Survived Heat Treatment Research Articles

Thermal Inactivation and Postthermal Treatment Growth during Storage of Multiple Salmonella Serotypes in Ground Beef as Affected by Sodium Lactate and Oregano Oil

We assessed the heat resistance of Salmonella in raw ground beef in both the absence and presence of sodium lactate, oregano oil, and in combinations of these 2 GRAS-listed ingredients, and determined their bactericidal or bacteriostatic activities during postthermal treatment storage at 15 degrees C. A cocktail of 8 serotypes of Salmonella spp. was inoculated into ground beef supplemented with sodium lactate (NaL) (1.5% and 3%) and/or oregano oil (0.5% and 1%) to obtain approximately 8 log CFU/g. The ground beef samples (3 g) were vacuum-packed and heated at 60, 65, or 71 degrees C in a circulating water bath for selected times to inactivate approximately 5 to 6 log CFU/g of the pathogen, and then stored at 15 degrees C for 15 and 30 d. Results show that especially at the lower cooking temperatures, addition of oregano oil increased the inactivation rate of Salmonella spp., whereas addition of NaL alone exhibited a protective effect against lethality and decreased the rate. Addition of combinations of oregano oil and NaL overcame this protective effect. During subsequent posttreatment storage for 15 d, Salmonella populations in the controls and in samples containing 0.5% oregano (60 and 65 degrees C) or 1% oregano oil (60 degrees C) increased to 4.5 to 6 log CFU/g. The values for all other samples were at or near undetectable levels. Results from the 30-d storage study were similar. These findings indicate that lactate and oregano oil may be used to render Salmonella spp. more susceptible to the lethal effect of heat and to inhibit growth of Salmonella spp. that survive heat treatments.

Growth, keratinolytic proteinase activity and thermotolerance of dermatophytes associated with alopecia in Uyo, Nigeria

Mycological research was conducted on the mycelial growth, keratinolytic proteinase activity and thermotolerance ofdermatophytes associated with alopecia patients in Uyo, Nigeria. The results revealed that Microsporum sp. - AP1, Epidermophyton sp. - AP2, Trichophyton rubrum - AP4, Trichophyton mentagrophytes - AP5 and a yeast Candida albicans - AP3 isolated exhibited variable growth and keratinase activity at different temperatures. Microsporum sp. - AP1 and T. mentagrophytes - AP5 survived heat treatment at 90 degrees C but exhibited best mycelial growth at 30 degrees C (with 53.41 mg/50 ml biomass dry weight) and 40 degrees C (with 61.32 mg/50 ml biomass dry weight) respectively, after incubation for 2 weeks. Trichophyton rubrum - AP4 and Epidermophyton sp. - AP2 could not survive heat treatment at 90 degrees C but grew better at 40 degrees C (with 38.52 mg/50 ml biomass dry weight) and 30 degrees C (with 48.32 mg/50 ml biomass dry weight) respectively, over the same incubation period, while C. albicans - AP3 grew better at 30 degrees C with 38.7 mg/50 ml biomass dry weight after 2 weeks, but failed to survive at 70 degrees C. All the isolates except Candida albicans - AP3 survived at 80 degrees C and exhibited great potential to elaborate keratinolytic enzymes, with T. mentagrophytes demonstrating the best potential at 30 degrees C and 40 degrees C. Higher temperatures tended to reduce keratinolytic activities and there were significant (P < 0.05) relationships between biomass weight and enzyme productivities of all the isolates except T. mentagrophytes. This indicates that in some dermatophytes keratinolytic proteinase activity is not a function of cell multiplicity. This plus the high thermostability of the enzymes are important attributes in the consideration of preventive and therapeutic methods against dermatophytes in the tropics.

Heat Treatment Effects on Strawberry Plant Survival and Angular Leaf Spot, Caused by Xanthomonas fragariae, in Nursery Production.

