Catalase activity during the recovery of heat-stressed Staphylococcus aureus MF-31.

Sublethal heating of Staphylococcus aureus produced little loss of catalase activity, but incubation of the injured cells in tryptic soy broth, with or without 10% NaCl added, produced an 85% decrease in catalase activity. Cells recovered in tryptic soy broth demonstrated increases in catalase levels after approximately 5 h, whereas in tryptic soy broth with 10% NaCl the levels remained low for at least 12 h. Thus, the loss of catalase activity during the recovery period was greater than during the heat treatment.     

Superoxide dismutase activity during recovery of thermally stressed Staphylococcus aureus MF-31.

Superoxide dismutase (SOD) activity was determined during the growth cycle of unheated and heat-injured cells of Staphylococcus aureus MF-31. SOd activity levels dropped in unheated cells during the lag phase, increased during logarithmic phase, and became constant in the stationary phase. Cells which were sublethally heated (52 degrees c, 20 min) in 100 mM phosphate buffer and subsequently allowed to recover in tryptic soy broth demonstrated an 85% decrease in SOD activity upon inoculation into recovery medium. As the injured cells repaired the heat-induced lesions and entered logarithmic growth, SOD levels rapidly increased. Heat-injured cells allowed to recover in tryptic soy broth plus 10% NaCl showed similar decreases in SOD activity levels. However, no subsequent increase was observed when specific activity was calculated based on milligrams of protein.     

Beneficial effects of catalase or pyruvate in a most-probable-number technique for the detection of Staphylococcus aureus.

The effects of the addition of catalase (EC 1.11.1.6) or pyruvate on the enumeration of Staphylococcus aureus in Trypticase soy broth with 10% NaCl were examined using a most-probable-number technique. Addition of catalase or pyruvate to the broth increased enumeration of all heat-stressed S. aureus strains tested. Increases were also observed with nonstressed cells. Catalase and pyruvate were similarly effective when added to Trypticase soy broth-10% NaCl in enumerating staphylococci naturally present in low-temperature-rendered ground-beef samples.     

Beneficial effects of catalase or pyruvate in a most-probable-number technique for the detection of Staphylococcus aureus.

The effects of the addition of catalase (EC 1.11.1.6) or pyruvate on the enumeration of Staphylococcus aureus in Trypticase soy broth with 10% NaCl were examined using a most-probable-number technique. Addition of catalase or pyruvate to the broth increased enumeration of all heat-stressed S. aureus strains tested. Increases were also observed with nonstressed cells. Catalase and pyruvate were similarly effective when added to Trypticase soy broth-10% NaCl in enumerating staphylococci naturally present in low-temperature-rendered ground-beef samples.     

Medium-dependent production of extracellular enterotoxins by non-O-1 Vibrio cholerae, Vibrio mimicus, and Vibrio fluvialis.

Fluid accumulation at 4 h in the intestines of suckling mice enabled us to distinguish non-O-1 Vibrio cholerae, V. mimicus, and V. fluvialis clinical isolates from environmental isolates. Enterotoxin production was culture medium dependent. Filtrates of cultures grown in tryptic soy broth without glucose but with added 0.5% NaCl did not exhibit marked enterotoxin activity in the assay. Culture filtrates of all clinical strains grown in brain heart infusion broth supplemented with 0.5% NaCl induced large amounts of fluid accumulation in mouse intestines. However, most environmental strains grown in brain heart infusion broth amended as described above were unable to induce fluid accumulation. The enterotoxin present in culture filtrates lost activity at 56 degrees C and appeared to be distinct from previously described virulence factors, including the well-described cholera toxin. The new enterotoxin could represent an important virulence mechanism common to all three species.     

Factors affecting hemolysin production byVibrio vulnificus

Factors affecting the hemolytic activity ofVibrio vulnificus cultures supernatant fluids against sheep erythrocytes were studied in order to optimize conditions and develop an assay system to assess the pathogenic potential of marineV. vulnificus strains. The heat-labile hemolysin produced during logarithmic growth ofV. vulnificus was produced optimally in heart infusion broth (HIB). Lesser amounts of hemolysin were produced in brain-heart infusion and tryptic soy broths. Hemolytic activity of HIB cultures decreased with increasing NaCl concentrations. Higher NaCl concentrations were found to affect hemolysin production but not its activity. The assay system described herein is a simple and rapid method that is being applied to the study of the pathogenic potential ofV. vulnificus strains from marine environments.     

