Inactivation Of Vegetative Cells Research Articles

Evaluation of the additive effects of volatile fatty acids and moderate heat treatment for enhancing the inactivation of vegetative cells and spores of Clostridium perfringens by flow cytometry

ObjectivesClostridium perfringens is a well-known spore-forming bacterium that can resist the environment. A mixture of volatile fatty acids or thermal treatments can interact with these bacteria. The aim of this study was to evaluate the effects of different volatile fatty acid concentrations and moderate heat treatment on Clostridium perfringens sporulation. MethodsA pure culture of Clostridium perfringens type A in Duncan Strong medium was treated with a mixture of volatile fatty acids at several concentrations. A thermal treatment was also tested. To evaluate the effects, a double staining method was employed, and treatments on Clostridium perfringens were analysed by flow cytometry. ResultsModerate heat treatment destroyed vegetative forms but had no effect on sporulating forms. Volatile fatty acids combined with moderate heat treatment inhibited Clostridium perfringens sporulation. ConclusionsThe use of flow cytometry as an original method for evaluating the treatment of Clostridium perfringens is of interest because of its simplicity, short time to obtain results, and the level of information provided on the microbial population (impact on metabolism). A combination of mild treatments (moderate heat treatment + volatile fatty acids) to decrease the Clostridium perfringens concentration when these bacteria sporulate is a very promising finding for inhibiting Clostridium perfringens propagation.

Genetic evidence for the interaction between Bacillus anthracis-encoded phage receptors and their cognate phage-encoded receptor binding proteins.

Bacteriophages such as γ and AP50c have been shown to infect strains of Bacillus anthracis with high specificity, and this feature has been exploited in the development of bacterial detection assays. To better understand the emergence of phage resistance, and thus the potential failure of such assays, it is important to identify the host and phage receptors necessary for attachment and entry. Using genetic approaches, the bacterial receptors of AP50c and γ have been identified as sap and GamR, respectively. A second AP50c-like phage, Wip1, also appears to use sap as a receptor. In parallel with this work, the cognate phage-encoded receptor binding proteins (RBPs) have also been identified (Gp14 for γ, P28 for AP50c, and P23 for Wip1); however, the strength of evidence supporting these protein-protein interactions varies, necessitating additional investigation. Here, we present genetic evidence further supporting the interaction between sap and the RBPs of AP50c and Wip1 using fluorescently tagged proteins and a panel of B. anthracis mutants. These results showed that the deletion of the sap gene, as well as the deletion of csaB, whose encoded protein anchors sap to the bacterial S-layer, resulted in the loss of RBP binding. Binding could then be rescued by expressing these genes in trans. We also found that the RBP of the γ-like prophage λBa03 relied on csaB activity for binding, possibly by a different mechanism. RBPλBa03 binding to B. anthracis cells was also unique in that it was not ablated by heat inactivation of vegetative cells, suggesting that its receptor is still functional following incubation at 98°C. These results extend our understanding of the diverse attachment and entry strategies used by B. anthracis phages, enabling future assay development.

Combination of radio frequency heating and antibacterial agents for microorganism control in the production of packaged tofu

The microorganism control in packaged tofu by radio frequency (RF) heating combined with antimicrobial agents was investigated. The inactivation of vegetative cells and spores by RF heating alone or in combination with antimicrobial agents was evaluated. Results showed that vegetative cells of typical food microorganisms, such as E.coli,S.aureus, and Bacillussubtilis, were completely inactivated by RF heating at 80 °C for 20 min, while no significant reduction in the population of spores was observed. Additionally, the growth of Bacillus spores was effectively inhibited by 0.312 mg/g of nisin. The total viable count (TVC) in RF-treated packaged tofu was below 2 log CFU/g during the storage at 4 or 37 °C for 10 days in the presence of nisin. Furthermore, the quality analysis indicated that the pH, texture, color, and volatile profile of samples were well maintained, which contributed to the favorable sensory acceptance of the consumers. These results suggested that effective microorganism control in packaged tofu was achieved by combining RF heating and nisin. The developed method was recommended to prevent spoilage of packaged tofu and extend its shelf-life.