Angular leaf spot is an important disease in strawberry nursery production. The European and Mediterranean Plant Protection Organization (EPPO) lists Xanthomonas fragariae as an A2 quarantine pathogen. Therefore, nurseries wishing to export plants to European countries must maintain phytosanitary standards to exclude X. fragariae. To help nurseries achieve these standards, heat treatment for killing or reducing the number of viable bacterial cells in strawberry crown tissue was investigated. First, the sensitivity of bacteria to heat was determined by dispensing 1-ml aliquots of standardized cell suspensions in microcentrifuge tubes for each of four isolates of X. fragariae, including the type culture, and submerging the tubes in water at 36, 40, 44, 48, 52, and 56°C for 0, 1, 2.5, 5, 10, 15, 30, 60, 120, 240, 360, and 480 min. Bacteria were transferred to growth medium to determine the proportion surviving heat treatment. Two trials were conducted in a greenhouse to determine the sensitivity of bare-root plants to heat treatment. In the first trial, plants of cvs. Camarosa and Diamante from two different nurseries were heat treated as follows: (i) plants placed in metallic mesh cages and immersed directly into water (industry standard, direct dip); (ii) plants sealed in a plastic bag and the bag immersed in water (bagged dry); or (iii) plants wetted in warm water, sealed in a plastic bag, and then immersed in water (bagged wet). Plants were treated at 44 or 48°C for 0, 60, 120, 180, and 240 min. In the second trial, plants of cvs. Camarosa, Camino Real, Diamante, Oso Grande, Strawberry Festival, and Ventana from a single nursery were subjected to the same treatments. In both trials, plants were potted after treatment and rated for growth characteristics. Results showed that populations of bacteria exposed to 56 and 52°C were killed completely after 15 and 60 min of exposure, respectively; both treatments killed plants. Bacterial populations exposed to 44°C for 4 h or 48°C for 2 h were reduced by 105 or 106 CFU/ml. The same treatments minimally affected vegetative growth of plants bagged dry or wet, but flowering was adversely affected. These heat treatments were selected for testing of nursery stock of several cultivars in field trials established at two locations in successive years. The survival rate among cultivars was similar to that observed in greenhouse trials, and angular leaf spot developed appreciably only in non-heat-treated control plots. Heat treatment of strawberry nursery stock is feasible and can be used to supplement standard production practices for producing pathogen-free nursery stock.

Insufficient radiofrequency ablation therapy may induce further malignant transformation of hepatocellular carcinoma

Radiofrequency ablation (RFA) for hepatocellular carcinoma (HCC) is a thermoablative technique to kill tumor tissue by generating areas of coagulative necrosis. Recent reports have raised concern that RFA may lead to a local recurrence of HCC with an aggressive phenotype and unfavorable prognosis, suggesting that RFA may induce further malignant transformation of HCC. However, the biological effects of RFA on HCC cells have not been directly analyzed. The aim of this study was to determine whether heat stress of the type associated with RFA induces malignant transformation of HCC. We assessed the sensitivity of three HCC cell lines (HepG2, Alexander, and Huh7) to heat treatment for 10 min. We then determined the temperature at which a heat-resistant subline can be generated. We established and expanded sublines that survived heat treatment. And their proliferation rates, heat sensitivities, and invasive capacities were further examined. All HepG2 died after 48 degrees C treatment, whereas 49 degrees C treatment was required to kill all Alexander and HuH7. We generated 20 sublines for each parental cell line. A HepG2 subline, HepG2#18, proliferated 100% faster than parental HepG2. Moreover, HepG2#18 survived after 50 degrees C treatment, whereas all parental HepG2 died after heat treatments at 48 degrees C or higher. Our results showed that even a single heat treatment could induce further transformation of an HCC cell line. Our results suggest that an insufficient treatment of HCC by RFA that enables survival of some cells might induce further malignant transformation in vivo.

Alicyclobacillus acidoterrestris: an increasing threat to the fruit juice industry?

