Geobacillus stearothermophilus STCC4517 spore suspensions showed survival curves with shoulder phenomena independent of sporulation temperature and pH, whose duration was an exponential function of treatment temperature.

Deviations in linearity in survival curves are common in inactivation kinetics during heat treatment. These might lead one to underestimate how effective thermal treatment is. In previous research we reported a relationship between decimal reduction time values (DT) and shoulder lengths (Sl) of survival curves which was characteristic of each microorganism. However, the impact of other factors such as sporulation temperature and pH of the treatment media on shoulder length is still not known. The objective of this research was to evaluate the effect of sporulation temperature (45, 55 and 65 °C) and pH (4.0, 5.0, 6.0 and 7.0) treatment has on the profile of survival curves and on the relationship between Sl/DT of G. stearothermophilus STCC 4517. The results obtained demonstrated that all the spore suspensions, independently of sporulation temperature and pH, showed survival curves with shoulder phenomena, whose duration was an exponential function of treatment temperature. Although both parameters had a significant effect on heat resistance, the relationship between the shoulder length and DT values was constant at all pHs for spores produced at the optimum sporulation temperature.

Identification of a New Heavy-Metal-Resistant Strain of Geobacillus stearothermophilus Isolated from a Hydrothermally Active Volcanic Area in Southern Italy.

Microorganisms thriving in hot springs and hydrothermally active volcanic areas are dynamically involved in heavy-metal biogeochemical cycles; they have developed peculiar resistance systems to cope with such metals which nowadays can be considered among the most permanent and toxic pollutants for humans and the environment. For this reason, their exploitation is functional to unravel mechanisms of toxic-metal detoxification and to address bioremediation of heavy-metal pollution with eco-sustainable approaches. In this work, we isolated a novel strain of the thermophilic bacterium Geobacillus stearothermophilus from the solfataric mud pool in Pisciarelli, a well-known hydrothermally active zone of the Campi Flegrei volcano located near Naples in Italy, and characterized it by ribotyping, 16S rRNA sequencing and mass spectrometry analyses. The minimal inhibitory concentration (MIC) toward several heavy-metal ions indicated that the novel G. stearothermophilus isolate is particularly resistant to some of them. Functional and morphological analyses suggest that it is endowed with metal resistance systems for arsenic and cadmium detoxification.

Walking dead: Permeabilization of heat-treated Geobacillus stearothermophilus ATCC 12980 spores under growth-preventing conditions.

Although heat treatment is probably the oldest and the most common method used to inactivate spores in food processes, the specific mechanism of heat killing of spores is still not fully understood. The purpose of this study is to investigate the evolution of the permeabilization and the viability of heat-treated spores during storage under growth-preventing conditions. Geobacillus stearothermophilus spores were heat-treated under various conditions of temperature and pH, and then stored under conditions of temperature and pH that prevent growth. Spore survival was evaluated by count plating immediately after heat treatment, and then during storage over a period of months. Flow cytometry analyses were performed to investigate the Syto 9 permeability of heat-treated spores. Sub-lethally heat-treated spores of G. stearothermophilus were physically committed to permeabilization after heat treatment. However, prolonged heat treatment may abolish the spore permeabilization and block heat-treated spores in the refractive state. However, viability loss and permeabilization during heat treatment seem to be two different mechanisms that occur independently, and the loss of permeabilization properties takes place at a much slower rate than spore killing. Under growth-preventing conditions, viable heat-treated spores presumably lose their viability due to the permeabilization phenomena, which makes them more susceptible to the action of adverse conditions precluding growth.

Bacterial metabolites from intra- and inter-species influencing thermotolerance: the case of Bacillus cereus and Geobacillus stearothermophilus.

