Heat-treated Spores Research Articles

Extension of growth/no growth predictive models for the preservation of low-acid pasteurized sauces by incorporating water activity and model validation in sauces

Combined preservation methods have awakened a growing interest in low-acid pasteurized sauces under ambient storage, aiming to produce more ‘natural’ foods with enhanced microbial stability. However, limited information and predictive models are available to assess the microbial stability of this kind of products, for which the spoilage is mainly caused by acid-tolerant spore-forming spoilage bacteria (ATSSB). In this study, a set of growth/no growth (G/NG) models developed previously (Sun et al., 2021a) for spores of two ATSSB strains (Bacillus velezensis and Bacillus subtilis) from pasteurized sauces were upgraded to incorporate the effect of water activity (aw). The growth from heat-treated spores (80 °C, 10 min) was assessed during three months in Nutrient Broth under 320 combinations of storage temperatures (22 and 30 °C), aw (0.95 and 0.965), pH (4.4–5.6), total acetic acid (0.0–0.3 % (w/w)), and total lactic acid (0.00–1.00 % (w/w)). Twelve replicates were tested for each combination. After merging the dataset collected previously at aw 0.98, a set of 4-variate (4V) models were developed for each strain under a single temperature after 30, 60 and 90 days separately. Additionally, the developed models were validated by challenge tests in different industrially produced low-acid pasteurized sauces for three months.The results showed that lowering aw significantly enhanced the antimicrobial effect of decreased pH and the addition of acetic and lactic acid against the ATSSB spores, while the enhancement was less evident at high acetic acid concentrations. In the challenge tests, the developed models exhibited accurate predictions for the sauces, with the % correct-predicted values ≥90 %. The developed models can be used in microbiological risk assessments or the innovation of preservation strategies for low-acid pasteurized sauces.

Modeling the combined effect of temperature, pH, acetic and lactic acid concentrations on the growth/no growth interface of acid-tolerant Bacillus spores

The application of minimal processing technologies has led to increased spoilage incidents in low-acid pasteurized sauces due to the outgrowth of acid-tolerant spore-forming spoilage bacteria (ATSSB). Controlling the germination and subsequent growth of ATSSB spores is vital to enhance the ambient storage stability of pasteurized sauces. This study developed and validated a set of growth/no growth (G/NG) models for spores of two ATSSB strains (Bacillus velezensis and Bacillus subtilis subsp. subtilis) isolated from pasteurized sauces. The G/NG data at two levels of temperature (22 and 30 °C) were collected in Nutrient Broth (aw = 0.98 adjusted with NaCl) by a full factorial design with five equidistant levels of pH (4.4–5.6), four concentrations of total acetic acid (0.0–0.3% (w/w)), and four concentrations of total lactic acid (0.00–1.00% (w/w)). The growth, starting from heat-treated (10 min 80 °C) spores, of each strain was assessed under 160 combinations by regular optical density measurements during three months. Twelve replicates were made for each combination.The developed models demonstrate that without organic acids even the lowest pH (4.4) allows a high growth possibility of the ATSSB spores, while acetic and lactic acids exhibit a significant antibacterial activity, which can be enhanced at decreased pH. The growth starting from B. subtilis spores can be inhibited for at least three months with 1.0% (w/w) total lactic acid in the water phase at both temperatures, which was not the case for B. velezensis, while 0.3% acetic acid achieves a full inhibition on both strains at 22 °C. With a combination of 0.3% acetic acid and 0.7% lactic acid, no growth should occur in the investigated range. This research is one of the first studies exploring the feasibility of ambient storage for low-acid pasteurized sauces eliminating preservatives such as benzoic and sorbic acids, and proves the synergistic effect of decreased pH and the presence of acetic and lactic acids on inhibiting bacterial growth from ATSSB spores.

