Structural insights into the specific interaction between Geobacillus stearothermophilus tryptophanyl-tRNA synthetase and antimicrobial Chuangxinmycin.

The potential antimicrobial compound Chuangxinmycin (CXM) targets the tryptophanyl-tRNA synthetase (TrpRS) of both Gram-negative and Gram-positive bacteria. However, the specific steric recognition mode and interaction mechanism between CXM and TrpRS is unclear. Here, we studied this interaction using recombinant GsTrpRS from Geobacillus stearothermophilus by X-ray crystallography and molecular dynamics (MD) simulations. The crystal structure of the recombinant GsTrpRS in complex with CXM was experimentally determined to a resolution at 2.06 Å. After analysis using a complex-structure probe, MD simulations, and site-directed mutation verification through isothermal titration calorimetry, the interaction between CXM and GsTrpRS was determined to involve the key residues M129, D132, I133, and V141 of GsTrpRS. We further evaluated binding affinities between GsTrpRS WT/mutants and CXM; GsTrpRS was found to bind CXM through hydrogen bonds with D132 and hydrophobic interactions between the lipophilic tricyclic ring of CXM and M129, I133, and V141 in the substrate-binding pockets. This study elucidates the precise interaction mechanism between CXM and its target GsTrpRS at the molecular level and provides a theoretical foundation and guidance for the screening and rational design of more effective CXM analogs against both Gram-negative and Gram-positive bacteria.

Assessing the Interactions of Statins with Human Adenylate Kinase Isoenzyme 1: Fluorescence and Enzyme Kinetic Studies.

Statins are the most effective cholesterol-lowering drugs. They also exert many pleiotropic effects, including anti-cancer and cardio- and neuro-protective. Numerous nano-sized drug delivery systems were developed to enhance the therapeutic potential of statins. Studies on possible interactions between statins and human proteins could provide a deeper insight into the pleiotropic and adverse effects of these drugs. Adenylate kinase (AK) was found to regulate HDL endocytosis, cellular metabolism, cardiovascular function and neurodegeneration. In this work, we investigated interactions between human adenylate kinase isoenzyme 1 (hAK1) and atorvastatin (AVS), fluvastatin (FVS), pravastatin (PVS), rosuvastatin (RVS) and simvastatin (SVS) with fluorescence spectroscopy. The tested statins quenched the intrinsic fluorescence of hAK1 by creating stable hAK1-statin complexes with the binding constants of the order of 104 M-1. The enzyme kinetic studies revealed that statins inhibited hAK1 with significantly different efficiencies, in a noncompetitive manner. Simvastatin inhibited hAK1 with the highest yield comparable to that reported for diadenosine pentaphosphate, the only known hAK1 inhibitor. The determined AK sensitivity to statins differed markedly between short and long type AKs, suggesting an essential role of the LID domain in the AK inhibition. Our studies might open new horizons for the development of new modulators of short type AKs.

The search for a microbiological inhibition method for the rapid, broad-spectrum and high-throughput screening of six kinds of antibiotic residues in swine urine.

In this study, a microbiological inhibition method for rapidly screening antibiotics in swine urine was established with an easy sample pre-treatment. The microbiological system consisted of an agar medium mixed with nutrients, sensitizers, a test bacterium (Geobacillus stearothermophilus ATCC12980) and pH indicator (bromocresol purple). It was observed that the detection limits of the test kit for twenty-eight common antimicrobial residues in urine, including ß-lactams, aminoglycosides, tetracyclines, sulfonamides, macrolides, and lincosamides, were less than or equal to the maximum residue limits of the kidney, as determined by the EU and China. Moreover, the false negative rate and the false positive rate, along with other performance indexes such as interassay coefficients of variation and shelf life of the kit, all met the standard requirements of the ISO13969:2003 guidelines. Additionally, our results were consistent with those using the gold-standard physical chemistry method, which suggest the proposed method is suitable for screening antibiotic residues.

Airborne Disinfection by Dry Fogging Efficiently Inactivates Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Mycobacteria, and Bacterial Spores and Shows Limitations of Commercial Spore Carriers.

