Lipopolysaccharide structure modulates cationic biocide susceptibility and crystalline biofilm formation in Proteus mirabilis.

Chlorhexidine (CHD) is a cationic biocide used ubiquitously in healthcare settings. Proteus mirabilis, an important pathogen of the catheterized urinary tract, and isolates of this species are often described as "resistant" to CHD-containing products used for catheter infection control. To identify the mechanisms underlying reduced CHD susceptibility in P. mirabilis, we subjected the CHD tolerant clinical isolate RS47 to random transposon mutagenesis and screened for mutants with reduced CHD minimum inhibitory concentrations (MICs). One mutant recovered from these screens (designated RS47-2) exhibited ~ 8-fold reduction in CHD MIC. Complete genome sequencing of RS47-2 showed a single mini-Tn5 insert in the waaC gene involved in lipopolysaccharide (LPS) inner core biosynthesis. Phenotypic screening of RS47-2 revealed a significant increase in cell surface hydrophobicity and serum susceptibility compared to the wildtype, and confirmed defects in LPS production congruent with waaC inactivation. Disruption of waaC was also associated with increased susceptibility to a range of other cationic biocides but did not affect susceptibility to antibiotics tested. Complementation studies showed that repression of smvA efflux activity in RS47-2 further increased susceptibility to CHD and other cationic biocides, reducing CHD MICs to values comparable with the most CHD susceptible isolates characterized. The formation of crystalline biofilms and blockage of urethral catheters was also significantly attenuated in RS47-2. Taken together, these data show that aspects of LPS structure and upregulation of the smvA efflux system function in synergy to modulate susceptibility to CHD and other cationic biocides, and that LPS structure is also an important factor in P. mirabilis crystalline biofilm formation.

De-repression of the smvA efflux system arises in clinical isolates of Proteus mirabilis and reduces susceptibility to chlorhexidine and other biocides.

Proteus mirabilis is a common pathogen of the catheterised urinary tract and often described as intrinsically resistant to the biocide chlorhexidine (CHD). Here we demonstrate that de-repression of the smvA efflux system has occurred in clinical isolates of P. mirabilis and reduces susceptibility to CHD and other cationic biocides. Compared to other isolates examined, P. mirabilis RS47 exhibited a significantly higher CHD MIC (≥512 µg/ml) and significantly greater expression of smvA. Comparison of the RS47 smvA and cognate smvR repressor with sequences from other isolates, indicated that RS47 encodes an inactivated smvR. Complementation of RS47 with a functional smvR from isolate RS50a (which exhibited the lowest smvA expression and lowest CHD MIC) reduced smvA expression by ∼59-fold, and markedly lowered the MIC of CHD and other cationic biocides. Although complementation of RS47 did not reduce MICs to concentrations observed in isolate RS50a, the significantly lower polymyxin B MIC of RS50a indicated that differences in LPS structure are also a factor in P. mirabilis CHD susceptibility. To determine if exposure to CHD can select for mutations in smvR, clinical isolates with the lowest CHD MICs were adapted to grow at increasing concentrations of CHD up to 512 µg/ml. Analysis of the smvR in adapted populations indicated that mutations predicted to inactivate smvR occurred following CHD exposure in some isolates. Collectively, our data show that smvA de-repression contributes to reduced biocide susceptibility in P. mirabilis, but differences in LPS structure between strains are also likely to be an important factor.

Bacterial biofilm formation on indwelling urethral catheters.

Urethral catheters are the most commonly deployed medical devices and used to manage a wide range of conditions in both hospital and community care settings. The use of long-term catheterization, where the catheter remains in place for a period >28 days remains common, and the care of these patients is often undermined by the acquisition of infections and formation of biofilms on catheter surfaces. Particular problems arise from colonization with urease-producing species such as Proteus mirabilis, which form unusual crystalline biofilms that encrust catheter surfaces and block urine flow. Encrustation and blockage often lead to a range of serious clinical complications and emergency hospital referrals in long-term catheterized patients. Here we review current understanding of bacterial biofilm formation on urethral catheters, with a focus on crystalline biofilm formation by P. mirabilis, as well as approaches that may be used to control biofilm formation on these devices. SIGNIFICANCE AND IMPACT OF THE STUDY: Urinary catheters are the most commonly used medical devices in many healthcare systems, but their use predisposes to infection and provide ideal conditions for bacterial biofilm formation. Patients managed by long-term urethral catheterization are particularly vulnerable to biofilm-related infections, with crystalline biofilm formation by urease producing species frequently leading to catheter blockage and other serious clinical complications. This review considers current knowledge regarding biofilm formation on urethral catheters, and possible strategies for their control.