The synergic interaction between environmental factors (pH and NaCl) and the physiological state (vegetative cells and spores) provides new possibilities for optimizing processes to manage risk of C. sporogenes spoilage

Clostridium sporogenes has been widely used as a surrogate for proteolytic C. botulinum for validating thermal processes in low-acid cans. To limit the intensity of heat treatments, industrials must use other ways of control as an association of acidic and saline environment after a low heat treatment. The probability of growth of pH (7–4.4), sodium chloride concentration (0–11%) and heat treatment (80°C-10 min; 100°C-1.5 min and 5.2 min) were studied on C. sporogenes PA 3679 spores and vegetative cells. Vegetative cells or heat-treated spores were inoculated in PYGm broth at 30 °C for 48 days in anaerobic conditions. Vegetative cells growth (pH 4.6–pH 4.5; 7%–8% NaCl) range is larger than the spore one (pH 5.2–pH 5.0; 6%–7% NaCl). Spores germination and outgrowth rage is decreased if the spores are heat-treated at 100 °C for 1.5 min (pH 5.5–5.3; 4%–5% NaCl) and 5.2 min (pH 5.7–5.3; 4%–5% NaCl). The C. sporogenes PA 3679 spores germination and outgrowth is impacted by their physiological state. The synergic interaction between environmental factors (pH and NaCl) and the physiological state (vegetative cells and spores) opening new possibilities for optimizing food formulation processes to manage the risks of C. sporogenes spoilage.

High pressure CO2 reduces the wet heat resistance of Bacillus subtilis spores by perturbing the inner membrane

Spores of wild-type Bacillus subtilis PS533 were treated by wet heat at 75 °C for 30 min, and high pressure CO2 (HPCD) at 6.5 MPa and 30 °C or 75 °C for 30 min. The spores were analyzed for wet heat resistance (85 °C, 90 °C, 95 °C) and typical germination events including DPA release and cortex hydrolysis, inner membrane permeability, and germination triggered by nutrient (L-valine and AGFK) or non-nutrient (dodecylamine and high pressure at 150 MPa or 550 MPa) germinants. The results showed that (i) HPCD-treated spores exhibited reduced wet heat resistance compared to the untreated or wet heat-treated spores; (ii) HPCD-treated spores did not undergo typical germination events such as DPA release or cortex hydrolysis compared to normally germinated spores; (iii) HPCD-treated spores released more metal ions and exhibited decreased ability to maintain DPA, indicating that the permeability of inner membrane of HPCD-treated spores was increased; (iv) HPCD-treated spores exhibited reduced germination rate when triggered by L-valine or 150 MPa, but increased germination rate when triggered by dodecylamine or 550 MPa, suggesting that the fluidity of the inner membrane of HPCD-treated spores might be increased. These results indicated that HPCD could reduce the wet heat resistance of spores, and this resistance decrease was probably due to the modification of the inner membrane caused by HPCD. Industrial relevanceThe extremely high wet heat resistance of spores makes them a significant problem in the thermal processing of foods. Thus, it of great interest to develop a process to reduce the wet heat resistance of spores. In this work, we found that HPCD can significantly reduce the wet heat resistance of B. subtilis spores, and this was achieved by perturbing the inner membrane of spores. These results can improve our understanding of the inactivation mechanism of spores by HPCD, and also provide an alternative approach for spore inactivation in foods.

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.

Recovery of Heat Treated Bacillus cereus Spores Is Affected by Matrix Composition and Factors with Putative Functions in Damage Repair.