Evaluation of the effectiveness of ultraviolet disinfection (254 nm) of aqueous solutions from phytopathogenic microorganisms

Relevance. Currently, greenhouse complexes use water for irrigation from open surface sources (ponds, lakes, rivers) and underground water from wells, adding a return (drainage) solution to it. At the same time, water from both open sources and drainage solution often becomes a source of emergence and spread of phytopathogenic infection in greenhouses, which brings economically significant crop losses. In this regard, the need to develop guaranteed methods and methods for the disinfection of drainage water and water from open sources is relevant. One of the methods of water disinfection is the use of ultraviolet irradiation, but for the effective use of irradiation, it is necessary to ensure the minimum effective UV dose. This UV dose is typical for each pathogen.Results. Work was carried out to experimentally establish doses of UV irradiation that allow complete inactivation of vegetative cells and spores of bacteria and fungi (Agrobacterium tumefaciens, Pythium debaryanum, Fusarium oxysporum, Verticillium dahliae, Phoma destructiva) in a solution prepared on the basis of distilled water. A dose of UV irradiation of 40 mJ/cm2 ensured complete inactivation of the bacterial pathogen, and a dose of 60 mJ/cm2 showed complete inactivation of the pathogens of the studied fungal infections.

Mechanism insights into bacterial sporulation at natural sphalerite interface with and without light irradiation: The suppressing role in bacterial sporulation by photocatalysis.

Unveiling the mechanisms of bacterial sporulation at natural mineral interfaces is crucial to fully understand the interactions of mineral with microorganism in aquatic environment. In this study, the bacterial sporulation mechanisms of Bacillus subtilis (B. subtilis) at natural sphalerite (NS) interface with and without light irradiation were systematically investigated for the first time. Under dark condition, NS was found to inactivate vegetative cells of B. subtilis and promote their sporulation simultaneously. The released Zn2+ from NS was mainly responsible for the bacterial inactivation and sporulation. With light irradiation, the photocatalytic effect from NS could increase the bacterial inactivation efficiency, while the bacterial sporulation efficiency was decreased from 8.1% to 4.5%. The photo-generated H2O2 and O2- played the major roles in enhancing bacterial inactivation and suppressing bacterial sporulation process. The intracellular synthesis of dipicolinic acid (DPA) as biomarker for sporulation was promoted by NS in dark, which was suppressed by the photocatalytic effect of NS with light irradiation. The transformation process from vegetative cells to spores was monitored by both 3D-fluerecence EEM and SEM observations. Compared with the NS alone system, the NS/light combined system induced higher level of intracellular ROSs, up-regulated antioxidant enzyme activity and decreased cell metabolism activity, which eventually led to enhanced inactivation of vegetative cells and suppressed bacterial sporulation. These results not only provide in-depth understanding about bacterial sporulation as a new mode of sub-lethal stress response at NS interface, but also shed lights on putting forward suitable strategies for controlling spore-producing bacteria by suppressing their sporulation during water disinfection.

Inactivation of vegetative cells, germinated spores, and dormant spores of Bacillus atrophaeus by pulsed electric field with fixed energy input

AbstractIncreasing the electric energy input during pulsed electric field (PEF) treatment is a straightforward technique to boost the efficiency of microbial inactivation. However, drawbacks such as electrode corrosion, arc formation, and high cost are also involved. This study aims to investigate the impact of medium conductivity (), electric field strength (), and the number of pulses () on the inactivation of vegetative cells, germinated spores, and dormant spores of Bacillus atrophaeus with fixed energy input. Our results indicated that (1) vegetative cells were susceptible to PEF treatment, and the relative importance of , , and was ; (2) germinated spores were more resistant to PEF treatment than vegetative cells, and the relative importance of , , and was ; and (3) similar to the thermal resistance property, dormant spores were also highly resistant to PEF treatment and were not significantly inactivated in our experiment setting. It is concluded that the degree of PEF resistance of cell at different physiological states may be related to its structure and size. This study added to the body of knowledge on selecting PEF treatment settings and gave insight into the scaling up of PEF applications in the food processing industry.Practical ApplicationsIn the food processing industry, eliminating contaminants such as bacterial spores is essential, and PEF processing is a common nonthermal technique. While the traditional method of increasing the electric energy input to increase microbial inactivation efficiency is beneficial, it is not cost‐effective. The results of this investigation demonstrated that decreasing spore resistance through germination followed by parameter‐optimized PEF treatments is a practical method for improving pasteurization efficiency. This novel technique might be used in the food sector.