Pseudomonas aeruginosa adapts to octenidine in the laboratory and a simulated clinical setting, leading to increased tolerance to chlorhexidine and other biocides.

BACKGROUND: Octenidine is frequently used for infection prevention in neonatal and burn intensive care units, where Pseudomonas aeruginosa has caused nosocomial outbreaks. AIM: To investigate the efficacy and impact of using octenidine against P. aeruginosa. METHODS: Seven clinical isolates of P. aeruginosa were exposed to increasing concentrations of octenidine over several days. Fitness, minimum bactericidal concentrations after 1 min, 5 min and 24 h, and minimum inhibitory concentrations (MICs) of a variety of antimicrobials were measured for the parental and octenidine-adapted P. aeruginosa strains. Octenidine and chlorhexidine MICs of a population of P. aeruginosa isolated from a hospital drain trap, exposed to a diluted octenidine formulation four times daily for three months, were also tested. FINDINGS: Some planktonic cultures of P. aeruginosa survived >50% of the working concentration of an in-use octenidine formulation at the recommended exposure time. Seven strains of P. aeruginosa stably adapted following continuous exposure to increasing concentrations of octenidine. Adaptation increased tolerance to octenidine formulations and chlorhexidine up to 32-fold. In one strain, it also led to increased MICs of antipseudomonal drugs. Subsequent to continuous octenidine exposure of a multi-species community in a simulated clinical setting, up to eight-fold increased tolerance to octenidine and chlorhexidine of P. aeruginosa was also found, which was lost upon removal of octenidine. CONCLUSION: Incorrect use of octenidine formulations may lead to inadequate decontamination, and even increased tolerance of P. aeruginosa to octenidine, with resulting cross-resistance to other biocides.

Growth media and assay plate material can impact on the effectiveness of cationic biocides and antibiotics against different bacterial species.

The effectiveness of several cationic disinfectants as well as colistin and polymyxin B were assessed under different growth conditions against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa strains. These conditions included different media (MH1, MH2, TSB and LB) and plate material (polypropylene and polystyrene). Results showed that Minimum inhibitory and bactericidal concentrations (MIC/MBC) values of colistin and polymyxin B were significantly lower on polypropylene plates when compared to polystyrene plates regardless of media used. There were also differences in MIC/MBC values to certain biocides e.g. chlorhexidine and octenidine particularly for S. aureus and E. coli strains, with polypropylene again showing lower values. Other biocides appear to be mostly unaffected by plate type. Whether biocide efficacy was altered by media composition was organism dependent with S. aureus and E. coli more affected than P. aeruginosa. Lower MIC values were more commonly associated with MH2 media and higher MIC values with TSB media for both polypropylene and polystyrene plates, although there were exceptions. Results obtained for standard strains were, in general, indicative for other S. aureus, E. coli and P. aeruginosa strains tested. This study demonstrates the importance of media composition and plate material on biocide effectiveness and highlights the need for optimized disinfectant testing methods. SIGNIFICANCE AND IMPACT OF THE STUDY: There are an increasing number of reports of bacterial strains that are multi-drug resistant. The use of biocides as part of infection control is crucial in helping to combat the spread of these particular strains. Unlike for antibiotics, there are few standardized measuring techniques to understand if an isolate has become more resistant to biocides. This study demonstrates the importance of media composition and plate material on variation and reporting of susceptibility of several bacterial species to specific cationic biocides. It is a useful comparison study to highlight the need to standardize biocide susceptibility testing.

Evaluation of Novel Process Indicators for Rapid Monitoring of Hydrogen Peroxide Decontamination Processes.