Bacterial metabolites with communicative functions could provide protection against stress conditions to members of the same species. Yet, information remains limited about protection provided by metabolites in Bacillus cereus and inter-species. This study investigated the effect of extracellular compounds derived from heat shocked (HS) and non-HS cultures of B. cereus and Geobacillus stearothermophilus on the thermotolerance of non-HS vegetative and sporulating B. cereus. Cultures of B. cereus and G. stearothermophilus were subjected to HS (42 or 65 °C respectively for 30 min) or non-HS treatments. Cells and supernatants were separated, mixed in a combined array, and then exposed to 50 °C for 60 min and viable cells determined. For spores, D values (85 and 95 °C) were evaluated after 120 h. In most cases, supernatants from HS B. cereus cultures added to non-HS B. cereus cells caused their thermotolerance to increase (D 50 12.2-51.9) when compared to supernatants from non-HS cultures (D 50 7.4-21.7). While the addition of supernatants from HS and non-HS G. stearothermophilus cultures caused the thermotolerance of non-HS cells from B. cereus to decrease initially (D 50 3.7-7.1), a subsequent increase was detected in most cases (D 50 18-97.7). In most cases, supernatants from sporulating G. stearothermophilus added to sporulating cells of B. cereus caused the thermotolerance of B. cereus 4810 spores to decline, whereas that of B. cereus 14579 increased. This study clearly shows that metabolites in supernatants from either the same or different species (such as G. stearothermophilus) influence the thermotolerance of B. cereus.

Die another day: Fate of heat-treated Geobacillus stearothermophilus ATCC 12980 spores during storage under growth-preventing conditions.

Geobacillus stearothermophilus spores are recognized as one of the most wet-heat resistant among aerobic spore-forming bacteria and are responsible for 35% of canned food spoilage after incubation at 55 °C. The purpose of this study was to investigate and model the fate of heat-treated survivor spores of G. stearothermophilus ATCC 12980 in growth-preventing environment. G. stearothermophilus spores were heat-treated at four different conditions to reach one or two decimal reductions. Heat-treated spores were stored in nutrient broth at different temperatures and pH under growth-preventing conditions. Spore survival during storage was evaluated by count plating over a period of months. Results reveal that G. stearothermophilus spores surviving heat treatment lose their viability during storage under growth-preventing conditions. Two different subpopulations were observed during non-thermal inactivation. They differed according to the level of their resistance to storage stress, and the proportion of each subpopulation can be modulated by heat treatment conditions. Finally, tolerance to storage stress under growth-preventing conditions increases at refrigerated temperature and neutral pH regardless of heat treatment conditions. Such results suggest that spore inactivation due to heat treatment could be completed by storage under growth-preventing conditions.

Ozone Gas as a Benign Sterilization Treatment for PLGA Nanofiber Scaffolds.

The use of electrospun nanofibers for tissue engineering and regenerative medicine applications is a growing trend as they provide improved support for cell proliferation and survival due, in part, to their morphology mimicking that of the extracellular matrix. Sterilization is a critical step in the fabrication process of implantable biomaterial scaffolds for clinical use, but many of the existing methods used to date can negatively affect scaffold properties and performance. Poly(lactic-co-glycolic acid) (PLGA) has been widely used as a biodegradable polymer for 3D scaffolds and can be significantly affected by current sterilization techniques. The aim of this study was to investigate pulsed ozone gas as an alternative method for sterilizing PLGA nanofibers. The morphology, mechanical properties, physicochemical properties, and response of cells to PLGA nanofiber scaffolds were assessed following different degrees of ozone gas sterilization. This treatment killed Geobacillus stearothermophilus spores, the most common biological indicator used for validation of sterilization processes. In addition, the method preserved all of the characteristics of nonsterilized PLGA nanofibers at all degrees of sterilization tested. These findings suggest that ozone gas can be applied as an alternative method for sterilizing electrospun PLGA nanofiber scaffolds without detrimental effects.

Added value of experts' knowledge to improve a quantitative microbial exposure assessment model--Application to aseptic-UHT food products.