Thermal inactivation kinetics of seven genera of vegetative bacterial pathogens common to the food chain are similar after adjusting for effects of water activity, sugar content and pH

A predictive model was made for the logarithm of the thermal decimal reduction time (logD) of Salmonella enterica (D = time to 90% reduction by inactivation). The model was fitted with multiple linear regression from 521 logD-values reported in literature for laboratory media and foods highly varying in water activity and pH. The single regression model with temperature as the only variable had a high residual standard error (RSE) of 0.883 logD and no predictive value (fraction of variance explained (R2) < 0.001). Adding water activity, sugar content and pH as predictors resulted in a model with a lower RSE of 0.458 logD and an adjusted R2 of 0.73. The model was validated by comparing 985 predicted with observed logD for S. enterica from other publications. The model was subsequently validated with 1498 published logD-values for inactivation of vegetative cells of nine other pathogenic bacteria genera (mainly Listeria monocytogenes, Escherichia coli, Clostridium perfringens, Cronobacter spp., Staphylococcus aureus, Yersinia enterocolitica) in or on a variety of laboratory media, meat, fish, dairy, nuts, fruits and vegetables. Regression analyses for validation with the 985 logD of S. enterica and 2483 logD of all genera show deviations from the expected slope of 1 (both 0.81) and the expected intercept of 0 (0.04 and 0.19 logD respectively). However, only 0.7% and 2% respectively of the new logD (expected: 0.5%) were observed above the 99% prediction interval of the original S. enterica model based on 521 logD. The findings suggest that i) the variability of thermal resistance of strains within species is larger than between genera and species; ii) one generic predictive model, also accounting for variability, suffices for designing the thermal inactivation of a variety of vegetative pathogenic bacteria in many food types.

Inactivation of Microorganisms in Foods by Ohmic Heating: A Review

Ohmic heating (OH) is an alternative food processing technology for effectively inactivating microorganisms that depends on the heat that has been generated when electrical current passes directly through food material. The advantages of OH for microbial inactivation include shorter heating time, more uniform heat distribution inside food, reduced nutrition losses, and higher energy efficiency. This review presents some published information regarding the inactivation of microorganisms by OH, including the major factors that influence the inactivation effectiveness of OH, the inactivation of vegetative cells and spores in foods by OH, the inactivation mechanisms of OH, and the challenges and prospects of OH for food processing. This information will improve the understanding of OH for inactivation of microorganisms and promote the application of OH in the food industry.

Inactivation of spores and vegetative cells of Bacillus subtilis and Geobacillus stearothermophilus by pulsed light

Abstract The effect of pulsed light (PL) on the inactivation of vegetative cells and spores of Bacillus subtilis and Geobacillus stearothermophilus at different cell densities was evaluated. The antimicrobial effect of PL decreased when population density increased, both for vegetative cells and spores of B. subtilis and G. stearothermophilus . For low cell densities, vegetative cells were more sensitive to PL than spores. However, lower reductions in vegetative cell counts were shown for higher cell densities, which could be attributed to the fact that vegetative cell suspensions transmitted less amount of light than spores. Concerning the resistance of both microorganisms, lower reduction in G. stearothermophilus than B. subtilis counts were found for the same cell density. When cell suspensions with similar light transmittance were compared, vegetative cells of B. subtilis were found to be more sensitive than the ones of G. stearothermophilus , while the spores of G. stearothermophilus were less resistant to PL.

