-ribotypes, independent of the concentrations tested for Virkon®, Newgenn®, Proceine 40® and Hibiscrub®. Whereas, for Steri7® and Biocleanse® the difference observed between the disinfectants were dependent on both PCR-ribotype and concentration. The oxidizing agent Perasafe® was consistently efficacious across all three PCR ribotypes at varying concentrations; with a consistent five Log10 reduction in spore titre. The PCR-ribotype and concentration dependent differences in the efficacy of the disinfectants in this study indicate that disinfectant choice is a factor for llimiting the survival and transmission of C. difficile spores in healthcare settings.
doi:10.1371/journal.pone.0025754
PMCID: PMC3201945  PMID: 22039420
18.  SleC Is Essential for Germination of Clostridium difficile Spores in Nutrient-Rich Medium Supplemented with the Bile Salt Taurocholate ▿  
Journal of Bacteriology  2009;192(3):657-664.
Clostridium difficile is the major cause of infectious diarrhea and a major burden to health care services. The ability of this organism to form endospores plays a pivotal role in infection and disease transmission. Spores are highly resistant to many forms of disinfection and thus are able to persist on hospital surfaces and disseminate infection. In order to cause disease, the spores must germinate and the organism must grow vegetatively. Spore germination in Bacillus is well understood, and genes important for this process have recently been identified in Clostridium perfringens; however, little is known about C. difficile. Apparent homologues of the spore cortex lytic enzyme genes cwlJ and sleB (Bacillus subtilis) and sleC (C. perfringens) are present in the C. difficile genome, and we describe inactivation of these homologues in C. difficile 630Δerm and a B1/NAP1/027 clinical isolate. Spores of a sleC mutant were unable to form colonies when germination was induced with taurocholate, although decoated sleC spores formed the same number of heat-resistant colonies as the parental control, even in the absence of germinants. This suggests that sleC is absolutely required for conversion of spores to vegetative cells, in contrast to CD3563 (a cwlJ/sleB homologue), inactivation of which had no effect on germination and outgrowth of C. difficile spores under the same conditions. The B1/NAP1/027 strain R20291 was found to sporulate more slowly and produce fewer spores than 630Δerm. Furthermore, fewer R20291 spores germinated, indicating that there are differences in both sporulation and germination between these epidemic and nonepidemic C. difficile isolates.
doi:10.1128/JB.01209-09
PMCID: PMC2812441  PMID: 19933358
19.  A test system for the biological safety cabinet 
Journal of Clinical Pathology  1974;27(7):585-589.
A simple, cheap and readily available test system for biological safety cabinets is described. It depends on the containment of an aerosol of Bacillus subtilis spores generated in a BIRD micronebulizer and the measurement of air flows with an anemometer. The system was set up to survey new equipment but equally valuable results have been obtained from tests during use. New units were often badly installed and used equipment was poorly maintained. It is suggested that any department which has a need for a biological safety cabinet must be in a position to test its function.
Images
PMCID: PMC475402  PMID: 4214380
20.  Efficacy of Dry Mist of Hydrogen Peroxide (DMHP) against Mycobacterium tuberculosis and use of DMHP for Routine Decontamination of Biosafety Level 3 Laboratories▿  
Journal of Clinical Microbiology  2008;46(9):2955-2958.
Mycobacterium tuberculosis is a major cause of morbidity and mortality worldwide and is becoming a greater concern due to the development of multidrug-resistant strains. M. tuberculosis can contaminate rooms, medical equipment, and research laboratories and has the propensity to be highly resistant to decontamination. The aim of this study was to determine the efficacy of room disinfection with a dry mist of hydrogen peroxide (DMHP) in a biosafety level 3 laboratory in the event of contamination with M. tuberculosis. The biological indicators (BIs) were comprised of presterilized cotton tissues on which amounts of about 107 CFU/ml of M. tuberculosis H37Ra were dried. The device (Sterinis; Gloster Sante Europe) provided a DMHP of 5% hydrogen peroxide during 25 min. Three experiments were performed. The viable bacteria were reduced by values of more than 5 log10, and no colony grew from any BI. In conclusion, DMHP shows promise as an effective and safe alternative to the currently used formaldehyde.