SummaryThe quality of food can be defined in various ways with processors and consumers considering flavour, odour and appearance as well as extended shelf life among the most important of its attributes. Spoilage of food by bacterial contamination may occur at any point during the processing, altering any one, or all, of these characteristics, rendering the product unusable. Alicyclobacillus spp. and Alicyclobacillus acidoterrestris, in particular, are emerging food spoilage organisms in the fruit juice and fruit juice products industry. Spores of the latter are able to survive heat treatments presently used in the industry and their elimination from products may be used as a measure of the effectiveness of any processing protocol to remove potential spoilage. This paper reviews the history, methods of detection, both traditional and rapid and the protocols that may be effective in controlling the growth of the organism and hence spoilage in the finished product.

Transactivation of Protein Expression by Rice HSP101 in Planta and Using Hsp101 as a Selection Marker for Transformation

Plant HSP101 has dual activities, first, in conferring thermotolerance, and secondly, in serving as a translational activator. In this study, we introduced Oryza sativa Hsp101 (osHsp101) cDNA into tobacco by Agrobacterium-mediated transformation. Stable integration and expression of the transgene into the tobacco genome was demonstrated by Southern and Western blot analysis. Overexpression of osHSP101 had no noticeable effect on growth or development of the transgenic plants. Homozygous T(2) transgenic plants with overexpressed osHSP101 survived heat treatment better than untransformed control plants. In addition, taking advantage of conferring basal thermotolerance by plant HSP101, we were able to demonstrate the feasibility of using osHsp101 as a selection marker and select the transformants under high temperature in tobacco leaf disc transformation mediated by Agrobacterium. Furthermore, transgenic tobacco plants with overexpressed osHSP101 were able to enhance luciferase expression up to 2.9-fold more than untransformed plants in the progeny of reciprocally crossed with omega-luciferase reporter lines.

Food poisoning associated with pumilacidin-producing Bacillus pumilus in rice

Food poisoning caused by other Bacillus species than B. cereus has been described, but the toxins involved have rarely been isolated. Endospores will survive heat treatment and will germinate and multiply in cooked foods producing toxins under appropriate conditions. We describe a small food poisoning outbreak where three people became ill after a dinner in a Chinese restaurant. Acute symptoms including dizziness, headache, chills and back pain developed during the meal, and a few hours later they got stomach cramps and diarrhoea which lasted for several days. Cooked, reheated rice was the prime suspect of the food poisoning, and from the rice large numbers of Bacillus pumilus were isolated. The isolated B. pumilus strain was found to produce a complex of lipopeptides known as pumilacidins with the highest amounts produced at 15 °C. This is the first report on isolation of a pumilacidin-producing B. pumilus strain from food implicated in food poisoning and characterization of the organism and the toxin complex involved.

A Probabilistic Modeling Approach in Thermal Inactivation: Estimation of Postprocess Bacillus cereus Spore Prevalence and Concentration

The survival of spore-forming bacteria is linked to the safety and stability of refrigerated processed foods of extended durability (REPFEDs). A probabilistic modeling approach was used to assess the prevalence and concentration of Bacillus cereus spores surviving heat treatment for a semiliquid chilled food product. This product received heat treatment to inactivate nonproteolytic Clostridium botulinum during manufacture and was designed to be kept at refrigerator temperature postmanufacture. As key inputs for the modeling, the assessment took into consideration the following factors: (i) contamination frequency (prevalence) and level (concentration) of both psychrotrophic and mesophilic strains of B. cereus, (ii) heat resistance of both types (expressed as decimal reduction times at 90°C), and (iii) intrapouch variability of thermal kinetics during heat processing (expressed as the time spent at 90°C). These three inputs were established as statistical distributions using expert opinion, literature data, and specific modeling, respectively. They were analyzed in a probabilistic model in which the outputs, expressed as distributions as well, were the proportion of the contaminated pouches (the likely prevalence) and the number of spores in the contaminated pouches (the likely concentration). The prevalence after thermal processing was estimated to be 11 and 49% for psychrotrophic and mesophilic strains, respectively. In the positive pouches, the bacterial concentration (considering psychrotrophic and mesophilic strains combined) was estimated to be 30 CFU/g (95th percentile). Such a probabilistic approach seems promising to help in (i) optimizing heat processes, (ii) identifying which key factor(s) to control, and (iii) providing information for subsequent assessment of B. cereus resuscitation and growth.