Airborne disinfection of high-containment facilities before maintenance or between animal studies is crucial. Commercial spore carriers (CSC) coated with 106 spores of Geobacillus stearothermophilus are often used to assess the efficacy of disinfection. We used quantitative carrier testing (QCT) procedures to compare the sensitivity of CSC with that of surrogates for nonenveloped and enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), mycobacteria, and spores, to an aerosolized mixture of peroxyacetic acid and hydrogen peroxide (aPAA-HP). We then used the QCT methodology to determine relevant process parameters to develop and validate effective disinfection protocols (≥4-log10 reduction) in various large and complex facilities. Our results demonstrate that aPAA-HP is a highly efficient procedure for airborne room disinfection. Relevant process parameters such as temperature and relative humidity can be wirelessly monitored. Furthermore, we found striking differences in inactivation efficacies against some of the tested microorganisms. Overall, we conclude that dry fogging a mixture of aPAA-HP is highly effective against a broad range of microorganisms as well as material compatible with relevant concentrations. Furthermore, CSC are artificial bioindicators with lower resistance and thus should not be used for validating airborne disinfection when microorganisms other than viruses have to be inactivated.IMPORTANCE Airborne disinfection is not only of crucial importance for the safe operation of laboratories and animal rooms where infectious agents are handled but also can be used in public health emergencies such as the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. We show that dry fogging an aerosolized mixture of peroxyacetic acid and hydrogen peroxide (aPAA-HP) is highly microbicidal, efficient, fast, robust, environmentally neutral, and a suitable airborne disinfection method. In addition, the low concentration of dispersed disinfectant, particularly for enveloped viral pathogens such as SARS-CoV-2, entails high material compatibility. For these reasons and due to the relative simplicity of the procedure, it is an ideal disinfection method for hospital wards, ambulances, public conveyances, and indoor community areas. Thus, we conclude that this method is an excellent choice for control of the current SARS-CoV-2 pandemic.

Dynamics of DNA nicking and unwinding by the RepC-PcrA complex.

The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s-1, while a typical velocity of 50 bp s-1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented.

Inactivation kinetics of Geobacillus stearothermophilus spores by a peracetic acid or hydrogen peroxide fog in comparison to the liquid form.

The aim of this study was to compare the sporicidal effect of the disinfectants peracetic acid (PAA) or hydrogen peroxide (H2O2) applied as a fog or as a liquid. The efficacy of fogging of the disinfectants was tested in a closed isolator cabinet using highly heat and chemical-resistant spores of Geobacillus stearothermophilus. Fogging of a 0.06% solution of PAA resulted in over 5-log reduction of spores in 10 min, whereas for PAA used in liquid form the same reduction was achieved in 4.5 min. The inactivation curves for fog and liquid were fitted using three different models (Linear with shoulder, Weibull, Gauss-Eyring). This showed a shoulder for the fog with an estimated length of 4.1 min, but the D values, calculated for the linear parts of the curves, were not significantly different (1.1 and 0.8 min for the PAA fog and solution, respectively). Similar results were obtained for a 12% H2O2 solution, albeit that H2O2 was less effective compared to PAA, requiring 60 min to reach 3-log reduction when applied as a fog, with an estimated shoulder of 18.5 min. Fogging of a 0.06% peracetic acid solution effectively inactivated G. stearothermophilus spores. Overall, the data show that fogging can be an effective method of applying disinfectants but that a shoulder in the inactivation curves should be considered in process design. This study provides inactivation kinetics for disinfection using PAA or H2O2-based fog, which can aid in selection and validation of process parameters for disinfection of contained areas by fogging.

Evaluation of a pulsed xenon ultraviolet light device for reduction of pathogens with biofilm-forming ability and impact on environmental bioburden in clinical laboratories.