In a previous study, a quantitative microbial exposure assessment (QMEA) model applied to an aseptic-UHT food process was developed [Pujol, L., Albert, I., Magras, C., Johnson, N. B., Membré, J. M. Probabilistic exposure assessment model to estimate aseptic UHT product failure rate. 2015 International Journal of Food Microbiology. 192, 124-141]. It quantified Sterility Failure Rate (SFR) associated with Bacillus cereus and Geobacillus stearothermophilus per process module (nine modules in total from raw material reception to end-product storage). Previously, the probabilistic model inputs were set by experts (using knowledge and in-house data). However, only the variability dimension was taken into account. The model was then improved using expert elicitation knowledge in two ways. First, the model was refined by adding the uncertainty dimension to the probabilistic inputs, enabling to set a second order Monte Carlo analysis. The eight following inputs, and their impact on SFR, are presented in detail in this present study: D-value for each bacteria of interest (B. cereus and G. stearothermophilus) associated with the inactivation model for the UHT treatment step, i.e., two inputs; log reduction (decimal reduction) number associated with the inactivation model for the packaging sterilization step for each bacterium and each part of the packaging (product container and sealing component), i.e., four inputs; and bacterial spore air load of the aseptic tank and the filler cabinet rooms, i.e., two inputs. Second, the model was improved by leveraging expert knowledge to develop further the existing model. The proportion of bacteria in the product which settled on surface of pipes (between the UHT treatment and the aseptic tank on one hand, and between the aseptic tank and the filler cabinet on the other hand) leading to a possible biofilm formation for each bacterium, was better characterized. It was modeled as a function of the hygienic design level of the aseptic-UHT line: the experts provided the model structure and most of the model parameters values. Mean of SFR was estimated to 10×10(-8) (95% Confidence Interval=[0×10(-8); 350×10(-8)]) and 570×10(-8) (95% CI=[380×10(-8); 820×10(-8)]) for B. cereus and G. stearothermophilus, respectively. These estimations were more accurate (since the confidence interval was provided) than those given by the model with only variability (for which the estimates were 15×10(-8) and 580×10(-8) for B. cereus and G. stearothermophilus, respectively). The updated model outputs were also compared with those obtained when inputs were described by a generic distribution, without specific information related to the case-study. Results showed that using a generic distribution can lead to unrealistic estimations (e.g., 3,181,000 product units contaminated by G. stearothermophilus among 10(8) product units produced) and emphasized the added value of eliciting information from experts from the relevant specialist field knowledge.

Effect of Essential Oils on Germination and Growth of Some Pathogenic and Spoilage Spore-Forming Bacteria.

The use of essential oils as a food preservative has increased due to their capacity to inhibit vegetative growth of some bacteria. However, only limited data are available on their effect on bacterial spores. The aim of the present study was to evaluate the effect of some essential oils on the growth and germination of three Bacillus species and Geobacillus stearothermophilus. Essential oils were chemically analyzed using gas chromatography and gas chromatography coupled to mass spectrometry. The minimal inhibitory and bactericidal concentrations of vegetative growth and spore germination were assessed using the macrodilution method. Germination inhibitory effect of treated spores with essential oils was evaluated on solid medium, while kinetic growth was followed using spectrophotometry in the presence of essential oils. Essential oil from Drypetes gossweileri mainly composed of benzyl isothiocyanate (86.7%) was the most potent, with minimal inhibitory concentrations ranging from 0.0048 to 0.0097 mg/mL on vegetative cells and 0.001 to 0.002 mg/mL on spore germination. Furthermore, essential oil from D. gossweileri reduced 50% of spore germination after treatment at 1.25 mg/mL, and its combination with other oils improved both bacteriostatic and bactericidal activities with additive or synergistic effects. Concerning the other essential oils, the minimal inhibitory concentration ranged from 5 to 0.63 mg/mL on vegetative growth and from 0.75 to 0.09 mg/mL on the germination of spores. Spectrophotometric evaluation showed an inhibitory effect of essential oils on both germination and outgrowth. From these results, it is concluded that some of the essential oils tested might be a valuable tool for bacteriological control in food industries. Therefore, further research regarding their use as food preservatives should be carried out.

Probabilistic exposure assessment model to estimate aseptic-UHT product failure rate.