Geobacillus stearothermophilus spores on stainless steel discs are routinely used as biological indicators for the validation of hydrogen peroxide bio-decontamination processes. Given ongoing concerns about the reliability and response time of biological indicators, we explored the potential for an enzyme-based approach to decontamination process evaluation. Thermostable adenylate kinase enzyme was coated onto a solid support and exposed to hydrogen peroxide vapour, in parallel with standard commercial 6-log biological indicators, during a series of vapour-phase hydrogen peroxide cycles in a flexible film isolator. The exposed biological indicators were enumerated to define the degree of kill at different time intervals and the results compared to the thermostable adenylate kinase values, as determined by measuring adenosine triphosphate produced by residual active enzyme. Both biological indicators and the thermostable adenylate kinase indicators exhibited a biphasic inactivation profile during the process. There was significant variance between individual cycles, with some cycles showing complete inactivation of the biological indicators to the limit of detection of the assay, within 6 min, whereas biological indicators in some cycles were inactivated at a time greater than 12 min. The log-kill of the biological indicators at intermediate time points were plotted and compared to the fully quantifiable measurements derived from the thermostable adenylate kinase indicators at the same time points. The results demonstrated very similar inactivation profiles for the enzyme and for the biological indicators, thus it was possible to define a relationship between relative light units measurement and biological indicator kill. This indicates that it is possible to use thermostable adenylate kinase measurement as a direct measure of vapour-phase hydrogen peroxide bio-decontamination performance, expressed in terms of log reduction. Because thermostable adenylate kinase measurement can be achieved within a few minutes of vapour-phase hydrogen peroxide cycle completion, compared with a minimum of 7 days for the evaluation of biological indicator growth, this offers a potentially valuable tool for rapid vapour-phase hydrogen peroxide bio-decontamination cycle development and subsequent re-qualification.LAY ABSTRACT: Pharmaceutical product manufacture is performed in controlled cleanroom and closed chamber environments (isolators) to reduce the risk of contamination. These environments undergo regular decontamination to control microbial contamination levels, using a range of methods, one of which is to vaporize hydrogen peroxide (a chemical disinfectant) into a gas or an aerosol and disperse it throughout the environment, killing any microorganisms present. Biological indicators, which consist of a small steel coupon carrying a population of bacterial spores that are more resistant to hydrogen peroxide than are most microorganisms, are placed within the environment, and then tested for growth following treatment to ensure the process was effective. Confirmation of growth/no growth (and therefore hydrogen peroxide cycle efficacy) can take up to 7 days, which significantly increases time and cost of developing and confirming cycle efficacy. This study tests whether a new technology which uses a robust enzyme, thermostable adenylate kinase, could be used to predict biological indicator growth. The study shows this method can be used to confirm hydrogen peroxide cycle efficacy, by predicting whether the BI is killed at a specific time point or not and results are obtained in a few minutes rather than 7 days. This potentially offers significant time and cost benefits.

Varying activity of chlorhexidine-based disinfectants against Klebsiella pneumoniae clinical isolates and adapted strains.

BACKGROUND: Control of multi-drug-resistant (MDR) organisms relies increasingly on the use of biocides, including chlorhexidine, to limit the risk of infection. The concentration and formulation of chlorhexidine can vary hugely between products. AIM: To establish the activity of chlorhexidine and in-use chlorhexidine formulations against 14 clinical Klebsiella pneumoniae strains isolated before and since the use of chlorhexidine became routine, and strains that have adapted following sublethal chlorhexidine exposure. METHODS: Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of five chlorhexidine-containing formulations were measured at 5min, 15min, 30min and 24h for the panel of K. pneumoniae strains. FINDINGS: After 5min, MBCs of five formulations varied from 0.006 to >50% working concentration (WC) or from 78 to 2500µg/mL chlorhexidine. For one formulation, MBCs were >50% WC for five of the 14 strains, and for another formulation, four of the 14 strains could resist 25% WC. NCTC 13368 was consistently most tolerant to chlorhexidine, whereas the strains isolated before the use of chlorhexidine became routine were more sensitive. One pre-chlorhexidine era and five modern strains increased MICs up to 16-fold following exposure to sublethal concentrations of chlorhexidine. A hand disinfectant with MBCs of 0.39% WC for all six of the wild-type strains, had MBCs of 50% WC for the chlorhexidine-adapted strains. CONCLUSION: Not all chlorhexidine formulations kill MDR K. pneumoniae after the recommended exposure time. Activity, especially against chlorhexidine-adapted strains, depends on additional ingredients. Careful formulation of chlorhexidine products is therefore important to maintain and enhance the activity of chlorhexidine products, and avoid potential breakdown in infection control.