Inactivation of thermoduric aerobic sporeformers in milk by ultrasonication

Thermoduric sporeformers can survive milk pasteurization and cause spoilages of dairy products. In the present study, ultrasonication was evaluated as a non-thermal processing technique to inactivate the thermally resistant vegetative cells of spore forming Bacillus spp. During the challenge studies, vegetative cells of Bacillus coagulans (ATCC® 12245), Anoxybacillus flavithermus (DSM 2641), Bacillus sporothermodurans (DSM 10599), Bacillus licheniformis (ATCC®6634), and Geobacillus stearothermophilus (ATCC® 15952) were studied for their survivability to batch pasteurization (63 °C/30 min) in skim and whole milk samples. Experiments were conducted to study the effect of ultrasonication alone or in combination with pasteurization on inactivation of vegetative cells of some thermoduric Bacillus spp. Effect of ultrasonication on milk pH, color, and alkaline phosphatase activity was also investigated. Vegetative cells of B. coagulans and A. flavithermus survived pasteurization treatment in both skim and whole milk samples. Ultrasonication at 80% amplitude for 10 min however, inactivated the vegetative cells of B. coagulans and A. flavithermus in skim milk by 4.53, and 4.26 logs, respectively. A combined treatment of pasteurization (63 °C/30 min) followed by ultrasonication completely eliminated approximately log 6 cfu/mL of these cells in skim milk. As visualized under the scanning electron microscope, ultrasonication physically disintegrated vegetative cells of sporeformers. Ultrasonication treatment caused significant reduction (P < 0.05) in brightness and greenness of milk; whereas, blueness (b*) of milk was increased. However, pH and alkaline phosphatase activity (P > 0.05) of treated skim milk were not affected.

막걸리에 접종한 Bacillus subtilis의 초고압에 대한 저항력

막걸리에서 분리된 B. subtilis의 특성을 파악하고자 영양유무(buffer와 TSB), pH(pH 7과 4, 막걸리 pH 4), adaptation 조건에 따라 초고압에 의한 저감 효과를 살펴보았다. B. subtilis 포자는 영양유무, pH에 관계없이 환경 적응력이 있었고, 초고압에 의한 저감효과도 미미하게 관찰되었다. B. subtilis 영양세포는 영양유무와 pH에 따라 대조구(non-treatment)의 균수 차이가 관찰되었고, 초고압에 의한 저감 효과도 다르게 나타났다. B. subtilis 영양세포는 저온에서 영양과 pH가 불리한 환경에서 endospore를 형성하여 적응을 하게 되고 초고압에 의한 저감효과는 감소하는 것으로 나타났다. 특히 막걸리에 접종한 경우는 buffer(pH 4)와 TSB(pH 4)보다 적응력이 약한 것으로 관찰되었다. B. subtilis 포자를 접종한 막걸리의 저장 실험 결과, 내열성이 있으며 균의 증식은 관찰되지 않았다. 결론적으로, 막걸리에서 B. subtilis는 대부분 포자상태로 존재하며, 포자는 초고압에 대한 저항력이 큰 것을 알 수 있었다. 그러나 초고압은 영양세포 사멸에 효과적이었다. In order to understand the effect of hydrostatic pressure (HP) on Bacillus subtilis isolated from makgeolli, the survival of B. subtilis after HP treatment (400 MPa for 5 min) in various substrates including phosphate buffer, tryptone soya broth at pH 7 and 4, and makgeolli at pH 4 was evaluated depending on bacterial forms (spores and vegetative cells) and adaptation conditions (<TEX>$25^{\circ}C$</TEX> for 3 h, or <TEX>$10^{\circ}C$</TEX> for 24 h). Spores were generally resistant to HP (<1 log reduction) regardless of conditions. In contrast, vegetative cells were generally susceptible to HP (up to 3 log reduction-except makgeolli) and were more susceptible after 3 h at <TEX>$25^{\circ}C$</TEX> compared to 24 h at <TEX>$10^{\circ}C$</TEX>. In vegetative cells inoculated makgeolli (7 log CFU/mL), the colonies were not detected after 24 h at <TEX>$10^{\circ}C$</TEX>. Consequently, B. subtilis in makgeolli easily existed as spores and the spores were resistant to HP. Results demonstrate that HP was more promising in the inactivation of vegetative cells.