Aseptic-Ultra-High-Temperature (UHT) products are manufactured to be free of microorganisms capable of growing in the food at normal non-refrigerated conditions at which the food is likely to be held during manufacture, distribution and storage. Two important phases within the process are widely recognised as critical in controlling microbial contamination: the sterilisation steps and the following aseptic steps. Of the microbial hazards, the pathogen spore formers Clostridium botulinum and Bacillus cereus are deemed the most pertinent to be controlled. In addition, due to a relatively high thermal resistance, Geobacillus stearothermophilus spores are considered a concern for spoilage of low acid aseptic-UHT products. A probabilistic exposure assessment model has been developed in order to assess the aseptic-UHT product failure rate associated with these three bacteria. It was a Modular Process Risk Model, based on nine modules. They described: i) the microbial contamination introduced by the raw materials, either from the product (i.e. milk, cocoa and dextrose powders and water) or the packaging (i.e. bottle and sealing component), ii) the sterilisation processes, of either the product or the packaging material, iii) the possible recontamination during subsequent processing of both product and packaging. The Sterility Failure Rate (SFR) was defined as the sum of bottles contaminated for each batch, divided by the total number of bottles produced per process line run (10(6) batches simulated per process line). The SFR associated with the three bacteria was estimated at the last step of the process (i.e. after Module 9) but also after each module, allowing for the identification of modules, and responsible contamination pathways, with higher or lower intermediate SFR. The model contained 42 controlled settings associated with factory environment, process line or product formulation, and more than 55 probabilistic inputs corresponding to inputs with variability conditional to a mean uncertainty. It was developed in @Risk and run through Monte Carlo simulations. Overall, the highest SFR was associated with G. stearothermophilus (380000 bottles contaminated in 10(11) bottles produced) and the lowest to C. botulinum (3 bottles contaminated in 10(11) bottles produced). Unsurprisingly, SFR due to G. stearothermophilus was due to its ability to survive the UHT treatment. More interestingly, it was identified that SFR due to B. cereus (17000 bottles contaminated in 10(11) bottles produced) was due to an airborne recontamination of the aseptic tank (49%) and a post-sterilisation packaging contamination (33%). A deeper analysis (sensitivity and scenario analyses) was done to investigate how the SFR due to B. cereus could be reduced by changing the process settings related to potential air recontamination source.

Genotypic and phenotypic characterization of foodborne Geobacillus stearothermophilus.

Geobacillus stearothermophilus is the main thermophilic spore former involved in flat sour spoilage of canned foods. Three typing methods were tested and applied to differentiate strains at intra-species level: panC sequence analysis, REP-PCR and M13-PCR. panC gene was highly conserved within the studied strains, suggesting a low intra-specific diversity. This was supported by REP-PCR primary assays and M13-PCR results. M13-PCR profile analysis succeeded in differentiating six closely related groups (at 79% threshold similarity) among 127 strains from a range of spoiled canned food products and from different canneries. Phenotypic traits were investigated among 20 selected strains representing groups and origins. Ranges of growth under different temperatures (from 40 °C to 70 °C), pH (from 5.0 to 6.5), NaCl concentrations (from 1 to 5%) and sporulation conditions poorly differed between strains, but wet heat resistance of spores showed a 20-fold variation between strains. Furthermore, in this study, strains that belonged to the same M13-PCR genetic group did not share phenotypic characteristics or common origin. The work emphasizes a low diversity within the G. stearothermophilus species but data from this study may contribute to a better control of G. stearothermophilus spoilage in canned food.

High-pressure thermal sterilization: food safety and food quality of baby food puree.

The benefits that high-pressure thermal sterilization offers as an emerging technology could be used to produce a better overall food quality. Due to shorter dwell times and lower thermal load applied to the product in comparison to the thermal retorting, lower numbers and quantities of unwanted food processing contaminants (FPCs), for example, furan, acrylamide, HMF, and MCPD-esters could be formed. Two spore strains were used to test the technique; Geobacillus stearothermophilus and Bacillus amyloliquefaciens, over the temperature range 90 to 121 °C at 600 MPa. The treatments were carried out in baby food puree and ACES-buffer. The treatments at 90 and 105 °C showed that G. stearothermophilus is more pressure-sensitive than B. amyloliquefaciens. The formation of FPCs was monitored during the sterilization process and compared to the amounts found in retorted samples of the same food. The amounts of furan could be reduced between 81% to 96% in comparison to retorting for the tested temperature pressure combination even at sterilization conditions of F0-value in 7 min.

Characterization of thermophilic bacilli from a milk powder processing plant.