The ability of spores to recover and grow out after food processing is affected by cellular factors and by the outgrowth conditions. In the current communication we studied the recovery and outgrowth of individually sorted spores in BHI and rice broth media and on agar plates using flow cytometry. We show that recovery of wet heat treated Bacillus cereus ATCC 14579 spores is affected by matrix composition with highest recovery in BHI broth or on rice agar plates, compared to BHI agar plates and rice broth. Data show that not only media composition but also its liquid or solid state affect the recovery of heat treated spores. To determine the impact of factors with putative roles in recovery of heat treated spores, specific genes previously shown to be highly expressed in outgrowing heat-treated spores were selected for mutant construction. Spores of nine B. cereus ATCC 14579 deletion mutants were obtained and their recovery from wet heat treatment was evaluated using BHI and rice broth and agar plates. Deletion mutant spores showed different capacity to recover from heat treatment compared to wild type with the most pronounced effect for a mutant lacking BC5242, a gene encoding a membrane protein with C2C2 zinc finger which resulted in over 95% reduction in recovery compared to the wild type in BHI broth. Notably, similar relative performance of wild type and mutants was observed using the other recovery conditions. We obtained insights on the impact of matrix composition and state on recovery of individually sorted heat treated spores and identified cellular factors with putative roles in this process. These results may provide leads for future developments in design of more efficient combined preservation treatments.

Identification of CdnL, a Putative Transcriptional Regulator Involved in Repair and Outgrowth of Heat-Damaged Bacillus cereus Spores.

Spores are widely present in the environment and are common contaminants in the food chain, creating a challenge for food industry. Nowadays, heat treatments conventionally applied in food processing may become milder to comply with consumer desire for products with higher sensory and nutritional values. Consequently subpopulations of spores may emerge that are sublethally damaged rather than inactivated. Such spores may germinate, repair damage, and eventually grow out leading to uncontrolled spoilage and safety issues. To gain insight into both the behaviour of damaged Bacillus cereus spores, and the process of damage repair, we assessed the germination and outgrowth performance using OD595 measurements and microscopy combined with genome-wide transcription analysis of untreated and heat-treated spores. The first two methods showed delayed germination and outgrowth of heat-damaged B. cereus ATCC14579 spores. A subset of genes uniquely expressed in heat-treated spores was identified with putative roles in the outgrowth of damaged spores, including cdnL (BC4714) encoding the putative transcriptional regulator CdnL. Next, a B. cereus ATCC14579 cdnL (BC4714) deletion mutant was constructed and assessment of outgrowth from heat-treated spores under food relevant conditions showed increased damage compared to wild type spores. The approach used in this study allows for identification of candidate genes involved in spore damage repair. Further identification of cellular parameters and characterisation of the molecular processes contributing to spore damage repair may provide leads for better control of spore outgrowth in foods.

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.

Influence of food matrix on outgrowth heterogeneity of heat damaged Bacillus cereus spores

Spoilage of heat treated foods can be caused by the presence of surviving spore-formers. It is virtually impossible to prevent contamination at the primary production level as spores are ubiquitous present in the environment and can contaminate raw products. As a result spore inactivation treatments are widely used by food producing industries to reduce the microbial spore loads. However consumers prefer mildly processed products that have less impact on its quality and this trend steers industry towards milder preservation treatments. Such treatments may result in damaged instead of inactivated spores, and these spores may germinate, repair, and grow out, possibly leading to quality and safety issues. The ability to repair and grow out is influenced by the properties of the food matrix. In the current communication we studied the outgrowth from heat damaged Bacillus cereus ATCC 14579 spores on Anopore membrane, which allowed following outgrowth heterogeneity of individual spores on broccoli and rice-based media as well as standard and mildly acidified (pH5.5) meat-based BHI. Rice, broccoli and BHI pH5.5 media resulted in delayed outgrowth from untreated spores, and increased heterogeneity compared to BHI pH7.4, with the most pronounced effect in rice media. Exposure to wet heat for 1min at 95°C caused 2 log inactivation and approximately 95% of the spores in the surviving fraction were damaged resulting in substantial delay in outgrowth based on the time required to reach a maximum microcolony size of 256 cells. The delay was most pronounced for heat-treated spores on broccoli medium followed by spores on rice media (both untreated and treated). Interestingly, the increase in outgrowth heterogeneity of heat treated spores on BHI pH7.4 was more pronounced than on rice, broccoli and BHI pH5.5 conceivably reflecting that conditions in BHI pH7.4 better support spore damage repair. This study compares the effects of three main factors, namely heat treatment, pH of BHI and the effect of food matrix highlighting the impact of different (model) food recovery media on outgrowth efficiency and heterogeneity of non-heat-treated and heat-damaged B. cereus spores.