Establishment of a multi-species biofilm model to evaluate chlorhexidine efficacy.

BACKGROUND: Chronic infections, for example, diabetic foot ulcers, have a large impact in terms of patient morbidity and mortality. These wounds are characterized by complex polymicrobial communities of bacteria, which may include a number of difficult-to-eradicate multidrug-resistant pathogens. AIM: To establish a multi-species biofilm model to test the efficacy of chlorhexidine and chlorhexidine-containing formulas in eradication of polymicrobial biofilms. METHODS: A Centers for Disease Control and Prevention bioreactor was used to establish a multi-species biofilm incorporating Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Enterococcus faecalis with equal numbers of each pathogen. This model was used to test the effectiveness of chlorhexidine at controlling the pre-formed biofilm. FINDINGS: Chlorhexidine digluconate (CHD) was added to the bioreactor at a range of concentrations. K. pneumoniae and P. aeruginosa survived within multi-species biofilms, up to and including 4% CHD, whereas S. aureus was reduced to below the level of detection at 1%. Wiping the biofilm-containing coupons from the bioreactor with chlorhexidine-containing medical wipes resulted in >3 to <4log10 reduction after 24h, for all species. When the coupons were embedded in a simulated wound bed, formed in an agar plate, CHD-containing medical dressings completely eliminated S. aureus (>8log10 reduction), but had minimal effect (<3log10) against the other species tested. CONCLUSION: The study demonstrates that the effectiveness of chlorhexidine may be limited in settings where it is required to act on multi-species biofilms. This may compromise the ability of chlorhexidine to control the infection and spread of these pathogens.

Study into the kinetic properties and surface attachment of a thermostable adenylate kinase.

A thermostable adenylate kinase (tAK) has been used as model protein contaminant on surfaces, so used because residual protein after high temperature wash steps can be detected at extremely low concentrations. This gives the potential for accurate, quantitative measurement of the effectiveness of different wash processes in removing protein contamination. Current methods utilise non-covalent (physisorbtion) of tAK to surfaces, but this can be relatively easily removed. In this study, the covalent binding of tAK to surfaces was studied to provide an alternative model for surface contamination. Kinetic analysis showed that the efficiency of the enzyme expressed as the catalytic rate over the Michaelis constant (kcat/KM) increased from 8.45±3.04 mM-1 s-1 in solution to 32.23±3.20 or 24.46±4.41 mM-1 s-1 when the enzyme was immobilised onto polypropylene or plasma activated polypropylene respectively. Maleic anhydride plasma activated polypropylene showed potential to provide a more robust challenge for washing processes as it retained significantly higher amounts of tAK enzyme than polypropylene in simple washing experiments. Inhibition of the coupled enzyme (luciferase/luciferin) system used for the detection of adenylate kinase activity, was observed for a secondary product of the reaction. This needs to be taken into consideration when using the assay to estimate cleaning efficacy.

Real-time microfluidic recombinase polymerase amplification for the toxin B gene of Clostridium difficile on a SlipChip platform.

Clostridium difficile is one of the key bacterial pathogens that cause infectious diarrhoea both in the developed and developing world. Isothermal nucleic acid amplification methods are increasingly used for identification of toxinogenic infection by clinical labs. For this purpose, we developed a low-cost microfluidic platform based on the SlipChip concept and implemented real-time isothermal recombinase polymerase amplification (RPA). The on-chip RPA assay targets the Clostridium difficile toxin B gene (tcdB) coding for toxin B, one of the proteins responsible for bacterial toxicity. The device was fabricated in clear acrylic using rapid prototyping methods. It has six replicate 500 nL reaction wells as well as two sets of 500 nL control wells. The reaction can be monitored in real-time using exonuclease fluorescent probes with an initial sample volume of as little as 6.4 µL. We demonstrated a limit of detection of 1000 DNA copies, corresponding to 1 fg, at a time-to-result of <20 minutes. This miniaturised platform for pathogen detection has potential for use in resource-limited environments or at the point-of-care because of its ease of use and low cost, particularly if combined with preserved reagents.

