Salmonella Survival on Pecans as Influenced by Processing and Storage Conditions

Survival of Salmonella senftenberg 775W, S. anatum, and S. typhimurium during exposure to currently practiced, as well as abusive, pecan processing and storage conditions was studied. Thermal treatments normally carried out during the processing of pecans are inadequate to consistently destory salmonellae in highly contaminated inshell nuts. Pecan nut packing tissue was toxic to salmonellae, thus affording some protection against high initial contamination and subsequent survival of the organisms. Examinations of inoculated inshell pecans stored at -18, -7, 5, and 21 C for up to 32 weeks revealed that the extent of survival was inversely correlated to the storage temperature. S. senftenberg 775W and S. anatum were not detectable on inshell nuts after 16 weeks of storage at 21 C. Little decrease in viable population of the three species was noted on inoculated pecan halves stored at -18, -7, and 5 C for 32 weeks. Due to organoleptic quality deterioration in pecan nutmeats at elevated temperatures, sterilization methods other than thermal treatment appear to be required for the elimination of viable salmonellae from pecan nuts.     

Improved Temperature-Gradient Incubator and the Maximal Growth Temperature and Heat Resistance of Salmonella

An improved all-metal temperature-gradient incubator produces its gradient by means of a bar permanently installed in a near-vertical position with its lower end in a cool constant-temperature water bath and with thermostatically controlled heaters near its top. Bolts hold the incubator in contact with the temperature-gradient bar, and polyurethane foam insulates the entire assemblage during use. Maximal growth temperatures of 34 representative strains of Salmonella were found to be between 43.2 and 46.2 C. In an agar medium with an initial level of 10⁶ cells per milliliter, no strain survived 50 C for 48 hr. S. senftenberg 775W showed no greater heat resistance at or near 48 C than did other species or other S. senftenberg strains. However, it was considerably more resistant than other strains at 55 C.     

Sporicidal properties of hydrogen peroxide against food spoilage organisms

The sporicidal properties of hydrogen peroxide were evaluated at concentrations of 10 to 41% and at temperatures of 24 to 76 C. The organisms tested and their relative resistance at 24 C to 25.8% H₂O₂ were: Bacillus subtilis SA 22 > B. subtilis var. globigii > B. coagulans > B. stearothermophilus > Clostridium sp. putrefactive anaerobe 3679 > S. aureus, with „D” values of 7.3, 2, 1.8, 1.5, 0.8., and 0.2 min, respectively. Heat shocking spores prior to hydrogen peroxide treatment decreased their resistance. Wet spores were more resistant than dry spores when good mixing was achieved during hydrogen peroxide treatment. Inactivation curves followed first-order kinetics except for a lag period where the inactivation rate was very slow. Increasing the H₂O₂ concentration and the temperature reduced the lag period.     

Thermal Resistance of Salmonellae Isolated from Dry Milk

Salmonella anatum, S. binza, S. cubana, S. meleagridis, S. newbrunswick, and S. tennessee isolated from dry milk, and S. senftenberg 775W were studied for heat resistance to determine whether these organisms would survive pasteurization as recommended by the 1965 Pasteurized Milk Ordinance of the U.S. Public Health Service. Thermal inactivation determinations were made on washed cells of the test microorganisms suspended in sterile whole milk. Excluding S. senftenberg, D values ranged from 3.6 to 5.7 sec at 62.8 C, from 1.1 to 1.8 sec at 65.6 C, and from 0.28 to 0.52 sec at 68.3 C. Corresponding values for S. senftenberg were 34.0, 10.0, 1.2, and 0.55 sec for respective exposure temperatures of 65.5, 68.3, 71.7, and 73.9 C. The present milk pasteurization processes as recommended by the Public Health Service will inactivate all seven strains of salmonellae studied, provided that the initial concentration does not exceed a calculated 3 × 10¹² salmonellae per ml of milk.     

Combined Effects of Ultrahigh Vacuum and Temperature on the Viability of Some Spores and Soil Organisms

Considerably fewer spores of Bacillus stearothermophilus, B. megaterium, and Clostridium sporogenes were recovered than were spores of B. subtilis var. niger and Aspergillus niger after 4 to 5 days at 53 and 60 C in ultrahigh vacuum. There were no significant differences in the recoveries of these five organisms at 25 C and atmospheric pressure, and after exposure to 25 and -190 C in vacuum. At 60 C, a far greater decrease in viability was demonstrated for B. stearothermophilus, B. megaterium, and C. sporogenes in ultrahigh vacuum than at atmospheric pressure. Viable B. subtilis var. niger spores were not detected in an initial 10⁷ spores after retention at 90 C and ultrahigh vacuum, and 10⁴ spores were viable after 5 days at 90 C and atmospheric pressure from an initial 10⁶ spores. Molds and actinomycetes in soil were particularly resistant up to 69 C in vacuum. Actinomycetes were the only soil organisms recovered so far at 120 C.     