Long-Term Survival of Bacillus Spores in Alcohol and Identification of 90% Ethanol as Relatively More Spori/Bactericidal

This study was taken up with a view to generate basic information on spore hardiness to ethanol in various Bacillus species and related genera, and to assess the effectiveness of different levels of ethanol as a bacterial disinfectant. Predominantly spore-bearing cultures of five Bacillus spp. (B. pumilus, B. subtilis, B. megaterium, B. fusiformis and B. flexus) that were isolated from the spent-alcohol used during plant tissue culture work were challenged with aqueous ethanol (25, 50, 60, 70, 80 and 90% v/v) in 1 ml volumes at 10¹⁰⁻¹¹ CFU ml⁻¹. Monitoring the spore endurance through spotting and plating revealed prolonged tolerance (>12 months) at different alcohol levels depending on the organism except in 90% where no survival was observed beyond 2-12 months. Spores of related genera like Paenibacillus and Lysinibacillus also showed long-term ethanol survival. Alcohol tolerance of spore-forming organisms depended on the extent of spores and spore hardiness, which in turn varied with the organism, strain, age of culture, growing conditions and other factors as authenticated with ATCC strains of B. pumilus and B. subtilis. Aqueous 90% ethanol caused instant inactivation of vegetative cells in different spore formers and twelve other non-sporulating Gram-positive and Gram-negative organisms tested. Taking into account both vegetative cells and spores, the appropriate concentration of ethanol as a disinfectant emerged to be 90% followed by absolute ethanol compared with the generally recommended 70-80% level.

Inactivation of Vegetative Cells by Continuous High‐Pressure Processing: New Insights on the Contribution of Thermal Effects and Release Device

Dynamic or continuous high-pressure processing of fluid foods has drawn significant interest as a microbial reduction process in the past decade, and many attempts have been made to better understand the mechanisms involved in that reduction. This study was intended to provide insight into the contributions of thermal effects and differences in pressure release components in the inactivation of 2 vegetative pathogen analogs--the Gram-positive Listeria innocua and the Gram-negative Escherichia coli. Fluids containing microbial loads of 10(8) or greater were subjected to continuous high-pressure processing at 200 to 210 MPa. Without active cooling of the release components, all fluids experienced a temperature rise in excess of 70 °C, thus occluding any pressure-related effects for all release components. Active cooling of the valve bodies of the 2 valve-style release components (a conical disruption valve and a micrometering valve) allowed the temperature rise to be abated enough to isolate the effects unique to a given valve. In Tryptic soy broth trials, the mean inactivation levels of E. coli between valves were similar--5.16 log and 5.33 log for the micrometering and conical disruption valves, respectively. When repeated with L. innocua, a similar inactivation level was observed in the conical disruption valve (5.1 log) but not the micrometering valve (3.02). Listeria innocua trials were also repeated using fluid whole milk, which showed a lower levels of inactivation--2.04 log for the micrometering valve and 2.51 log for the conical valve. This paper compares some of the most common pressure release components used in continuous high-pressure processing and attempts to isolate the contributions of thermal effects from those of pressure and shear. This information is important to those seeking to compare and evaluate the effectiveness of a proposed set of process parameters for microbial inactivation. Further, the ability to reduce the extreme nature of the temperature rise has the potential to expand the use of process into more temperature sensitive products.

Effects of microwave and ultrasonic wave treatment on inactivation of Alicyclobacillus

SummaryThe objective of this study was to determine the effects of microwave (MW), ultrasonic wave (UW) and their combination treatments for the same length of time (10 or 30 min) on the inactivation of vegetative cells of three different strains of Alicyclobacillus, (Alicyclobacillus acidiphilus DSM 14558T, Alicyclobacillus acidoterrestris DSM 3922T and Alicyclobacillus cycloheptanicus DSM 4006T) in a laboratory medium. The results showed that A. acidoterrestris DSM 3922T seemed to be the most susceptible strain. It was also found that when MW or UW was employed separately, UW was more effective and the effectiveness of MW and UW was improved as the power and exposure time increased. By comparison, UW treatment followed by MW was significantly better than the treatment of MW followed by UW. Furthermore, combined MW and UW treatment had a markedly better effect than separate MW processing, but had a less effect than UW alone, except that the inactivation of A. cycloheptanicus DSM 4006T was significantly increased (P &lt; 0.05) by the 30‐min treatment of UW (400 W for 20 min) + MW (900 W for 10 min).