Modeling the recovery of heat-treated Bacillus licheniformis Ad978 and Bacillus weihenstephanensis KBAB4 spores at suboptimal temperature and pH using growth limits.

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory.

Fate of Bacillus anthracis during production of laboratory-scale cream cheese and homemade-style yoghurt

The viability of Bacillus anthracis during production and storage of cream cheese and yoghurt was evaluated. Experimental cheeses were manufactured from whole milk inoculated with a suspension of B. anthracis vegetative cells and spores at a final concentration of 104 cfu/ml. Lactic acid bacteria (LAB) and lab ferment were used to induce milk ripening and milk coagulation. The pH-value of the contaminated milk dropped below 4.5 within the first 6 h and the amount of LAB increased by approximately 2-logs. During cheese production and storage at 5–9 °C for 24 days no growth of B. anthracis was observed. The amount of vegetative cells and spores fluctuated by 1-log. Inoculation of whole milk with heat-treated spores at 104 cfu/ml resulted in a slight increase of vegetative cell counts during the first 6 h. This indicated that germination occurred, but replication of vegetative cells was still inhibited in the produced cheese. Incubation of cheeses at room temperature or heating after milk coagulation strongly reduced the amount of LAB but had no effect on the growth behaviour of B. anthracis. The vegetative cell and spore content remained steady at 104 cfu/100 mg. During yoghurt production the pH-value decreased within 5 h below 5 and growth of B. anthracis was inhibited throughout storage. A pH-value of 5 or less is likely a critical factor to control the growth of B. anthracis. However, spores remained viable in experimental cream cheeses and yoghurts and are a potential risk of infection.

Quantification of the impact of single and multiple mild stresses on outgrowth heterogeneity of Bacillus cereus spores

Outgrowth heterogeneity of bacterial spore populations complicates both prediction and efficient control of spore outgrowth. In this study, the impact of mild preservation stresses on outgrowth of Bacillus cereus ATCC 14579 spores was quantified during the first stages of outgrowth. Heterogeneity in outgrowth of heat-treated (90°C for 10min) and non-heat-treated germinated single spores to the maximum micro-colony stage of 256 cells was assessed by direct imaging on Anopore strips, placed on BHI plates at pH7 and pH5.5, without and with added NaCl or sorbic acid (HSA). At pH7 non-heated and heat-treated germinated spores required 6h to reach the maximum microcolony stage with limited heterogeneity, and these parameters were only slightly affected with both types of spores when incubated at pH7 with added NaCl. Notably, the most pronounced effects were observed during outgrowth of spores at pH5.5 without and with added NaCl or HSA. Non-heat-treated germinated spores showed again efficient outgrowth with limited heterogeneity reaching the maximum microcolony stage after 6h at pH5.5, which increased to 12h and 16h with added NaCl and HSA, respectively. In contrast, heat-treated spores displayed a strong delay between initial germination and swelling and further outgrowth at pH5.5, resulting in large heterogeneity and low numbers of fastest growers reaching the maximum microcolony stage after 10, 12 and 24h, without and with added NaCl or HSA, respectively. This work shows that Anopore technology provides quantitative information on the impact of combined preservation stresses on outgrowth of single spores, showing that outgrowth of germinated heat-treated spores is significantly affected at pH5.5 with a large fraction of spores arrested in the early outgrowth stage, and with outgrowing cells showing large heterogeneity with only a small fraction committed to relatively fast outgrowth.