Time-Temperature Effects on Salmonellae and Staphylococci in Foods: II. Thermal Death Time Studies

Thermal death time studies were conducted at 5 F intervals from 130 to 150 F with strains of salmonellae and enterotoxigenic staphylococci. Heat-resistant Salmonella senftenberg strain 775W, Staphylococcus aureus strains 196E and Ms149, and non-heat-resistant Salmonella manhattan were studied in custard, chicken à la king, and ham salad.

The F₁₄₀ values (minutes of exposure at 140 F required to effect 100% destruction) were as follows: S. senftenberg 775W in custard 78, and chicken à la king 81.5; S. manhattan in custard 19, and chicken à la king 3.1; S. aureus 196E in custard 59, and chicken à la king 47; S. aureus Ms149 in custard 53, and chicken à la king 40.

The end points of survival-kill at all the test temperatures for both salmonellae and staphylococci in ham salad were considerably less than for the other foods studied.

D₁₄₀ values (minutes of exposure at 140 F required to effect a 90% reduction in numbers) were also calculated from the data and presented.

Values for z_F and z_D (slope of the thermal-death-time and decimal-reduction-time curves) are also presented and discussed in relation to type of food, organism, and temperature.

These data indicate that heating perishable foods of the type studied to 150 F and holding every particle of food at this temperature for at least 12 min reduces 10 million or less salmonellae or staphylococci per gram to nondetectable levels.

The same degree of destruction is achieved in similarly contaminated foods when held at 140 F for 78 to 83 min.

On the basis of the calculation procedures employed, it is estimated that 45-min exposure at 140 F would be necessary to reduce 1,000 organisms per gram to nondetectable levels.

     

Resistivity of Spores to Ultraviolet and γ Radiation while Exposed to Ultrahigh Vacuum or at Atmospheric Pressure

Viability studies were conducted on microbial spores subjected to ultrahigh vacuum (UHV) in the 10^-9 to 10^-10 torr range. After 5 to 7 days in vacuum, they were exposed to ultraviolet (UV) or to γ radiation either while still under vacuum or in the presence of dried air. Among the four test organisms subjected to UHV and ultraviolet radiation, Aspergillus niger was the most resistant; Bacillus megaterium, B. subtilis var. niger, and B. stearothermophilus were about equally less resistant. All four spores were more sensitive to ultraviolet radiation when UHV-dried than when desiccant-dried. Of the four test organisms subjected to UHV and γ radiation, B. megaterium proved to be the most resistant; A. niger was the least resistant; and the remaining two organisms were of intermediate resistivity. All four organisms were less radiation resistant when UHV-dried than when irradiated in their normally hydrated state, and all showed an increased radiosensitivity after vacuum drying when oxygen was present. In addition, spores of B. subtilis var. niger and A. niger were less radiosensitive when UHV-dried and irradiated in vacuum than when “wet” and irradiated in air, whereas the reverse relationship was observed for the remaining two organisms. Based on the fact that microbial contaminants can be readily shielded from UV light by soils, metal particles, etc., and considering that the levels of ionizing radiations reported to be present in interstellar space are generally lower than those used in these experiments, the decrease in radioresistivity imparted by UHV drying is not of a sufficient magnitude to sterilize dependably portions of a spacecraft while on a mission.     

Degradation of nicosulfuron by Bacillus subtilis YB1 and Aspergillus niger YF1

The optimal degrading conditions for the nicosulfuron degradation by Bacillus subtilis YB1 and Aspergillus niger YF1, and site of their action on nicosulfuron were studied. The results showed that the degradation efficiency of free cells of B. subtilis YB1 and A. niger YF1 was respectively 87.9 and 98.8% in basic medium III containing 2 mg/l of nicosulfuron after inoculation with 1 ml of culture containing 2.3 × 10⁷ CFU ml^?1 and incubation for 5 days at 35°C. Moreover, the degradation rate of nicosulfuron by the mixture of microorganisms was much higher than for every of them taken separately in the same conditions. The mass spectrometric analysis of the products degraded by B. subtilis YB1 revealed that the sulfonylurea bridge in nicosulfuron molecule had been broken. Extracellular (EXF) and endocellular (ENF) fractions obtained from bacterium and fungus were tested for the ability to degrade nicosulfuron. The degradation efficiency of fractions extracted from B. subtilis YB1 was 66.8% by EXF and 15.8% by ENF, but neither EXF nor ENF extracted from A. niger YF1 had the activity of degrading nicosulfuron.     