Microbiological aspects of thermally processed foods

The knowledge requirements of the food microbiologist working with thermally processed products are very challenging; particularly in relation to minimally processed foods. In the past, the minimum criteria for sterilized canned foods was very well-defined, however, the diversity of food products, packages and related heat processes are now very difficult to summarize in terms of minimum requirements. The overall heating characteristics of these products can be complex, particularly for mixed particulate products or multicompartment packages. Similarly, the death kinetics of the contaminating micro-organisms can be markedly different dependent on the composition of the food product. These factors combined with the consumer demands for 'fresher' foods has resulted in product, process and package development and innovation progressing at a much faster rate than in the past. While food safety is the most important factor, there are also other issues such as shelf-life and visual quality that have strong market influences and must therefore also be considered by the microbiologist.

CONTROL OF CLOSTRIDIUM PERFRINGENS IN A MODEL ROAST BEEF BY SALTS OF ORGANIC ACIDS DURING CHILLING1

ABSTRACTControl of Clostridium perfringens germination and outgrowth by the following salts of organic acids, sodium lactate [Purasal™S/SP (Purasal); 1.50, 3.00 and 4.80%], sodium lactate supplemented with sodium diacetate [Purasal™ Opti.form™ (Optiform), 1.50, 3.00 and 4.80%], buffered sodium citrate [Ional™ (Ional), 0.75, 1.00 and 1.30]) and buffered sodium citrate supplemented with sodium diacetate [Ional Plus™ (Ional Plus), 0.75, 1.00 and 1.30%] was evaluated during continuous chilling of a model roast beef product. Beef rounds were ground through an 1/8′’ plate and NaCl, potato starch and potassium tetra pyrophosphate were added to final concentrations of 0.85, 0.25 and 0.20%, respectively, and mixed. Portions (250 g) of the meat were mixed with either Purasal (1.5, 3.0 or 4.8%), Optiform (1.5, 3.0 or 4.8%), Ional (0.75, 1.0 or 1.3%) or Ional Plus (0.75, 1.0 or 1.3%) along with a control that did not have any added antimicrobials. Each product (10 g) inoculated with C. perfringens spores (ca. 2.2 log10 spores/g) was packaged into vacuum bags (2 in. × 3 in.), vacuum sealed, heated to 60C within 1 h, and subsequently chilled from 54.4C to 7.2C in 18 or 21 h following exponential chilling rates. Products were sampled immediately after cooking to enumerate the C. perfringens populations (spores surviving heat treatment) and subsequent to chilling (total C. perfringens populations, including spores and vegetative cells resulting from germination and outgrowth of the spores). Chilling of cooked, model ground roast beef resulted in germination and outgrowth of C. perfringens spores; the population densities increased by 4.13 and 4.40 log10 CFU/g, following 18 and 21 h chill rates, respectively. Incorporation of Purasal (1.5–4.8%), Optiform (1.5–4.8%), Ional and Ional Plus (0.75–1.3%) substantially (P ± 0.05) inhibited germination and outgrowth of C. perfringens spores. Incorporation of antimicrobial ingredients resulted in ± 1.0 log10 CFU/g increase of the pathogen, except for model roast beef with Ional Plus at 0.75% concentration, following 18 h chilling rate. Similar results were obtained when 21 h chilling rate was followed, with roast beef containing ingredients (at all the concentrations) resulting in either reductions or ± 1.0 log10 CFU/g growth in total C. perfringens populations, except for Purasal and Ional Plus at 1.5 and 0.75% concentrations, respectively. Use of sodium salts of organic acids in formulation of model roast beef can reduce the risk of C. perfringens spore germination and outgrowth during extended chilling rates.

Growth and heat resistance kinetic variation among various isolates of Salmonella and its application to risk assessment.