Evaluation of the effectiveness of hydrogen-peroxide-based disinfectants on biofilms formed by Gram-negative pathogens.

BACKGROUND: Hydrogen peroxide (H2O2)-based disinfectants are widely used in a number of different healthcare settings to control bacterial colonization and contamination, and reduce the risk of cross-infection. Efficacy tests of these formulations are performed on planktonic cultures, although it is well known that biofilms are the dominant form of bacterial contamination and more difficult to eradicate. AIM: To determine if the biofilms of three different Gram-negative pathogens associated with multi-drug-resistant phenotypes can be eradicated effectively using different H2O2-based disinfectants. METHODS: Planktonic cultures and single-species 24-h biofilms of seven strains of Acinetobacter spp., seven strains of Klebsiella pneumoniae and seven strains of Pseudomonas aeruginosa, including clinical isolates, were exposed to working concentrations of H2O2 and H2O2-based formulations for 1 min to 24h. Survival was monitored. FINDINGS: The levels of susceptibility of planktonic cultures to unformulated and formulated H2O2 were similar in all organisms and strains tested, with minimum inhibitory concentrations ranging from 0.5 to 20mM H2O2. However, biofilms showed up to 266-fold less sensitivity to H2O2 and its formulations. The level of reduced susceptibility correlated with the strain's propensity to form biofilm, and differed between species. The two formulations with additional acidic active ingredients performed better at short exposure times, whereas ethanol-containing products required longer exposure times to be effective. CONCLUSION: Biofilms of a significant number of clinical isolates of multi-drug-resistant nosocomial pathogens are not susceptible to working concentrations of several H2O2-based disinfectants. This may compromise the ability to control these pathogens with such products.

Application of rapid read-out cleaning indicators for improved process control in hospital sterile services departments.

BACKGROUND: Heightened awareness of the importance of cleaning has led to an emphasis on automated systems for the decontamination of re-usable medical devices. The authors have previously described an enzymatic indicator system, based on thermostable adenylate kinases (tAK), for quantitative monitoring of automated cleaning processes within hospital sterile services departments (SSDs). AIM: To evaluate tAK indicators for routine process monitoring across a range of SSDs with different cleaning chemistries and different automated washer disinfectors (AWDs). METHODS: tAK indicator devices and alternative industry test indicators were included in five independent cleaning cycles in each of eight different AWDs. Residual tAK post wash was determined by a coupled luciferase assay using a modified hygiene monitoring system. FINDINGS: In all cases, with the exception of a single test, the alternative indicators showed that cleaning had been adequate. They were not able to discriminate between the performance of different processes. In contrast, the tAK indicators were able to resolve differences in the performance of processes across the different SSDs. Where the tAK indicators identified cleaning to the limits of detection of the assay, this demonstrated a log10 enzyme removal factor of >5.69. CONCLUSION: The results suggest that tAK indicators are suitable for providing improved process control for automated cleaning processes, being able to distinguish between wash performance in different hospital settings and between individual process runs. This technology is believed to be a useful addition to routine AWD performance qualification when used as a daily or weekly test.

A quantitative assessment of residual protein levels on dental instruments reprocessed by manual, ultrasonic and automated cleaning methods.