Sporicidal Activity of Peracetic Acid and β-Propiolactone at Subzero Temperatures

A method was developed to evaluate and measure the sporicidal activity of peracetic acid (PAA) and β-propiolactone (BPL) at subzero temperatures as low as -40 C. Bacillus subtilis var. niger spores were used as the test organism. Both PAA and BPL were sporicidal at low temperatures, with PAA the more active. The temperature coefficients of the two chemicals are generally low over a range of 20 to -20 C, but increase significantly at temperatures below this. Results showed an initial lag in the PAA death rates that was directly dependent on the temperature. BPL did not show this lag time.     

Screening and selection of bacteria inhibiting white spot syndrome virus infection to Litopenaeus vannamei

A total of 173 bacterial strains were isolated from different sources at different regions such as fermented foods, shrimp guts, sea water, mangrove water, and sediments. These bacteria were screened against white spot syndrome virus (WSSV) infection in Palaemon paucidens. Based on mortality, white spot level, and healthiness, three bacterial strains were selected and identified using 16S rRNA gene sequencing. These bacterial strains were Bacillus subtilis KA1, B. licheniformis KA2, and B. subtilis KA3. WSSV challenge test in pilot scale was conducted using Litopenaeus vannamei with B. subtilis KA1 and B. subtilis KA3. The survival ratio of shrimp was 0% for WSSV control after 17th days, 84% for B. subtilis KA1 plus WSSV after 26th days, and 28% for B. subtilis KA3 with WSSV after 26th days. B. subtilis KA1 showed good growth at 18–37 °C in with and without 3% NaCl, and therefore can be applied to aquaculture at low to high temperatures. B. subtilis KA1 produced protease and lipase which can increase digestion to shrimp; exhibited antibacterial activity against Vibrio parahaemolyticus; and significantly increased the survival of WSSV challenged shrimps.     

Improved Large-Volume Sampler for the Collection of Bacterial Cells from Aerosol

A modified large-volume sampler was demonstrated to be an efficient device for the collection of mono-disperse aerosols of rhodamine B and poly-disperse aerosols of bacterial cells. Absolute efficiency for collection of rhodamine B varied from 100% with 5-μm particles to about 70% with 0.5-μm particles. The sampler concentrated the particles from 950 liters of air into a flow of between 1 and 2 ml of collecting fluid per min. Spores of Bacillus subtilis var. niger were collected at an efficiency of about 82% compared to the collection in the standard AGI-30 sampler. In the most desirable collecting fluids tested, aerosolized cells of Serratia marcescens, Escherichia coli, and Aerobacter aerogenes were collected at comparative efficiencies of approximately 90, 80, and 90%, respectively. The modified sampler has practical application in the study of aerosol transmission of respiratory pathogens.     

Genes encoding thymidylate synthases A and B in the genus Bacillus are members of two distinct families

Bacillus subtilis strain 168 is known to possess two genes that encode thymidylate synthases, thyA and thyB. We have identified genes similar to the thyA and thyB genes in several Bacillus strains by Southern hybridization and by DNA amplification with sequence-specific primers. Analysis of thyA genes cloned from B. subtilis W23 strain 2A6, B. subtilis ATCC6633, B. amyloliquefaciens S18 and B. atrophaeus S223 reveals that they are very similar to the thyA genes from B. subtilis 168 and its phage φ3T, but differ considerably from the majority of known prokaryotic and eukaryotic thymidylate synthases.     

Heat resistance of Salmonella senftenberg 775W at various sucrose concentrations in distilled water