The abilities of cells of a particular type of bacteria to leave lag phase and begin the process of dividing or surviving heat treatment can depend on the serotypes or strains of the bacteria. This article reports an investigation of serotype-specific differences in growth and heat resistance kinetics of clinical and food isolates of Salmonella. Growth kinetics at 19 degrees C and 37 degrees C were examined in brain heart infusion broth and heat resistance kinetics for 60 degrees C were examined in beef gravy using a submerged coil heating apparatus. Estimates of the parameters of the growth curves suggests a small between-serotype variance of the growth kinetics. However, for inactivation, the results suggest a significant between-serotype effect on the asymptotic D-values, with an estimated between-serotype CV of about 20%. In microbial risk assessment, predictive microbiology is used to estimate growth and inactivation of pathogens. Often the data used for estimating the growth or inactivation kinetics are based on measurements on a cocktail--a mixture of approximately equal proportions of several serotypes or strains of the pathogen being studied. The expected growth or inactivation rates derived from data using cocktails are biased, reflecting the characteristics of the fastest growing or most heat resistant serotype of the cocktail. In this article, an adjustment to decrease this possible bias in a risk assessment is offered. The article also presents discussion of the effect on estimating growth when stochastic assumptions are incorporated in the model. In particular, equations describing the variation of relative growth are derived, accounting for the stochastic variations of the division of cells. For small numbers of cells, the expected value of the relative growth is not an appropriate "representative" value for actual relative growths that might occur.

The resistance to heat of thermo-resistant streptococci attached to stainless steel in the presence of milk.

Skim milk residues had a significant impact on the sensitivity to heat of a dairy isolate of the thermo-resistant, Streptococcus thermophilus. Cells of S. thermophilus (H) suspended in water or in milk had D values at 60 degrees C of 2.0 and 14 min, respectively. Cells of S. thermophilus (H) attached to stainless steel in the presence of water or milk had D values at 60 degrees C of 2.2 and 8.1 min, respectively. The attached cells in both experiments were heat-treated in the presence of water. The increase in heat resistance could not be fully attributed to individual components (caseinate or whey) in the milk. The potential for thermo-resistant streptococci to survive heat treatment in a dairy manufacturing plant is therefore greater than may be expected for the organism in less complex environments.

The resistance to heat of thermo-resistant streptococci attached to stainless steel in the presence of milk

Skim milk residues had a significant impact on the sensitivity to heat of a dairy isolate of the thermo-resistant, Streptococcus thermophilus. Cells of S. thermophilus (H) suspended in water or in milk had D values at 60 degrees C of 2.0 and 14 min, respectively. Cells of S. thermophilus (H) attached to stainless steel in the presence of water or milk had D values at 60 degrees C of 2.2 and 8.1 min, respectively. The attached cells in both experiments were heat-treated in the presence of water. The increase in heat resistance could not be fully attributed to individual components (caseinate or whey) in the milk. The potential for thermo-resistant streptococci to survive heat treatment in a dairy manufacturing plant is therefore greater than may be expected for the organism in less complex environments.

Characterization and growth of Bacillus spp. in heat-treated cream with and without nisin.

To study the germination and growth of both inoculated and naturally occurring Bacillus strains in heat-treated cream with and without nisin. In heat-treated cream (90 degrees C for 15 min) stored at 8 degrees C, growth was dominated by naturally occurring Bacillus strains such as Bacillus pumilus and B. licheniformis. Only six of the 52 isolated strains were B. cereus/thuringiensis. All of the B. cereus strains, but none of the other strains, produced enterotoxin when tested with the TECRA and reverse passive latex agglutination kits. Bacterial growth during storage of the cream at 8 or 10 degrees C was completely inhibited by low concentrations of nisin. The high number of Bacillus strains surviving the heat treatment represent a risk for heat-treated food that contains cream. The safety of the cream, for instance in "ready-to-eat" products, can be improved by the addition of low concentrations of nisin. Spores of several Bacillus species may survive heat treatment of cream, but low concentration of nisin with inhibit germination and growth.

Johne's Disease and Milk: Do Consumers Need to Worry?