Combined Effects of Water Activity, Solute, and Temperature on the Growth of Vibrio parahaemolyticus

Vibrio parahaemolyticus was grown at 36 C in tryptic soy broth (pH 7.8) containing added levels of NaCl ranging from 0.5 to 7.9% (wt/wt). The fastest generation time was 16.4 min in tryptic soy broth containing 2.9% NaCl (TSBS) which corresponded to a water activity (a(w)) of 0.992 (+/-0.005). Tryptic soy broth containing lower or higher levels of NaCl resulted in higher or lower a(w), respectively, and slower generation times. Growth was measured turbidimetrically at 36 C in TSBS containing added amounts of NaCl, KCl, glucose, sucrose, glycerol, or propylene glycol. The solutes used to reduce a(w) to comparable levels resulted in extended lag times of varied magnitude, dissimilar growth rates, and different cell numbers. Reduction of a(w) with glycerol was less inhibitory to growth than similar a(w) reductions with NaCl and KCl. Sucrose, glucose, and propylene glycol generally had the greatest effect on extending the lag times of V. parahaemolyticus when the addition of these solutes was made to establish similar a(w) levels lower than 0.992. Minimal a(w) for growth at 15, 21, 29, and 36 +/- 0.2 C for each of four strains of V. parahaemolyticus was tested in TSBS containing added solutes. Reduced a(w) was generally most tolerable at 29 C, whereas higher minimal a(w) for growth was required at 15 C. Solutes added to TSBS to achieve reduction in a(w), minimal a(w) for growth after 20 days, and incubation temperatures were as follows: glycerol, 0.937, 29 C; KCl, 0.945, 29 C; NaCl, 0.948, 29 C; sucrose, 0.957, 29 and 36 C; glucose, 0.983, 21 C; and propylene glycol, 0.986, 29 C. Each of the four strains tested responded similarly to investigative conditions. It appears that minimal a(w) for growth of V. parahaemolyticus depends upon the solute used to control a(w).     

Survival and virulence of Salmonella enterica serovar enteritidis filaments induced by reduced water activity

Salmonella enterica serovar Enteritidis strain E40 filaments were developed under conditions of a reduced water activity (a(w)) of 0.95 in tryptic soy broth (TSB) or tryptic soy agar (TSA) supplemented with 8% or 7% NaCl, respectively. Filament formation was accompanied by an increase of biomass without an increase in CFU and was affected by incubation temperature and the physical milieu. The greatest amount of filaments was recovered from TSA with 7% NaCl and incubation at 30°C. Within 2 h of transfer to fresh TSB, filaments started to septate into normal-sized cells, resulting in a rapid increase in CFU. S. Enteritidis E40 filaments were not more tolerant of low- or high-temperature stresses than nonfilamented control cells. However, there was greater survival of filaments in 10% bile salts after 24 to 48 h of incubation, during pH 2.0 acid challenge for 10 min, and under desiccation on stainless steel surfaces at 25°C and 75.5% relative humidity for 7 days. S. Enteritidis E40 filaments invaded and multiplied within Caco-2 human intestinal epithelial cells to a similar degree as control cells when a comparable CFU of filaments and control cells was used. S. Enteritidis E40 filaments established a successful infection in mice via intragastric inoculation. The filaments colonized the gastrointestinal tract and disseminated to the spleen and liver at levels comparable to those attained by control cells, even when animals were inoculated with 10- to 100-fold fewer CFU. To our knowledge this is the first demonstration of virulence of stress-induced Salmonella filaments in vitro and in vivo. Formation of filaments by Salmonella in food products and food processing environments is significant to food safety, because detection and quantitation of the pathogen may be compromised. The finding that these filaments are virulent further enhances their potential public health impact.     