BACKGROUND: Biofilm-forming organisms can persist on surfaces in hospital clinical laboratories and potentially lead to nosocomial infections. Therefore, effective decontamination procedures are essential for reducing infections. In this study, we investigated an alternative to often ineffective manual cleaning methods, a pulsed xenon ultraviolet (PX-UV) light device. We evaluated PX-UV effect on biofilm formation ability of pathogens and also evaluated PX-UV effectiveness on environmental bioburden in clinical laboratories. METHODS: We selected and identified P. aeruginosa PA47, Staphylococcus aureus B1, and K. pnenumoniae CR52 from clinic isolates. Biofilm-forming ability and effectiveness of PX-UV in killing these biofilm forming strains on surfaces was evaluated. The central laboratory, the clinical microbiology laboratory, and the clinical immunology laboratory were chosen for testing environmental bioburden. Air samples and high-touch surface specimens in the three laboratories were obtained before and after routine manual cleaning, and after 6 min of PX-UV disinfection. The cultured microbes were then identified with MALDI- TOF-MS. RESULTS: We found that P. aeruginosa PA47, Staphylococcus aureus B1, and K. pnenumoniae CR52 were able to form robust biofilms, and that PX-UV significantly reduced colony counts of these strains on all surfaces tested. PX-UV reduced the bioburden of air samples and eliminated bioburden on surfaces. All microbes identified in the clinical laboratories were pathogenic and consisted of cocci, rods, and fungi. CONCLUSIONS: The PX-UV device effectively reduced pathogens with biofilm-forming ability on surfaces, and the environmental bioburden was also significantly reduced by PX-UV. PX-UV is a viable option for protecting staff and decreasing rates of laboratory-acquired infections.

A microbiological inhibition method for the rapid, broad-spectrum, and high-throughput screening of 34 antibiotic residues in milk.

In this study, we developed a microbiological inhibition method for the rapid screening of antibiotics in milk with Geobacillus stearothermophilus ATCC12980 as an indicator bacterium and an easy sample pretreatment. We observed that the limits of detection of the kit for 34 common antibiotic residues in milk, including ß-lactams (13), aminoglycosides (6), tetracyclines (4), sulfonamides (6), macrolides (4), lincosamides (1), were lower than or close to the maximum residue limits formulated by the European Union and China. Moreover, the false-positive rate was 1% and the false-negative rates were less than 5%. The ruggedness of the method (the reproducibility of detection capability of different batches of medium) met requirements at determined levels and residual limits. The shelf life of the kit was more than 6 mo at 4°C. Additionally, we observed good correlations between the kit results and ultra-high-performance liquid chromatography-tandem mass spectrometry results for incurred milk (samples taken from animals treated with antibiotics according to the pre-slaughter medication data), which indicated that the kit was reliable for screening antibiotics in incurred samples. In conclusion, the kit has a broad application potential with high sensitivity, specificity, and reproducibility, stability, and reliability, combined with simple operation, low cost, and high-throughput capacity.

Combined calorimetric gas- and spore-based biosensor array for online monitoring and sterility assurance of gaseous hydrogen peroxide in aseptic filling machines.

A combined calorimetric gas- and spore-based biosensor array is presented in this work to monitor and evaluate the sterilization efficacy of gaseous hydrogen peroxide in aseptic filling machines. H2O2 has been successfully measured under industrial conditions. Furthermore, the effect of H2O2 on three different spore strains , namely Bacillus atrophaeus, Bacillus subtilis and Geobacillus stearothermophilus, has been investigated by means of SEM, AFM and impedimetric measurements. In addition, the sterilization efficacy of a spore-based biosensor and the functioning principle are addressed and discussed: the sensor array is convenient to be used in aseptic food industry to guarantee sterile packages.

Whole room disinfection with hydrogen peroxide mist to control Listeria monocytogenes in food industry related environments.