Mycobacterium paratuberculosis, an acid-fast bacillus that causes enteritis in ruminants, has been suggested as an etiological agent of Crohn's disease in humans. The mode of transmission is unclear; however, some evidence suggests that humans may become infected via contaminated milk. Currently, it is not known whether commercial pasteurization effectively kills M. paratuberculosis in contaminated raw milk. Using a laboratory-scale pasteurizer unit designed to simulate the high-temperature, short-time method (72°C, 15sec) currently used by commercial dairies, we previously demonstrated that treatment of raw milk inoculated with 104 to 106 cfu of M. paratuberculosis/ml reduced numbers to an undetectable level. However, M. paratuberculosis is an intracellular pathogen that resides within the macrophages of the host and evades destruction. We subsequently performed further experiments examining heat treatment of milk inoculated with mammary gland macrophages containing ingested M. paratuberculosis. Heat treatment of these samples under high-temperature, short-time conditions demonstrated that the macrophage does not protect the organism because we were unable to recover any viable M. paratuberculosis from the samples. Conversely, other researchers have demonstrated that a residual population of M. paratuberculosis may survive heat treatment of milk. In addition, a recent news report stated that viable M. paratuberculosis organisms have been cultured from retail-ready milk in Ireland. A summary of past and current studies concerning this issue along with a discussion of methodologies used to recover M. paratuberculosis from experimentally inoculated milk will be presented in this paper.

Changes in heat tolerance of Escherichia coli O157:H7 after exposure to acidic environments

Acid-adapted bacterial cells are known to have enhanced tolerance to various secondary stresses. However, a comparison of heat tolerance of acid-adapted and acid-shocked cells of Escherichia coli O157:H7 has not been reported. D - and z -values of acid-adapted, acid-shocked, and control cells of an unusually heat-resistant strain (E0139) of E. coli O157:H7, as well as two other strains of E. coli O157:H7, were determined based upon the number of cells surviving heat treatment at 52, 54 or 56°C in tryptic soy broth (pH 7·2) for 0, 10, 20 or 30 min. The unusual heat tolerance of E. coli O157:H7 strain E0139 was confirmed. D -values for cells from 24-h cultures were 100·2, 28·3, and 6·1 min at 52, 54 and 56°C, respectively, with a z -value of 3·3°C. The highest D -values of other E. coli O157:H7 strains were 13·6 and 9·2 min at 52 and 54°C, respectively, whereas highest D -values of non-O157:H7 strains were 78·3 and 29·7 min at 52 and 54°C. D -values of acid-adapted cells were significantly higher than those of unadapted and acid-shocked cells at all temperatures tested. In a previous study, we observed that both acid-adapted cells and acid-shocked cells of strain E0139 had enhanced acid tolerance. This suggests that different mechanisms protect acid-adapted and acid-shocked cells against subsequent exposure to heat or an acidic environment. The two types of cells should be considered separately when evaluating survival and growth characteristics upon subsequent exposure to different secondary stress conditions.

Heat resistance of Yersinia enterocolitica grown at different temperatures and heated in different media

In the range of 4–20°C, growth temperature did not influence the heat resistance at 54–66°C for Yersinia enterocolitica at pH 7 in citrate phosphate buffer. However, when cells were grown at 37°C, the D 62 increased from 0.044 to 0.17 min. This increase was constant at all heating temperatures tested ( z=5.7–5.8). Growth temperature did not influence the proportion of heat-damaged cells after a heat treatment, as measured by their response to a 2% of sodium chloride added to the recovery medium. The sensitivity of heat treated cells to nisin or lysozyme depended on growth temperature: Whereas the number of cells grown at 4°C surviving heat treatment was the same regardless of the presence of 100 IU/ml of nisin or 100 μg/ml of lysozyme in the recovery medium, that of cells grown at 37°C was, in these media, lower. The pH of maximum heat resistance in citrate phosphate buffer was pH 7 for cells grown at 37°C, but pH 5 for those grown at 4°C. In both suspensions the magnitude of the effect of pH on heat resistance was constant at all heating temperatures. For cells grown at 4°C the heat resistance at 54–66°C, in skimmed milk or pH 7 buffer, was the same. For cells grown at 37°C this also applied for heat treatment at 66°C but at 56°C the heat resistance in skimmed milk was higher.