doi:10.1128/JCM.00250-08
PMCID: PMC2546749  PMID: 18632916
21.  An evaluation of the infection control potential of a UV clinical podiatry unit 
Background
Infection control is a key issue in podiatry as it is in all forms of clinical practice. Airborne contamination may be particularly important in podiatry due to the generation of particulates during treatment. Consequently, technologies that prevent contamination in podiatry settings may have a useful role. The aims of this investigation were twofold, firstly to determine the ability of a UV cabinet to protect instruments from airborne contamination and secondly to determine its ability to remove microbes from contaminated surfaces and instruments.
Method
A UV instrument cabinet was installed in a University podiatry suite. Impact samplers and standard microbiological techniques were used to determine the nature and extent of microbial airborne contamination. Sterile filters were used to determine the ability of the UV cabinet to protect exposed surfaces. Artificially contaminated instruments were used to determine the ability of the cabinet to remove microbial contamination.
Results
Airborne bacterial contamination was dominated by Gram positive cocci including Staphylococcus aureus. Airborne fungal levels were much lower than those observed for bacteria. The UV cabinet significantly reduced (p < 0.05) the observed levels of airborne contamination. When challenged with contaminated instruments the cabinet was able to reduce microbial levels by between 60% to 100% with more complex instruments e.g. clippers, remaining contaminated.
Conclusions
Bacterial airborne contamination is a potential infection risk in podiatry settings due to the presence of S. aureus. The use of a UV instrument cabinet can reduce the risk of contamination by airborne microbes. The UV cabinet tested was unable to decontaminate instruments and as such could pose an infection risk if misused.
doi:10.1186/1757-1146-7-17
PMCID: PMC3942763  PMID: 24576315
Infection control; UV; Bacteria; Fungi; Dermatophytes; Contamination
22.  Aerial Dissemination of Clostridium difficile spores 
Background
Clostridium difficile-associated diarrhoea (CDAD) is a frequently occurring healthcare-associated infection, which is responsible for significant morbidity and mortality amongst elderly patients in healthcare facilities. Environmental contamination is known to play an important contributory role in the spread of CDAD and it is suspected that contamination might be occurring as a result of aerial dissemination of C. difficile spores. However previous studies have failed to isolate C. difficile from air in hospitals. In an attempt to clarify this issue we undertook a short controlled pilot study in an elderly care ward with the aim of culturing C. difficile from the air.
Methods
In a survey undertaken during February (two days) 2006 and March (two days) 2007, air samples were collected using a portable cyclone sampler and surface samples collected using contact plates in a UK hospital. Sampling took place in a six bedded elderly care bay (Study) during February 2006 and in March 2007 both the study bay and a four bedded orthopaedic bay (Control). Particulate material from the air was collected in Ringer's solution, alcohol shocked and plated out in triplicate onto Brazier's CCEY agar without egg yolk, but supplemented with 5 mg/L of lysozyme. After incubation, the identity of isolates was confirmed by standard techniques. Ribotyping and REP-PCR fingerprinting were used to further characterise isolates.
Results
On both days in February 2006, C. difficile was cultured from the air with 23 samples yielding the bacterium (mean counts 53 – 426 cfu/m3 of air). One representative isolate from each of these was characterized further. Of the 23 isolates, 22 were ribotype 001 and were indistinguishable on REP-PCR typing. C. difficile was not cultured from the air or surfaces of either hospital bay during the two days in March 2007.