AIMS: To determine whether strains of Geobacillus stearothermophilus isolated from a milk powder manufacturing plant were different in their ability to form biofilms and produce spores. In addition, this study evaluated whether there were other physiological characteristics that could differentiate these strains. METHODS AND RESULTS: Ten G. stearothermophilus strains and one Anoxybacillus species were isolated from a milk powder manufacturing plant. A microtitre plate assay was used to show that these strains differed in their abilities to form biofilms and produce spores. Scanning electron microscopy showed differences in the biofilm morphologies of three of the G. stearothermophilus strains. Biochemical profiling, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and fatty acid profiling further showed that they had distinct characteristics. CONCLUSIONS: These G. stearothermophilus strains, isolated from the same environment, showed differences in their ability to form biofilms and produce endospores. Based on the multiple characterization methods used in this study, these strains of G. stearothermophilus isolated from one manufacturing plant are diverse. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences in the ability of G. stearothermophilus to form biofilms and produce spores may influence the cleaning method used to control the growth of thermophilic bacilli in a dairy processing environment.

Quantitative assessment of the risk of microbial spoilage in foods. Prediction of non-stability at 55 °C caused by Geobacillus stearothermophilus in canned green beans.

Microbial spoilage of canned foods by thermophilic and highly heat-resistant spore-forming bacteria, such as Geobacillus stearothermophilus, is a persistent problem in the food industry. An incubation test at 55 °C for 7 days, then validation of biological stability, is used as an indicator of compliance with good manufacturing practices. We propose a microbial risk assessment model predicting the percentage of non-stability due to G. stearothermophilus in canned green beans manufactured by a French company. The model accounts for initial microbial contaminations of fresh unprocessed green beans with G. stearothermophilus, cross-contaminations in the processing chain, inactivation processes and probability of survival and growth. The sterilization process is modeled by an equivalent heating time depending on sterilization value F0 and on G. stearothermophilus resistance parameter z(T). Following the recommendations of international organizations, second order Monte-Carlo simulations are used, separately propagating uncertainty and variability on parameters. As a result of the model, the mean predicted non-stability rate is of 0.5%, with a 95% uncertainty interval of [0.1%; 1.2%], which is highly similar to data communicated by the French industry. A sensitivity analysis based on Sobol indices and some scenario tests underline the importance of cross-contamination at the blanching step, in addition to inactivation due to the sterilization process.

Kinetics of germination of individual spores of Geobacillus stearothermophilus as measured by raman spectroscopy and differential interference contrast microscopy.

Geobacillus stearothermophilus is a gram-positive, thermophilic bacterium, spores of which are very heat resistant. Raman spectroscopy and differential interference contrast microscopy were used to monitor the kinetics of germination of individual spores of G. stearothermophilus at different temperatures, and major conclusions from this work were as follows. 1) The CaDPA level of individual G. stearothermophilus spores was similar to that of Bacillus spores. However, the Raman spectra of protein amide bands suggested there are differences in protein structure in spores of G. stearothermophilus and Bacillus species. 2) During nutrient germination of G. stearothermophilus spores, CaDPA was released beginning after a lag time (T(lag)) between addition of nutrient germinants and initiation of CaDPA release. CaDPA release was complete at T(release), and DT(release) (T(release) - T(lag)) was 1-2 min. 3) Activation by heat or sodium nitrite was essential for efficient nutrient germination of G. stearothermophilus spores, primarily by decreasing T(lag) values. 4) Values of T(lag) and T(release) were heterogeneous among individual spores, but DT(release) values were relatively constant. 5) Temperature had major effects on nutrient germination of G. stearothermophilus spores, as at temperatures below 65°C, average T(lag) values increased significantly. 6) G. stearothermophilus spore germination with exogenous CaDPA or dodecylamine was fastest at 65°C, with longer T(lag) values at lower temperatures. 7) Decoating of G. stearothermophilus spores slowed nutrient germination slightly and CaDPA germination significantly, but increased dodecylamine germination markedly. These results indicate that the dynamics and heterogeneity of the germination of individual G. stearothermophilus spores are generally similar to that of Bacillus species.

Effect of dielectric and liquid on plasma sterilization using dielectric barrier discharge plasma.