Summary The heat resistance of Salmonella senftenberg 775 W, NCTC 9959, has been determined in distilled water pH 6.5 at sucrose concentrations up to 2.20 mol l^–1 at temperatures between 63 and 70°C. Surviving cells were counted on minimal and enriched agar media to investigate the influence of the various nutrients on the recovery of heat injured cells. At various sucrose concentrations and temperatures multiphasic exponential parts of inactivation curves were found. Systematic differences between the recovery media depended on sucrose concentration, temperature and phase of exponential inactivation. At 60°C and sucrose concentrations between 0.52 and 1.82 mol l^–1 the relationship between inactivation rate and sucrose concentration could be described by the equation ln k₅=ln k₀-_T [sucrose]. The activation energy of thermal inactivation reactions, substantially decreased when sucrose (1.82 mol l^–1) was added to the heating menstruum. The activation energies in different recovery agars were of the same order, which suggests that the critical sites in heat inactivation are not key enzymes of the synthetic pathways of amino-acids and nucleotides. The differences between activation energies, calculated for cells of the various exponential phases of inactivation in both non-sucrose and 1.82 mol sucrose per 1 heating media, were also small, further suggesting that these critical sites are the same in cells from the various phases. Compared to published data on the heat resistance of S. senftenberg 775 W, we found a decreased resistance in a non-sucrose medium but an equal or increased resistance, depending on the phase of exponential inactivation, at a sucrose concentration of 1.82 mol l^–1.     

Ethylene Oxide Gaseous Sterilization: I. Concentration and Temperature Effects

The relationships of reaction temperature and concentration of gaseous ethylene oxide to the time required for inactivation of air-dried Bacillus subtilis var. niger spores are more complex than previously reported. A plot of temperature vs. the logarithm of “thermochemical death time” (TCDT) resulted in a straight line between 18 and 57 C for systems of “high” ethylene oxide concentration. The TCDT values were independent of ethylene oxide concentrations above certain temperature-dependent limits. A given ethylene oxide concentration produced a TCDT curve identical in the upper temperature regions with that for higher concentrations. As the temperature was lowered beyond a critical point, this curve diverged from that for higher concentrations, as a straight line of lesser slope. Thus, a series of curves exists for a range of ethylene oxide concentrations. They are characterized by two segments, both logarithmic, intersecting at a critical temperature for each concentration. The intersecting point is at a temperature inversely related to the ethylene oxide gas concentration. The temperature quotient for the high temperature segments of all systems was 1.8. This value was characteristic for ethylene oxide concentrations of 440 and 880 mg/liter at temperatures above 40.6 and 33.4 C, respectively. Below these critical temperatures, the Q₁₀ values for the respective systems were 3.2 and 2.3.     

Ethylene Oxide Gaseous Sterilization: II. Influence of Method of Humidification

The duration of the equilibration period between admission of water vapor and subsequent introduction of gaseous ethylene oxide to an evacuated sterilizer chamber was studied with respect to its effect on the inactivation of spores of Bacillus subtilis var. niger under simulated practical conditions. Introduction of a water-adsorbing cotton barrier between the spores and an incoming gas mixture of water vapor and ethylene oxide caused a marked increase in the observed thermochemical death time of the spore populations. This effect was negated by admission of water vapor one or more minutes prior to introduction of ethylene oxide gas. Increases in temperature and relative humidity of the system promoted passage of water vapor through the cotton barriers and diminished their effect.     

Relation of the Heat Resistance of Salmonellae to the Water Activity of the Environment

The effect of water activity (a_w) on the heat resistance of eight strains of Salmonella was studied. Heat resistance of the organisms increased as the a_w of the heating menstruum was reduced. Sucrose afforded the cells a greater degree of protection than did fructose, glycerol, and sorbitol. A direct correlation between a_w and heat resistance could not be established over the range of a_w levels tested in this study. There was variation among the strains of salmonellae in the magnitude of the increase in heat resistance as the a_w level was reduced. All strains of Salmonella tested showed a greater increase in heat resistance than S. senftenberg 775W as the environment became drier. Washed cells had D values 25 to 75% lower than unwashed cells. Prior growth of the organisms in media with a reduced a_w increased the heat resistance of the organisms when glycerol, but not when sucrose, was the controlling substance.     

Defense Responses and Changes in Symbiotic Gut Microflora in the Colorado Potato Beetle <Emphasis Type="Italic">Leptinotarsa decemlineata</Emphasis> under the Effect of Endophytic Bacteria from the Genus Bacillus

Phytophagous insects and host plants have a complex of microsymbionts and make up a united co-evolving system with them. Microsymbiotic complexes are actively involved in stress responses of macrosymbionts. We established that a treatment of potato plants with endophytic bacterial strains Bacillus thuringiensis var. thuringiensis-5689, B. th. var. kurstaki-5351, and Bacillus subtilis 26D decreased the survival rate of the plant feeder, Colorado potato beetle Leptinotarsa decemlineata Say. The B. th. strains suppressed phenoloxidase and acetylcholinesterase activities in the beetle hemolymph. An antagonistic relationship was found between endophytic bacteria B. subtilis 26D and beetle symbiotic bacteria from the genera Acinetobacter and Enterobacter, with the former being able to suppress the growth of endophytic colonies. The recombinant B. subtilis strain 26D Cry, containing the B. th. var. kurstaki δ-endotoxin cry1Ia gene, combined the ability of the original B. subtilis 26D strain to suppress the development of beetle symbionts and immune responses with a production of the Cry toxin, thus leading to a high mortality of the phytophage.     