Membrane permeabilization and cellular death of Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae as induced by high pressure carbon dioxide treatment

In this study, the relationship between (irreversible) membrane permeabilization and loss of viability in Escherichia coli, Listeria monocytogenes and Saccharomyces cerevisiae cells subjected to high pressure carbon dioxide (HPCD) treatment at different process conditions including temperature (35–45 °C), pressure (10.5–21.0 MPa) and treatment time (0–60 min) was examined. Loss of membrane integrity was measured as increased uptake of the fluorescent dye propidium iodide (PI) with spectrofluorometry, while cell inactivation was determined by viable cell count. Uptake of PI by all three strains indicated that membrane damage is involved in the mechanism of HPCD inactivation of vegetative cells. The extent of membrane permeabilization and cellular death increased with the severity of the HPCD treatment. The resistance of the three tested organisms to HPCD treatment changed as a function of treatment time, leading to significant tailing in the survival curves, and was dependent on pressure and temperature. The results in this study also indicated a HPCD-induced damage on nucleic acids during cell inactivation. Transmission electron microscopy showed that HPCD treatment had a profound effect on the intracellular organization of the micro-organisms and influenced the permeability of the bacterial cells by introducing pores in the cell wall.

MALDI-TOF Mass Spectrometry Compatible Inactivation Method for Highly Pathogenic Microbial Cells and Spores

Identification of microorganisms, specifically of vegetative cells and spores, by intact cell mass spectrometry (ICMS) is an emerging new technology. The technique provides specific biomarker profiles which can be employed for bacterial identification at the genus, species, or even at the subspecies level holding the potential to serve as a rapid and sensitive identification technique in clinical or food microbiology and also for sensitive detection of biosafety level (BSL) 3 microorganisms. However, the development of ICMS as an identification technique for BSL-3 level microorganisms is hampered by the fact that no MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) compatible inactivation procedure for microorganisms, and particularly for bacterial endospores, has been evaluated so far. In this report we describe a new methodology for effective inactivation of microorganisms which is compatible with the analysis of microbial protein patterns by MALDI-TOF mass spectrometry. The main challenge of this work was to define the conditions that ensure microbial inactivation and permit at the same time comprehensive analysis of microbial protein patterns. Among several physical, chemical, and mechanical inactivation procedures, inactivation by trifluoroacetic acid (TFA) proved to be the best method in terms of bactericidal capacity and information content of the mass spectra. Treatment of vegetative cells by 80% TFA alone for 30 min assured complete inactivation of microbial cells under all conditions tested. For spore inactivation, the "TFA inactivation protocol" was developed which is a combination of TFA treatment with basic laboratory routines such as centrifugation and filtering. This MALDI-TOF/ICMS compatible sample preparation protocol is simple and rapid (30 min) and assures reliable inactivation of vegetative cells and spores of highly pathogenic (BSL-3) microorganisms.

Inactivation of vegetative cells, but not spores, of Bacillus anthracis, B. cereus, and B. subtilis on stainless steel surfaces coated with an antimicrobial silver- and zinc-containing zeolite formulation.

Stainless steel surfaces coated with paints containing a silver- and zinc-containing zeolite (AgION antimicrobial) were assayed in comparison to uncoated stainless steel for antimicrobial activity against vegetative cells and spores of three Bacillus species, namely, B. anthracis Sterne, B. cereus T, and B. subtilis 168. Under the test conditions (25 degrees C and 80% relative humidity), the zeolite coating produced approximately 3 log(10) inactivation of vegetative cells within a 5- to 24-h period, but viability of spores of the three species was not significantly affected.