Listeria monocytogenes surviving daily cleaning and disinfection is a challenge for many types of food industries. In this study, it was tested whether whole room disinfection (WRD) with H2O2 mist could kill L. monocytogenes under conditions relevant for the food industry. Survival of a mixture of four L. monocytogenes strains exposed to H2O2 mist was investigated in a 36 m3 room. A commercial machine produced H2O2 mist by pumping a 5% H2O2 solution containing 0.005% silver through a nozzle, and breaking the liquid up in droplets using pressurized air. When a suspension of bacteria in 0.9% NaCl applied on stainless steel coupons was exposed to WRD with H2O2 mist, a >5 log reduction (LR) of L. monocytogenes was observed. Similar reductions were observed in all tests with conditions between 12 and 20 °C, H2O2 concentrations of 35-80 ppm and 1-2 h exposure. It was shown that the H2O2 in the mist dissolved and accumulated in the liquid on the steel, and acted against L. monocytogenes in the liquid phase. At high cell concentrations, the effect was reduced if cells were pregrown at highly aerated conditions. The anti-listerial effect was robust against protein and fat, but the effect was quenched by raw meat and raw salmon, probably due to high catalase activity. The effect of whole room disinfection with H2O2 against dried L. monocytogenes cells was 1-2 LR, however the effect of air-drying by itself lead to 3-4 LR. When biofilms were exposed to WRD, no surviving L. monocytogenes were observed on stainless steel, however for L. monocytogenes on a PVC conveyor belt material, there were surviving bacteria, with about 2 LR. Screening of 54 L. monocytogenes strains for growth susceptibility to H2O2 showed that their sensitivity to H2O2 was very similar, thus WRD with H2O2 are likely to be robust against strain variation in susceptibility to H2O2. Production of H2O2 mist resulted in increased room humidity, and this may limit the maximum H2O2 concentration achievable, especially at low temperatures. The results in this study show that whole room disinfection with H2O2 may have potential to control L. monocytogenes in the food industry, however intervention studies in the food industry are needed to verify the effect in practical use.

Physicochemical Solubility of and Biological Sensitivity to Long-Chain Alcohols Determine the Cutoff Chain Length in Biological Activity.

The cutoff phenomenon associated with the effectiveness of long-chain alcohols in the induction of anesthesia is also observed for various antimicrobial activities, although the mechanism has remained unknown for over eight decades. The minimum inhibitory concentrations at 25°C for budding yeast growth exponentially decreased with increasing chain length of n-alcohols (C2-C12), whereas alcohols ≥C13 lost the inhibitory effect. Thus, growth inhibition by n-alcohols obeys the Meyer-Overton correlation up to C12 and exhibits a cutoff phenomenon. The densities of n-alcohols are low, and the melting point and hydrophobicity increase with chain length. C13 and C14 inhibited yeast growth at 39.8°C, above their melting points. Alcohols ≤C14 inhibited thermophilic bacterial growth at 50°C, whereas C16 inhibited it at 67.5°C, above their melting points. Thus, the high melting points of long-chain alcohols contribute to the cutoff phenomenon. C14 did not effectively inhibit yeast growth in a static culture at 39.8°C, in contrast to a shaking culture, in which the low density-dependent concentration gradient was eliminated. The duration of the transient growth inhibition of yeast by C12 was prolonged by sonication, which prevented hydrophobic aggregation. Therefore, a nonuniform distribution owing to low density and high hydrophobicity contributes to the cutoff. C14 inhibited the growth at 25°C of the pdr1,3,5 mutant, defective in multidrug efflux pumps, whereas C12 did not inhibit the growth of yeast overexpressing PDR5, indicating that the sensitivity to long-chain alcohols contributed to the cutoff. A balance between the physicochemical solubility of and the biological sensitivity to long-chain alcohols determines the cutoff chain length.

Improvement of Vivarium Biodecontamination through Data-acquisition Systems and Automation.