Plasma sterilization offers a faster, less toxic and versatile alternative to conventional sterilization methods. Using a relatively small, low temperature, atmospheric, dielectric barrier discharge surface plasma generator, we achieved ≥ 6 log reduction in concentration of vegetative bacterial and yeast cells within 4 minutes and ≥ 6 log reduction of Geobacillus stearothermophilus spores within 20 minutes. Plasma sterilization is influenced by a wide variety of factors. Two factors studied in this particular paper are the effect of using different dielectric substrates and the significance of the amount of liquid on the dielectric surface. Of the two dielectric substrates tested (FR4 and semi-ceramic (SC)), it is noted that the FR4 is more efficient in terms of time taken for complete inactivation. FR4 is more efficient at generating plasma as shown by the intensity of spectral peaks, amount of ozone generated, the power used and the speed of killing vegetative cells. The surface temperature during plasma generation is also higher in the case of FR4. An inoculated FR4 or SC device produces less ozone than the respective clean devices. Temperature studies show that the surface temperatures reached during plasma generation are in the range of 30°C-66 °C (for FR4) and 20 °C-49 °C (for SC). Surface temperatures during plasma generation of inoculated devices are lower than the corresponding temperatures of clean devices. pH studies indicate a slight reduction in pH value due to plasma generation, which implies that while temperature and acidification may play a minor role in DBD plasma sterilization, the presence of the liquid on the dielectric surface hampers sterilization and as the liquid evaporates, sterilization improves.

A meta-analysis accounting for sources of variability to estimate heat resistance reference parameters of bacteria using hierarchical Bayesian modeling: Estimation of D at 121.1 °C and pH 7, zT and zpH of Geobacillus stearothermophilus.

Predicting microbial survival requires reference parameters for each micro-organism of concern. When data are abundant and publicly available, a meta-analysis is a useful approach for assessment of these parameters, which can be performed with hierarchical Bayesian modeling. Geobacillus stearothermophilus is a major agent of microbial spoilage of canned foods and is therefore a persistent problem in the food industry. The thermal inactivation parameters of G. stearothermophilus (D(ref), i.e.the decimal reduction time D at the reference temperature 121.1°C and pH 7.0, z(T) and z(pH)) were estimated from a large set of 430 D values mainly collected from scientific literature. Between-study variability hypotheses on the inactivation parameters D(ref), z(T) and z(pH) were explored, using three different hierarchical Bayesian models. Parameter estimations were made using Bayesian inference and the models were compared with a graphical and a Bayesian criterion. Results show the necessity to account for random effects associated with between-study variability. Assuming variability on D(ref), z(T) and z(pH), the resulting distributions for D(ref), z(T) and z(pH) led to a mean of 3.3 min for D(ref) (95% Credible Interval CI=[0.8; 9.6]), to a mean of 9.1°C for z(T) (CI=[5.4; 13.1]) and to a mean of 4.3 pH units for z(pH) (CI=[2.9; 6.3]), in the range pH 3 to pH 7.5. Results are also given separating variability and uncertainty in these distributions, as well as adjusted parametric distributions to facilitate further use of these results in aqueous canned foods such as canned vegetables.

Sporicidal efficacy of genipin: a potential theoretical alternative for biomaterial and tissue graft sterilization.

Terminal sterilization of musculoskeletal allografts by gamma radiation minimizes the risk of disease transmission but impairs allograft mechanical properties. Commonly employed crosslinking agents can sterilize tissues without affecting mechanical properties adversely; however, these agents are toxic. Genipin is reported to be a benign crosslinking agent that strengthens mechanical properties of tissues; however, the antimicrobial capacity of genipin is largely unknown. The present study's aims were: (1) to assess the sporicidal potential of genipin, (2) to improve antimicrobial capacity by changing chemical and physical treatment conditions. To establish genipin's sterilization potential Bacillus subtilis var. niger spore strips were treated with 0-10% genipin in PBS or in 1:1 DMSO:PBS up to 72 h at room temperature (RT). Sterilizing doses and concentrations of genipin were used to treat B. pumilus and Geobacillus stearothermophilus spores to assess broader spectrum sporicidal activity of genipin. Scanning electron microscopy (SEM) was performed to evaluate gross morphological changes after genipin treatment. Optimal sterilization conditions were determined by evaluating the effects of temperature (RT-50 °C), DMSO:PBS ratio (0:100-100:0), and treatment duration (24-72 h) on B. subtilis. Genipin penetration of full thickness bovine patellar tendon and cortical bone specimens was observed to assess the feasibility of the agent for treating grafts. Initial studies showed that after 72 h of treatment at RT with 0.63-10% genipin/DMSO:PBS B. subtilis spore strips were sterilized; 0.63% genipin/PBS did not sterilize spore strips at 72 h at RT. Genipin doses and concentrations that sterilized B. subtilis spore strips sterilized B. pumilus and G. stearothermophilus spore strips. SEM revealed no gross morphological differences between untreated and treated spores. Treatment optimization resulted in sterilization within 24 h with 100% PBS, and DMSO facilitated sporicidal activity. Genipin penetrated full thickness patellar tendon specimens and 3.72 ± 0.58 mm in cortical bone specimens. Genipin sterilizes B. subtilis, B. pumilus, and G. stearothermophilus spore strips. It penetrates soft and hard tissues at doses previously shown to be non-toxic and to improve mechanical strength in collagen-rich soft tissues. Further studies are indicated to assess genipin's effects on the mechanical properties of genipin-sterilized grafts, the ability of genipin to eradicate infectious species other than spores, and to assess whether sterilant activity persists after penetrating tissues and biomaterials.