Foam separation of bacteria with a cationic surfactant

An experimental investigation is presented of the foam separation of six species of bacteria: Escherichia coli, Serratia marcescens, Proteus vulgaris, Pseudomonas fluorescens, Bacillus cereus, and Bacillus subtilis var niger. A cationic surfactant, ethylhexadeeyldimethylammonium bromide is used and results are evaluated in terms of total cell count, using a membrane filtration technique. From similar neutral distilled water suspensions of the pure cultures (approximately 10⁷ cells/ml.) and using the same operating conditions, ratios of cell concentrations in the residual suspensions to those in the initial suspensions range from 0.0013 for Bacillus subtilis var niger to 0.25 for Serratia marcescens. The presence of bacteria, compared to pure surfactant solutions, produces lower collapsed foam volumes; the foam volumes have a strong influence on the separations achieved with the various species, with enrichment ratios ranging from 27 to 3088 and residual ratios ranging from 0.001 to 0.247.     

Protein synthesis in Bacillus subtilis. I. Hydrodynamics and in vitro functional properties of ribosomes from B. subtilis W168.

On the basis of their sedimentation properties, the ribosomal particles in crude extracts of Bacillus subtilis W168 are characterized as pressure-sensitive couples, pressure-resistant couples, or non-associating subunits. Pressure-sensitive couples dissociate into subunits, yielding a peak at 60 S in the gradient profile, on sedimentation at high speed in the presence of 10 to 15 mm-Mg²⁺. Under the same conditions, pressure-resistant couples sediment at 70 S. Under certain conditions, pressure-resistant couples apparently aggregate, possibly in 70 S · 70 S dimers. Procedures are described for the isolation of pressure-sensitive couples from B. subtilis. The isolated couples are shown by chemical fixation experiments to require approximately twice the Mg²⁺ concentration required by Escherichia coli couples to remain associated at atmospheric pressure.All three types of B. subtilis ribosome incorporate amino acids into acid-insoluble material in the presence of B. subtilis cellular RNA, B. subtilis ribosomal salt wash fraction, and E. coli post-ribosomal supernatant. Overall incorporation, dependence on added RNA, and dependence on salt wash fraction are greatest with pressure-sensitive couples. The products of protein synthesis in vitro stimulated by total B. subtilis RNA appear to be a low molecular weight subset of the proteins synthesized most abundantly in vivo. Incubation of pressure-sensitive couples with cellular RNA from B. subtilis, fMet-tRNA_f^Met, ribosomal salt wash fraction and GTP results in their conversion to pressure-resistant couples, with concomitant and stoichiometric binding of fMet-tRNA to the 70 S species. It is concluded that in B. subtilis as in E. coli, pressure-sensitive couples are “vacant”, while pressure-resistant couples are “complexed” with messenger RNA. fMet-tRNA-bearing complexed couples are interpreted as initiation complexes in which ribosomes have bound mRNA, presumably at initiation sites. Their formation in vitro is strictly dependent on RNA, salt wash fraction and fMet-tRNA when vacant ribosomal couples are used.     

Comparison of Responses to Double-Strand Breaks between Escherichia coli and Bacillus subtilis Reveals Different Requirements for SOS Induction

DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell. E. coli strains incapable of SOS induction were sensitive to ionizing radiation. In striking contrast, we found that in B. subtilis both ionizing radiation and a site-specific double-strand break causes induction of prophage PBSX and SOS gene expression in only a small subpopulation of cells. These results show that double-strand breaks provoke global SOS induction in E. coli but not in B. subtilis. Remarkably, RecA-GFP focus formation was nearly identical following ionizing radiation challenge in both E. coli and B. subtilis, demonstrating that formation of RecA-GFP foci occurs in response to double-strand breaks but does not require or result in SOS induction in B. subtilis. Furthermore, we found that B. subtilis cells incapable of inducing SOS had near wild-type levels of survival in response to ionizing radiation. Moreover, B. subtilis RecN contributes to maintaining low levels of SOS induction during double-strand break repair. Thus, we found that the contribution of SOS induction to double-strand break repair differs substantially between E. coli and B. subtilis.