Thermal Effects on Surface Structures and Properties of Bacillus anthracis Spores on Nanometer Scales

Bacterial spores, one of the hardiest forms of life known, can survive severe environmental stresses such as high temperature. Using thermal atomic force microscopy (AFM), we show that the surface structures and properties of Bacillus anthracis spores when exposed to elevated temperatures undergo substantial changes on nanometer scales. Thermal-blister-like nanostructures, which grow in size with increasing temperature, are formed on the spore surface when it is heated by a thermal tip. Although thermal damage to the spore surface is persistent upon cooling heat-treated spores to room temperature, thermal effects on surface properties of the spores are complex. The thermally induced nanostructures show a lower surface-tip adhesion and a higher modulus than the surrounding spore surface. The overall trend is for the adhesion to decrease with increasing temperature. However, the adhesion of heat-treated spores may be smaller than, equal to, or larger than that of untreated spores, depending upon the degree of surface damage induced by heat. Although the overall spore dimensions show few changes during and after heat treatment, the size of the spore substructures decreases significantly. In addition, we demonstrate a nanoscratch AFM method for imaging the subsurface structures of spores.

Effects of wet heat treatment on the germination of individual spores of Clostridium perfringens

To analyse the effect of wet heat treatment on nutrient and non-nutrient germination of individual spores of Clostridium perfringens. Raman spectroscopy and differential interference contrast (DIC) microscopy were used to monitor the dynamic germination of individual untreated and wet heat-treated spores of Cl.perfringens with various germinants. When incubated in water at 90-100°C for 10-30min, more than 90% of spores were inactivated but 50-80% retained their Ca(2+) -dipicolinic acid (CaDPA). The wet heat-treated spores that lost CaDPA exhibited extensive protein denaturation as seen in the 1640-1680cm(-1) (amide I) and 1230-1340cm(-1) (amide III) regions of Raman spectra, while spores that retained CaDPA showed partial protein denaturation. Wet heat-treated spores that retained CaDPA germinated with KCl or l-asparagine, but wet heat treatment increased values of T(lag) , ΔT(release) and ΔT(lys) , during which spores initiated release of the majority of their CaDPA after mixing with germinant, released >90% of their CaDPA and completed the decrease in their DIC intensity because of cortex hydrolysis, respectively. Untreated Cl.perfringens spores lacking the essential cortex-lytic enzyme (CLE), SleC, exhibited longer T(lag) and ΔT(release) values during KCl germination than wild-type spores and germinated poorly with CaDPA. Wet heat-treated wild-type spores germinating with CaDPA or dodecylamine exhibited increased T(lag) , ΔT(release) and ΔT(lys) values, as did wet heat-treated sleC spores germinating with dodecylamine. (i) Some proteins important in Cl.perfringens spore germination are damaged by wet heat treatment; (ii) the CLE SleC or the serine protease CspB that activates SleC might be germination proteins damaged by wet heat; and (iii) the CaDPA release process seems likely to be damaged by wet heat. This study provides information on the germination of individual Cl.perfringens spores and improves the understanding of effects of wet heat treatment on spores.

Identification and Characterization of a Spore-Like Morphotype in Chronically Starved Mycobacterium avium Subsp. Paratuberculosis Cultures

Mycobacteria are able to enter into a state of non-replication or dormancy, which may result in their chronic persistence in soil, aquatic environments, and permissive hosts. Stresses such as nutrient deprivation and hypoxia provide environmental cues to enter a persistent state; however, a clear definition of the mechanism that mycobacteria employ to achieve this remains elusive. While the concept of sporulation in mycobacteria is not novel, it continues to spark controversy and challenges our perceptions of a non-replication. We investigated the potential role of sporulation in one-year old broth cultures of Mycobacterium subsp. paratuberculosis (MAP).We show that dormant cultures of MAP contain a mix of vegetative cells and a previously unknown morphotype resembling a spore. These spore-like structures can be enriched for using sporulating media. Furthermore, purified MAP spore forms survive exposure to heat, lysozyme and proteinase K. Heat-treated spores are positive for MAP 16SrRNA and IS900. MAP spores display enhanced infectivity as well as maintain acid-fast characteristics upon germination in a well-established bovine macrophage model. This is the first study to demonstrate a new MAP morphotype possessing spore-like qualities. Data suggest that sporulation may be a viable mechanism by which MAP accomplishes persistence in the host and/or environment. Thus, our current understanding of mycobacterial persistence, pathogenesis, epidemiology and rational drug and vaccine design may need to be reevaluated.