Study of the influence of sporulation conditions on heat resistance of Geobacillus stearothermophilus used in the development of biological indicators for steam sterilization.

Biological indicators are important tools in infection control via sterilization process monitoring. The use of a standardized spore crop with a well-defined heat resistance will guarantee the quality of a biological indicator. Ambient factors during sporulation can affect spore characteristics and properties, including heat resistance. The aim of this study is to evaluate the main sporulation factors responsible for heat resistance in Geobacillus stearothermophilus, a useful biological indicator for steam sterilization. A sequence of a three-step optimization of variables (initial pH, nutrient concentration, tryptone, peptone, beef extract, yeast extract, manganese sulfate, magnesium sulfate, calcium chloride and potassium phosphate) was carried out to screen those that have a significant influence on heat resistance of produced spores. The variable exerting greatest influence on G. stearothermophilus heat resistance during sporulation was found to be the initial pH. Lower nutrient concentration and alkaline pH around 8.5 tended to enhance decimal reduction time at 121 °C (D(121°C)). A central composite design enabled a fourfold enhancement in heat resistance, and the model obtained accurately describes positive pH and negative manganese sulfate concentration influence on spore heat resistance.

Spore germination based assay for monitoring antibiotic residues in milk at dairy farm.

Spore germination based assay involves the transformation of dormant spores of Bacillus stearothermophilus 953 into active vegetative cells. The inhibition of germination process specifically in presence of antibiotic residues was used as a novel approach for monitoring target contaminants in milk. The indicator organism i.e., B. stearothermophilus 953 was initially allowed to sporulate by seeding in sporulation medium and incubating at 55 °C for 18 ± 2 h. The spores exhibited a typical chain behavior as revealed through phase contrast microscopy. The minimal medium inoculated with activated spores was incubated at 64 °C for 2-3 h for germination and outgrowth in presence of specific germinant mixture containing dextrose, whey powder and skimmed milk powder added in specific ratio along with reconstituted milk as negative control and test milk samples. The change in color of the medium from purple to yellow was used as criteria for detection of antibiotic residues in milk. The efficiency of the developed assay was evaluated through a surveillance study on 228 samples of raw, pasteurized and dried milks and results were compared with AOAC approved microbial receptor assay. The presence of antibiotic level was 10.08 % at Codex maximum residual limit having false positive result only in 0.43 % of the samples. The results of the present investigation suggest that developed spore based assay can be a practical solution to dairy industry for its application at farm level, milk processing units, independent testing and R & D centres in order to comply with the legal requirements set by Codex.

Solvent removal and spore inactivation directly in dispensing vials with supercritical carbon dioxide and sterilant.

A process is described using supercritical carbon dioxide to extract organic solvents from drug solutions contained in 30-mL serum vials. We report drying times of less than 1 h with quantitative recovery of sterile drug. A six-log reduction of three spore types used as biological indicators is achieved with direct addition of peracetic acid to a final concentration of approximately 5 mM (~0.04 %) to the drug solution in the vial. Analysis of two drugs, acetaminophen and paclitaxel, indicated no drug degradation as a result of the treatment. Furthermore, analysis of the processed drug substance showed that no residual peracetic acid could be detected in the final product. We have demonstrated an effective means to simultaneously dry and sterilize active pharmaceutical ingredients from organic solvents directly in a dispensing container.