Biodecontamination is important for eliminating pathogens at research animal facilities, thereby preventing contamination within barrier systems. We enhanced our facility's standard biodecontamination method to replace the traditional foggers, and the new system was used effectively after creating bypass ducts in HVAC units so that individual rooms could be isolated. The entire system was controlled by inhouse-developed supervisory control and data-acquisition software that supported multiple cycles of decontamination by equipment, which had different decontamination capacities, operated in parallel, and used different agents, including H2O2 vapor and ClO2 gas. The process was validated according to facility mapping, and effectiveness was assessed by using biologic (Geobacillus stearothermophilus) and chemical indicator strips, which were positioned before decontamination, and by sampling contact plates after the completion of each cycle. The results of biologic indicators showed 6-log reduction in microbial counts after successful decontamination cycles for both agents and found to be compatible with clean-room panels including commonly used materials in vivarium such as racks, cages, trolleys, cage changing stations, biosafety cabinets, refrigerators and other equipment in both procedure and animal rooms. In conclusion, the automated process enabled users to perform effective decontamination through multiple cycles with realtime documentation and provided additional capability to deal with potential outbreaks. Enabling software integration of automation improved quality-control systems in our vivarium.

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.

Nitrogen gas plasma treatment of bacterial spores induces oxidative stress that damages the genomic DNA.

Gas plasma, produced by a short high­voltage pulse generated from a static induction thyristor power supply [1.5 kilo pulse/sec (kpps)], was demonstrated to inactivate Geobacillus stearothermophilus spores (decimal reduction time at 15 min, 2.48 min). Quantitative polymerase chain reaction and enzyme­linked immunosorbent assays further indicated that nitrogen gas plasma treatment for 15 min decreased the level of intact genomic DNA and increased the level of 8-hydroxy-2'-deoxyguanosine, a major product of DNA oxidation. Three potential inactivation factors were generated during operation of the gas plasma instrument: Heat, longwave ultraviolet-A and oxidative stress (production of hydrogen peroxide, nitrite and nitrate). Treatment of the spores with hydrogen peroxide (3x2­4%) effectively inactivated the bacteria, whereas heat treatment (100˚C), exposure to UV-A (75­142 mJ/cm2) and 4.92 mM peroxynitrite (•ONOO­), which is decomposed into nitrite and nitrate, did not. The results of the present study suggest the gas plasma treatment inactivates bacterial spores primarily by generating hydrogen peroxide, which contributes to the oxidation of the host genomic DNA.

Evaluating different concentrations of hydrogen peroxide in an automated room disinfection system.

UNLABELLED: A comparative study was made on the efficacy of 5, 10 and 35% weight by weight (w/w) hydrogen peroxide solutions when applied using an automated room disinfection system. Six-log biological indicators of methicillin-resistant Staphylococcus aureus (MRSA) and Geobacillus stearothermophilus were produced on stainless steel coupons and placed within a large, sealed, environmentally controlled enclosure. Five percent hydrogen peroxide was distributed throughout the enclosure using a Bioquell hydrogen peroxide vapour generator (BQ-50) for 40 min and left to reside for a further 200 min. Biological indicators were removed at 10-min intervals throughout the first 120 min of the process. The experiment was repeated for 10 and 35% hydrogen peroxide solutions. Five percent and 10% hydrogen peroxide solutions failed to achieve any reduction of MRSA, but achieved full kill of G. stearothermophilus spores at 70 and 40 min respectively. Thirty-five percent hydrogen peroxide achieved a 6-log reduction of MRSA after 30 min and full kill of G. stearothermophilus at 20 min. The concentration of 5% hydrogen peroxide within the enclosure after the 200-min dwell was measured at 9·0 ppm. This level exceeds the 15-min Short Term Exposure Limit (STEL) for hydrogen peroxide of 2·0 ppm. Users of automated hydrogen peroxide disinfection systems should review system efficacy and room re-entry protocols in light of these results. SIGNIFICANCE AND IMPACT OF THE STUDY: This research allows hospital infection control teams to consider the impact and risks of using low concentrations of hydrogen peroxide for disinfection within their facilities, and to question automated room disinfection system providers on the efficacy claims they make. The evidence that low concentration hydrogen peroxide solutions do not rapidly, autonomously break down, is in contradiction to the claims made by some hydrogen peroxide equipment providers and raises serious health and safety concerns. Facilities using hydrogen peroxide systems that claim autonomous break down of hydrogen peroxide should introduce monitoring procedures to ensure rooms are safe for re-entry and patient occupation.