DNA Damage and DNA Damage Responses in THP-1 Monocytes after Exposure to Spores of either Stachybotrys chartarum or Aspergillus versicolor or to T-2 toxin

We have characterized cell death in THP-1 cells after exposure to heat-treated spores from satratoxin G-producing Stachybotrys chartarum isolate IBT 9631, atranone-producing S. chartarum isolate IBT 9634, and sterigmatocystin-producing Aspergillus versicolor isolate IBT 3781, as well as the trichothecenes T-2 and satratoxin G. Spores induced cell death within 3-6 h, with Stachybotrys appearing most potent. IBT 9631 induced both apoptosis and necrosis, while IBT 9634 and IBT 3781 induced mostly necrosis. T-2 toxin and satratoxin G caused mainly apoptosis. Comet assay +/- formamidopyrimidine DNA glycosylase showed that only the spore exposures induced early (3h) oxidative DNA damage. Likewise, only the spores increased the formation of reactive oxygen species (ROS), suggesting that spores as particles may induce ROS formation and oxidative DNA damage. Increased Ataxia Telangiectasia Mutated (ATM) phosphorylation, indicating DNA damage, was observed after all exposures. The DNA damage response induced by IBT 9631 as well as satratoxin G was characterized by rapid (15 min) activation of p38 and H2AX. The p38 inhibitor SB 202190 reduced IBT 9631-induced H2AX activation. Both IBT 9631 and T-2 induced activation of Chk2 and H2AX after 3 h. The ATM inhibitor KU 55933, as well as transfection of cells with ATM siRNA, reduced this activation, suggesting a partial role for ATM as upstream activator for Chk2 and H2AX. In conclusion, activation of Chk2 and H2AX correlated with spore- and toxin-induced apoptosis. For IBT 9631 and satratoxin G, additional factors may be involved in triggering apoptosis, most notably p38 activation.

Postharvest hot water treatment for the control of Thielaviopsis black rot of pineapple

Abstract Black rot of pineapple (Chalara paradoxa (De Seyen.) Sacc.) is a common postharvest problem in many countries. Consumer resistance to the use of fungicides has precipitated the need for alternative means of controlling the disease. In vitro studies on heat-treated (50 °C for 3 min) spores of the pathogen showed a mean colony count of 11 ± 0.7, following 48 h incubation at 28 °C, while plates with heat-treated spores at 54 and 58 °C showed a mean colony count of 1 ± 1.0 and 1 ± 0.7, respectively. Pineapples inoculated with 104 spores/ml, C. paradoxa, followed by a hot water dip treatment at 54 °C for 3 min were free of disease when stored at 10 °C for 21 days followed by 48 h at an ambient temperature (28 ± 2 °C). Inoculated dip-treated fruit held at 28 ± 2 °C for 6 days also remained healthy. However, characteristic symptoms of the disease were observed in fruit that were inoculated and held as controls under similar storage conditions, with no hot water dip treatment. No significant difference occurred between hot water dip-treated and untreated controls with respect to flesh and shell colour of fruit, ascorbic acid levels and titratable acidity. Mean ascorbic acid level was 18.8 mg/100 g in fruit stored at 10 °C compared with 9.3 mg/100 g in fruit stored at 28 ± 2 °C, irrespective of whether they were inoculated or non-inoculated fruit. A significant difference (p

MALDI-TOF-MS analysis of bacterial spores: Wet heat-treatment as a new releasing technique for biomarkers and the influence of different experimental parameters and microbiological handling