Conclusion
This pilot study produced clear evidence of sporadic aerial dissemination of spores of a clone of C. difficile, a finding which may help to explain why CDAD is so persistent within hospitals and difficult to eradicate. Although preliminary, the findings reinforce concerns that current C. difficile control measures may be inadequate and suggest that improved ward ventilation may help to reduce the spread of CDAD in healthcare facilities.
doi:10.1186/1471-2334-8-7
PMCID: PMC2245947  PMID: 18218089
23.  In-hospital source of airborne Penicillium species spores. 
Between 16 July and 1 October 1984, prospectively monitored corridor air samples from a bone marrow transplant station revealed a marked increase in airborne thermotolerant Penicillium spores. Simultaneous cultures of outside air showed lower spore counts, which were unchanged before, during, and after the corridor outburst, establishing that the source was within the hospital. Although the corridor was equipped with recirculating high-efficiency particulate air filtration units which provided 16 air changes per h, the mean corridor air count rose to 64.4 thermotolerant Penicillium CFU/m3 during the outburst period. The in-hospital source was ultimately traced to rotting cabinet wood enclosing a sink with leaking pipes in the medication room. It produced approximately 5.5 X 10(5) thermotolerant Penicillium CFU/h. In a patient room supplied by corridor air, an in-room recirculating high-efficiency particulate air filter reduced the mean thermotolerant Penicillium count to 2.2 CFU/m3. No patient illness or colonization occurred as a result of this event, although the cabinet wood, after sterilization, was shown to sustain abundant growth of Aspergillus fumigatus and Aspergillus flavus. Wet organic substrates should be avoided in hospital areas with immunosuppressed patients.
Images
PMCID: PMC265796  PMID: 3539981
24.  Decontamination Options for Bacillus anthracis-Contaminated Drinking Water Determined from Spore Surrogate Studies ▿  
Applied and Environmental Microbiology  2010;76(19):6631-6638.
Five parameters were evaluated with surrogates of Bacillus anthracis spores to determine effective decontamination alternatives for use in a contaminated drinking water supply. The parameters were as follows: (i) type of Bacillus spore surrogate (B. thuringiensis or B. atrophaeus), (ii) spore concentration in suspension (102 and 106 spores/ml), (iii) chemical characteristics of the decontaminant (sodium dichloro-S-triazinetrione dihydrate [Dichlor], hydrogen peroxide, potassium peroxymonosulfate [Oxone], sodium hypochlorite, and VirkonS), (iv) decontaminant concentration (0.01% to 5%), and (v) exposure time to decontaminant (10 min to 1 h). Results from 138 suspension tests with appropriate controls are reported. Hydrogen peroxide at a concentration of 5% and Dichlor or sodium hypochlorite at a concentration of 2% were highly effective at spore inactivation regardless of spore type tested, spore exposure time, or spore concentration evaluated. This is the first reported study of Dichlor as an effective decontaminant for B. anthracis spore surrogates. Dichlor's desirable characteristics of high oxidation potential, high level of free chlorine, and a more neutral pH than that of other oxidizers evaluated appear to make it an excellent alternative. All three oxidizers were effective against B. atrophaeus spores in meeting the EPA biocide standard of greater than a 6-log kill after a 10-min exposure time and at lower concentrations than typically reported for biocide use. Solutions of 5% VirkonS and Oxone were less effective as decontaminants than other options evaluated in this study and did not meet the EPA's efficacy standard for a biocide, although they were found to be as effective for concentrations of 102 spores/ml. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult.
doi:10.1128/AEM.01136-10
PMCID: PMC2950457  PMID: 20709855
25.  Observations on a new disinfectant 
Journal of Clinical Pathology  1969;22(4):496-500.
A disinfectant formula (Resiguard) containing picloxydine digluconate, benzalkonium chloride, and a detergent was examined for antibacterial activity.
Studies in vitro with simulated organic contamination were followed by realistic in-use tests employing items of anaesthetic and urological equipment heavily infected with Pseudomonas aeruginosa and contaminated with blood. These showed that a dilution of 1/80 was reliably bactericidal to this resistant organism.
It is concluded that Resiguard is a valuable disinfectant which may reliably be employed in the cleaning and disinfection of items of delicate, non-autoclavable, hospital equipment.
PMCID: PMC474220  PMID: 4978998

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