OBJECTIVE: To assess residual protein on dental instruments cleaned in general dental practice by manual, manual plus ultrasonic and automated washer disinfector (AWD) processes. DESIGN AND SETTING: Instruments submitted by 30 dental surgeries in the South West of England. SUBJECTS (MATERIALS) AND METHODS: Instruments analysed were matrix bands, associated retaining clips, diamond and stainless steel burs, extraction forceps and hand scalers. Each instrument was visually assessed under magnification for residual debris. Residual protein was extracted by immersion in detergent and sonication. A collection of used but uncleaned instruments of each type (n = 177) was also analysed for adherent protein using ophthalaldehyde/N-acetylcysteine reagent. MAIN OUTCOME MEASURES: Residual protein levels allowed comparisons to be made on the effectiveness of different cleaning processes. RESULTS: One thousand, three hundred and four instruments were analysed. Observational data demonstrated several shortcomings in cleaning chemistries and operation of the AWD. For uncleaned instruments, median residual protein levels ranged from 0.4 µg (stainless steel burs) to 462 µg (extraction forceps). Following manual washing, median protein levels ranged from 0.3-78 µg; for manual plus ultrasonic washing, levels ranged from 9-39 µg and AWD levels ranged from 0.3-27 µg. Manual washing combined with ultrasonic cleaning was significantly less effective than the other two processes (p <0.008). AWDs reduced the variability in the cleaning process. No correlation was found between visual scoring and residual protein determination. CONCLUSION(S): There was a wide variation in residual protein levels both within and between different methods and instruments and this underlines the complexity of this process.

Neuroticism as a common dimension in the internalizing disorders.

BACKGROUND: Several theories have posited a common internalizing factor to help account for the relationship between mood and anxiety disorders. These disorders are often co-morbid and strongly covary. Other theories and data suggest that personality traits may account, at least in part, for co-morbidity between depression and anxiety. The present study examined the relationship between neuroticism and an internalizing dimension common to mood and anxiety disorders. METHOD: A sample of ethnically diverse adolescents (n=621) completed self-report and peer-report measures of neuroticism. Participants also completed the Structured Clinical Interview for DSM-IV (SCID). RESULTS: Structural equation modeling showed that a single internalizing factor was common to lifetime diagnosis of mood and anxiety disorders, and this internalizing factor was strongly correlated with neuroticism. Neuroticism had a stronger correlation with an internalizing factor (r=0.98) than with a substance use factor (r=0.29). Therefore, neuroticism showed both convergent and discriminant validity. CONCLUSIONS: These results provide further evidence that neuroticism is a necessary factor in structural theories of mood and anxiety disorders. In this study, the correlation between internalizing psychopathology and neuroticism approached 1.0, suggesting that neuroticism may be the core of internalizing psychopathology. Future studies are needed to examine this possibility in other populations, and to replicate our findings.

Thermostable adenylate kinase technology: a new process indicator and its use as a validation tool for the reprocessing of surgical instruments.

Adenylate kinase (tAK), a thermostable enzyme, was assessed as a possible means of providing a quantitative measure of cleaning efficacy suitable for validating the performance of an automated washer disinfector (AWD) during routine use. Two indicator formulations were developed using either a commercially available washer disinfector soil or a protein-based soil. Each indicator consisted of 100 microg (in test soil) of tAK dried on to a steel or plastic surface. These indicators were placed in each basket of a washer disinfector and processed alongside soiled surgical instruments during a standard day's operation. After processing, remaining tAK activity was detected using a rapid enzyme assay (2 min detection time) in a handheld hygiene monitor. The amount of tAK remaining on each indictor after a full AWD cycle was found to range from 0.1 to 0.4 ng, which represented a mean log(10) removal of 5.8+/-0.3. There was no statistical difference in the residual tAK activity between individual runs or the position of the indicator in the machine. The tAK indicator was also used to analyse the protein removal within each component of the wash cycle. These results demonstrated that all phases of the wash process contributed to the removal of the protein load, with the main wash alone being responsible for 3.6-4.0 log(10) reductions in protein activity. We propose that a quantitative cleaning index using such rapid readout indicator devices would provide a valuable addition to the methodologies for validating cleaning processes.

Quantitative measurement of the efficacy of protein removal by cleaning formulations; comparative evaluation of prion-directed cleaning chemistries.