Plasma Decontamination: A Case Study on Kill Efficacy of Geobacillus stearothermophilus Spores on Different Carrier Materials.

A new technology to the pharmaceutical field is presented: surface decontamination by plasmas The technology is comparable to established barrier systems like e-beam, volatile hydrogen peroxide, or radiation inactivation of microbiological contaminations. This plasma technology is part of a fully automated and validated syringe filling line at a major pharmaceutical company and is in production operation. Incoming pre-sterilized syringe containers ("tubs") are processed by plasma, solely on the outside, and passed into the aseptic filling isolator upon successful decontamination. The objective of this article is to present the operating principles and develop and establish a validation routine on the basis of standard commercial biological indicators. Their decontamination efficacies are determined and correlated to the actual inactivation efficacy on the pharmaceutical packaging material.The reference setup is explained in detail and a short presentation of the cycle development and the relevant plasma control parameters is given, with a special focus on the in-process monitor determining the cycle validity. Different microbial inactivation mechanisms are also discussed and evaluated for their contribution and interaction to enhance plasma decontamination. A material-dependent inactivation behavior was observed. In order to be able to correlate the tub surface inactivation of Geobacillus stearothermophilus endospores to metallic biological indicators, a comparative study was performed. Through consistently demonstrating the linear inactivation behavior between the different materials, it becomes possible to develop an effective and time-saving validation scheme. LAY ABSTRACT: The challenge in new decontamination systems lies in a thorough validation of the inactivation efficacy under different operating regimes. With plasma, as an ionized gas, a new barrier concept is introduced into pharmaceutical aseptic processing of syringes. The presented system operates in vacuum and only decontaminates the outer surface of pre-sterilized syringe containers ("tubs"), before they are transferred into the aseptic area. The plasma does not penetrate into the tub. This article discusses the phase from development and test germ selection, across the identified sporicidal mechanisms, to a proposal for a validation scheme on the basis of commercially available biological indicators. A special focus is placed on an extensive investigation to establish a link between the tub surface microbial kill (polystyrene and Tyvek(and (2)) ) and biological indicator inactivation (stainless steel). Additionally, a rationale is developed on how an optical in-process monitor can be applied to establish a validatable limit on the base of the predetermined inactivation data of Geobacillus stearothermophilus endospores.

A sterilization system using ultraviolet photochemical reactions based on nitrous oxide and oxygen gases.

Active oxygen species (AOS) generated under ultraviolet (UV) lamps can be applied for various industrial processes owing to extremely strong oxidative abilities. We have already reported on an application of the AOS for a sterilization process of microorganisms. Here, a sterilization method using active oxygen generated under ultraviolet (UV) lamps introducing nitrous oxide (N2O) and oxygen gases into a vacuum chamber was investigated. Nitrogen dioxide (NO2) gas was readily produced from N2O by UV photochemical reactions under the low-pressure mercury lamp and then used to sterilize medical devices. We compared the ability of the N2O gas to sterilize Geobacillus stearothermophilus spores with those of conventional methods. Successful sterilization of spores on various biological indicators was achieved within 60 min, not only in sterilization bags but also in a lumen device.

Sterilization Effect of Wet Oxygen Plasma in the Bubbling Method.

A new low-temperature sterilization method to replace the ethylene oxide gas sterilization is needed. Strong bactericidal effects of OH and O2H radicals are well known. The purpose of this study was to evaluate the sterilization effect of wet oxygen ("O2+H2O") plasma in the bubbling method, confirming the effect of humidity. Sterility assurance was confirmed by using a biological indicator (Geobacillus stearothermophilus ATCC7953, Namsa, USA). One hundred and eight samples (10(5) spores/carrier) were divided into three groups of 36 in each for treatment with a different type of gas (O2, O2+H2O, Air+H2O). Plasma processing was conducted using a plasma ashing apparatus (13.56 MHz, PACK-3(®), Y. A. C., Japan) under various gas pressures (13, 25, 50 Pa) and gas flows (50, 100, 200 sccm). Fixed plasma treatment parameters were power at 150 W, temperature of 60 ℃, treatment time of 10 min. The samples after treatment were incubated in trypticase soy broth at 58 ℃ for 72 h. The negative culture rate in the "O2+H2O" group was significantly (Mantel-Haenszel procedure, p<0.001) higher than in the other gas groups. It is suggested that the significant sterilization effect of the "O2+H2O" group depends on the bubbling method which is the method of introducing vapor into the chamber. The bubbling method seems able to generate OH and O2H radicals in a stable way.