Comparison of selective agar media and enrichment broths for recovering heat-stressedEscherichia coliO157:H7 from ground beef

Media for detecting and enumerating healthy as well as heat-injured cells ofEscherichia coliO157:H7 in foods are highly desired. This study was conducted to evaluate the performance of eight selective and two non-selective direct plating agar media for their ability to recoverE. coliO157:H7 cells from unheated and heated ground beef, and to compare the ability of five enrichment broths to recoverE. coliO157:H7 cells from heated ground beef. Ground beef was incoulated withE. coliO157:H7 and heated at 56°C for up to 30min. Each agar was evaluated for its ability to support colony formation byE. coliO157:H7 surviving heat treatment, and each enrichment broth was evaluated for its ability to recover low numbers of surviving cells. Of the selective media tested, modified eosin methylene blue agar (MEMB) and RainbowTMagar O157 supported recovery of significantly (P≤0·05) higher numbers of heat-stressed cells ofE. coliO157:H7, regardless of heating time. CHROMagarTMO157, sorbitol MacConkey agar (SMA) supplemented with cefixime and potassium tellurite (CT-SMAC), and SMA supplemented with cefixime and rhamnose (CR-SMAC) performed less favorably, even in recovering cells ofE. coliO157:H7 that had not been subjected to heat stress. SMA and BCMTMO157:H7 agar were similar to CT-SMAC and CR-SMAC in their ability to recoverE. coliO157:H7 from heated beef. Tryptone bile X-glucuronide (TBX) agar performed significantly better than these media, but was inferior to MEMB agar and RainbowTMagar O157:H7. Enrichment using tryptone soya broth with novobiocin or a procedure using brain–heart infusion and tryptone phosphate broths recovered the highest population of heat stressedE. coliO157:H7. EZ ColiTMenrichment broth was inferior to other broths in resuscitating injured cells and supporting subsequent growth.

Thermal response of Heterorhabditis bacteriophora transformed with the Caenorhabditis elegans hsp70 encoding gene.

A heat-shock response is induced when cells are exposed to temperatures slightly higher than their optimal physiological temperature. This response is based on the synthesis of heat-shock proteins encoded by the heat-shock genes. A correlation between the increased thermotolerance and production of 70-kDa heat-shock protein (hsp70) has been observed in many organisms. We tested this hypothesis by transferring a Caenorhabditis elegans heat-inducible hsp70 A-encoding gene into the entomopathogenic nematodes Heterorhabditis bacteriophora Hp88. Successful transformation of the gene was confirmed by Southern blot hybridization and polymerase chain reaction. Our blot studies showed that the transgenic nematodes contained five to ten copies per genome of the introduced hsp70 A gene. hsp70 mRNA transcripts were detected in both wild-type and transgenic nematodes. Transcripts increased severalfold in transgenic nematodes upon heat shock. Infective juveniles of both transgenic and wild-type nematodes that exposed to a sublethal heat treatment (35 degrees C) for 2 h followed by a normally lethal heat treatment (40 degrees C) for 1 h. More than 90% of transgenic nematodes survived heat treatment, compared to 2% to 3% of the wild-type strain. Our observations establish that overexpression of hsp70 A gene resulted an enhanced thermotolerance in the transgenic nematodes. The transgenic nematodes displayed normal growth and development.

Combinations of Heat Treatment and Sodium Chloride That Prevent Growth from Spores of Nonproteolytic Clostridium botulinum

The ability of spores of nonproteolytic Clostridium botulinum to survive heat treatment and subsequently produce turbidity at 10°C in the presence of NaCl was quantified for different incubation times. Spores of nonproteolytic C. botulinum strain Eklund 17B were heated at 75°C for up to 4 min or 90°C for up to 30 min and subsequently incubated at l0°C in PYGS broth containing 1.5%. 3.0%, or 4.0% NaCl (wt/vol) with or without 10 μg hen egg white lysozyme ml−1. Heat treatment at 90°C for 30 min or incubation at 10°C in the presence of 4.0% NaCl did not prevent growth from up to 1.4 × l05 spores. Heat treatment at 90°C for 15 min and incubation in the presence of 4.0% NaCl did prevent growth in the same conditions.