The stability of the infectious agent causing variant Creutzfeldt-Jakob disease (vCJD) has highlighted the importance of cleaning surgical instruments for controlling potential spread of iatrogenic CJD. In this study, thermostable adenylate kinases (tAKs) in test soil were coated on to stainless steel and these surrogate agents used to evaluate the efficacy of a range of cleaning chemistries in a bench-top washer disinfector (btWD), or as a pre-soak either with or without subsequent treatment by btWD. Two tAKs were tested initially for ease of removal, the most persistent being Sulfolobus acidocaldarius-derived tAK which was used for evaluating the cleaning chemistries. Conventional chemistries were generally more effective when used in a btWD than as pre-soaks. Cleaning efficacy improved when pre-soaks were followed by treatment with intermediate performing enzymes, demonstrating greater than additive effect on residual tAK activity. Three of the four prion-directed chemistries reduced residual tAK activity to below the limit of quantification (LoQ) by more than 4.8 log(10); <175pg tAK remaining as a pre-soak alone. A conventional alkaline cleaning product also reduced residual tAK activity to below the LoQ but only when used in a btWD. tAK soil dried on to the device was removed less efficiently than tAK soil still moist on the device, with a 320-fold and 28-fold increase in residual tAK activity for pre-soak and btWD, respectively. The study demonstrated the potential for a tAK indicator to describe the effectiveness of protein removal using different chemistries or treatment processes.

Decontamination of prion protein (BSE301V) using a genetically engineered protease.

A previous study has demonstrated the potential of alkaline proteases to inactivate bovine spongiform encephalopathy (BSE301V). Here we explored the use of MC3, a genetically engineered variant of Bacillus lentus subtilisin. MC3 was used to digest BSE301V infectious mouse brain homogenate (iMBH). MC3 eliminated all detectable 6H4-immunoreactive material at pH 10 and 12; however, Proteinase K was only partially effective at pH 12. When bioassayed in VM mice, MC3- and Proteinase K-digested iMBH gave respectively 66.6% and 22.7% survival rates. Using a titration series for disease incubation, this equates to a >7log reduction in infectivity for MC3 and >6log reduction for Proteinase K. This study demonstrates the potential for thermostable proteases to be developed as effective inactivation processes for prion agents in healthcare management.

Implications for Creutzfeldt-Jakob disease (CJD) in dentistry: a review of current knowledge.

This review explores our current understanding of the risks of (variant) Creutzfeldt-Jakob disease transmission via dental practice, and whether they merit the rigorous enforcement of improved standards of instrument cleaning and decontamination. The recognition of prions as novel infectious agents in humans has caused significant concern among the public and medical professionals alike. Creutzfeldt-Jakob disease (CJD) in humans has been shown to be transmissible via several routes, including transplantation, contaminated medical products, and via neurosurgery. While the likelihood of transmission via dentistry is undoubtedly very low, this may be amplified considerably by unknown risk factors, such as disease prevalence (particularly in the UK), altered tissue distribution of vCJD, and the failure of decontamination processes to address the inactivation of prions adequately. Since current diagnostic techniques are unable to detect PrP(Sc) in human dental tissues, there is limited evidence for the presence of infectivity. Given these uncertainties, the control of risk by reinforced and improved decontamination practices seems the most appropriate response.

Cleanability of dental instruments--implications of residual protein and risks from Creutzfeldt-Jakob disease.

Cleaning of dental instruments is the first line of control in reducing the adherent bioburden. The threat of vCJD and the difficulty in removing prion protein has provided a new challenge for cleaning surgical and dental instruments. Prion proteins are also more resistant to many disinfection and sterilisation techniques. A number of different methods are currently available in primary care for cleaning instruments including manual washing, ultrasonic cleaners and washer disinfectors. Manual cleaning of dental instruments is time-consuming, introduces operator error and the risk of puncture wounds, is not reproducible and does not completely remove debris from instruments. Ultrasonic baths are significantly more effective than hand cleaning alone and are currently used by the majority of dental surgeries (often as an adjunct to manual cleaning). Automated washer-disinfectors appear to provide a validated, reliable and reproducible procedure for disinfection and sterilisation of dental instruments to ensure both the safety of patients and dental staff. Dental instruments that are difficult to clean are frequently contaminated with tissue debris after routine reprocessing and cannot be excluded as a potential transmission risk for infectious agents, including prions. The transmission of vCJD via dentistry is considered to be low risk, however, the Department of Health (DoH) has recently advised dentists to ensure that endodontic reamers and files are treated as single-use as a precautionary basis in order to further reduce any risk of vCJD transmission.