Short wet heat-treatment is presented as a new technique to release high-mass biomarkers to obtain strain-specific fingerprints of intact bacterial spores by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Wet heat-treatment was applied for several minutes (3-30) by two techniques using either a screw-cap tube submerged in a glycerol bath at 120 degrees C or an Eppendorff-tube submerged in a water bath at 100 degrees C. Both techniques turned out to be successful for releasing high-mass biomarkers. The influence of different experimental parameters and microbiological handling on the peak pattern of the released high-mass biomarkers was studied. While the sporulation medium, the applied washing procedure, and the choice of matrix crucially influenced the peak pattern, other parameters like storage conditions were found to be insignificant. A protocol of optimized experimental conditions for MALDI-MS of wet heat-treated spores is presented.

Luminometric and differential scanning calorimetry (DSC) studies on heat - and radiation inactivation of Bacillus Subtilis luxAB spores

A bioluminescent derivative of Bacillus subtilis containing a plasmid encoding a luxAB fusion under control of a vegetative promoter and gives bioluminescence upon addition of an exogenous long-chain aldehyde has been used as test organism. Its spore populations have been produced and their heat- and radiation survival curves established. Heat-sensitization effect of pre-irradiation of spores was proven not only by colony counting but also with differential scanning calorimetry. Under a linearly programmed temperature increase, the heat destruction of spores surviving 2.5 kGy gamma irradiation resulted in at a few centigrade lower temperature than that of untreated spores. Heat denaturation endotherms in the DSC-thermogram of irradiated spores were shifted to lower temperatures as well. Comparative turbidimetric, luminometric and phase-contrast microscopic studies of untreated, heat-treated and irradiated spore populations showed that the kinetics of germination and the light emission during germination of radiation-inactivated spores were the same as those of untreated spores, revealing that the pre-formed luciferase enzyme packaged into the spores during sporulation remained intact after an irradiation dose causing 90% decrease in number of colony forming spores. Therefore, in contrast to heat-treated spores, the initial bioluminescence reading upon germination of irradiated spores does not reflect the viable count of their population.

Differences in Detected Fluorescence Among Several Bacterial Species Measured with a Direct-Reading Particle Sizer and Fluorescence Detector

Naturally-contained fluorescing chemicals (such as riboflavin or NADPH) can be used to detect the presence of biological organisms. A new instrument from TSI Incorporated measures fluorescence of particles using an ultraviolet laser operating at an excitation wavelength of 355 nm. We have employed this instrument (Model 3312 Ultraviolet Aerodynamic Particle Sizer (tm) Spectrometer) to assess the degree of fluoresence associated with a variety of biological aerosols. Nonfluorescent and fluorescent latex sphere and sodium chloride aerosols were first used to assure proper operation of the instrument and to obtain correct instrument settings. Biological aerosols were then generated by combining organisms with double distilled and filtered water in a Collison nebulizer operated at low pressure. After passage through a charge neutralizer and dilution with humidified air (45%RH), the aerosol was measured downstream for both particle size and fluorescence distributions. Bacterial aerosols generated include Bacillus subtilis subsp. niger (spores and vegetative cells), Staphylococcus epidermidis, Eschericia coli, and Mycobacterium abscessus (a surrogate for M. tuberculosis). Cladosporium spp. fungal spores were also evaluated, and the effect of heat treatment on fluorescence was tested using B. subtilis spores. For each test the percentage of organisms that produced a fluorescence signal above a threshold was recorded. The organisms demonstrated considerable differences in percent fluorescence, ranging from means of 11% for S. epidermidis to 44% for B. subtilis spores. Vegetative cells of B. subtilis were generally less fluorescent (mean of 33%) than the spores, while the highest level of fluorescence was associated with heat-treated spores (averaging about 75%). This instrument has some potential for use in settings where immediate detection of biological organisms is important. Work remains to be done on understanding the effect on fluorescence of organism viability, presence of nonbiological particles, and interferences from mixtures.