Study of the combined effect of electro-activated solutions and heat treatment on the destruction of spores of Clostridium sporogenes and Geobacillus stearothermophilus in model solution and vegetable puree.

The combined effect of heat treatment and electro-activated solution (EAS) on the heat resistance of spores of Clostridium sporogenes and Geobacillus stearothermophilus was assessed under various heating and exposure time combinations. The acid and neutral EAS showed the highest inhibitory activity, indicating that these solutions may be considered as strong sporicidal disinfectants. These EAS were able to cause a reduction of ≥6 log of spores of C. sporogenes at 60 °C in only 1 min of exposition. For G. stearothermophilus spores, a reduction of 4.5 log was observed at 60 °C in 1 min, while in 5 min, ≥7 log CFU/ml reduction was observed. Inoculated puree of pea and corn were used as a food matrix for the determination of the heat resistance of these spores during the treatments in glass capillaries. The inactivation kinetics of the spores was studied in an oil bath. Combined treatment by EAS and temperature demonstrated a significant decrease in the heat resistance of C. sporogenes. The D100°C in pea puree with NaCl solution was 66.86 min while with acid and neutral EAS it was reduced down to 3.97 and 2.19 min, respectively. The spore of G. stearothermophilus displayed higher heat resistance as confirmed by other similar studies. Its D130°C in pea puree showed a decrease from 1.45 min in NaCl solution down to 1.30 and 0.93 min for acid and neutral EAS, respectively. The differences between the spores of these species are attributable to their different sensitivities with respect to pH, Redox potential and oxygen.

Metronomic chemotherapy in the neoadjuvant setting: results of two parallel feasibility trials (TraQme and TAME) in patients with HER2+ and HER2− locally advanced breast cancer

Neoadjuvant chemotherapy has practical and theoretical advantages over adjuvant chemotherapy strategy in breast cancer (BC) management. Moreover, metronomic delivery has a more favorable toxicity profile. The present study examined the feasibility of neoadjuvant metronomic chemotherapy in two cohorts [HER2+ (TraQme) and HER2− (TAME)] of locally advanced BC. Twenty patients were prospectively enrolled (TraQme, n=9; TAME, n=11). Both cohorts received weekly paclitaxel at 100 mg/m2 during 8 weeks followed by weekly doxorubicin at 24 mg/m2 for 9 weeks in combination with oral cyclophosphamide at 100 mg/day (fixed dose). The HER2+ cohort received weekly trastuzumab. The study was interrupted because of safety issues. Thirty-six percent of patients in the TAME cohort and all patients from the TraQme cohort had stage III BC. Of note, 33% from the TraQme cohort and 66% from the TAME cohort displayed hormone receptor positivity in tumor tissue. The pathological complete response rates were 55% and 18% among patients enrolled in the TraQme and TAME cohorts, respectively. Patients in the TraQme cohort had more advanced BC stages at diagnosis, higher-grade pathological classification, and more tumors lacking hormone receptor expression, compared to the TAME cohort. The toxicity profile was also different. Two patients in the TraQme cohort developed pneumonitis, and in the TAME cohort we observed more hematological toxicity and hand-foot syndrome. The neoadjuvant metronomic chemotherapy regimen evaluated in this trial was highly effective in achieving a tumor response, especially in the HER2+ cohort. Pneumonitis was a serious, unexpected adverse event observed in this group. Further larger and randomized trials are warranted to evaluate the association between metronomic chemotherapy and trastuzumab treatment.