The immune response of Taiwan abalone Haliotis diversicolor supertexta and its susceptibility to Vibrio parahaemolyticus at different salinity levels

Addition of NaCl at 2.5% to 3.5% to tryptic soy broth (TSB) significantly increased the growth of Vibrio parahaemolyticus. Taiwan abalone Haliotis diversicolor supertexta held in 30 per thousand seawater were injected with V. parahaemolyticus grown in TSB containing NaCl at 0.5, 1.5, 2.5, 3.5 and 4.5% at a dose of 1.6 x 10(5)colony-forming units (cfu) abalone(-1). After 48 h, the cumulative mortality was significantly higher for the abalone challenged with V. parahaemolyticus grown in 2.5% than those grown in 0.5 and 1.5% NaCl. In other experiments, abalones held in 30 per thousand seawater were injected with TSB-grown V. parahaemolyticus (1.6 x 10(5)cfu abalone(-1)), and then transferred to 20, 25, 30 and 35 per thousand seawater. All abalones held in 20 per thousand were killed in 48 h. The mortality of V. parahaemolyticus-injected abalone held in 30 per thousand was significantly lower over 24-120 h. Abalone held in 30 per thousand seawater and then transferred to 20, 25, 30 and 35 per thousand were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency of V. parahemolyticus after 24 and 72 h. The THC increased directly related with salinity levels. Phenoloxidase activity, respiratory burst, phagocytic activity and clearance efficiency of V. parahaemolyticus decreased significantly for the abalone in 20, 25 and 35 per thousand. It is concluded that the abalone transferred from 30 per thousand to 20, 25 and 35 per thousand had reduced immune ability and decreased resistance against V. parahaemolyticus infection.     

Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice

Studies were done to determine the interacting effects of pH, NaCl, temperature, and time on growth, survival, and death of two strains of Listeria monocytogenes. Viable population of the organism steadily declined in heat-sterilized cabbage stored at 5 degrees C for 42 days. In contrast, the organism grew on raw cabbage during the first 25 days of a 64-day storage period at 5 degrees C. Growth was observed in heat-sterilized unclarified cabbage juice containing less than or equal to 5% NaCl and tryptic phosphate broth containing less than or equal to 10% NaCl. Rates of thermal inactivation increased as pH of clarified cabbage juice heating medium was decreased from 5.6 to 4.0. At 58 degrees C (pH 5.6), 4 X 10(6) cells/mL were reduced to undetectable levels within 10 min. Thermal inactivation rates in clarified cabbage juice (pH 5.6) were not significantly influenced by the presence of up to 2% NaCl; however, heat-stressed cells had increased sensitivity to NaCl in tryptic soy agar recovery medium. Cold enrichment of heat-stressed cells at 5 degrees C for 21 days enhanced resuscitation. Results indicate that L. monocytogenes can proliferate on refrigerated (5 degrees C) raw cabbage which, in turn, may represent a hazard to health of the consumer. Heat pasteurization treatments normally given to cabbage juice or sauerkraut would be expected to kill any L. monocytogenes cells which may be present.     

Effect of free-radical scavengers on enumeration of thermally stressed cells of Staphylococcus aureus MF-31.

Exposure of crude cell lysates of Staphylococcus aureus MF-31 to 5 or 10 mM hydrogen peroxide resulted in a linear decrease in superoxide dismutase activity. Approximately 13% of the superoxide dismutase activity was lost after 16 min. Thermally stressed and nonstressed cells were exposed to a photochemically generated exogenous flux of superoxide radicals (O2.-). The death of thermally stressed cells was linear with time. Addition of superoxide dismutase or catalase to the O2.- generating system resulted in protection of thermally stressed and nonstressed cells, with the protective effect being greater for thermally stressed cells. Incorporation of O2-, hydroxyl radical, or singlet oxygen scavengers or antioxidants to tryptic soy agar containing 7.5% NaCl did not increase the enumeration of thermally stressed cells.     

Sustained Photoproduction of Ammonia from Nitrate by Anacystis nidulans

Exposure of crude cell lysates of Staphylococcus aureus MF-31 to 5 or 10 mM hydrogen peroxide resulted in a linear decrease in superoxide dismutase activity. Approximately 13% of the superoxide dismutase activity was lost after 16 min. Thermally stressed and nonstressed cells were exposed to a photochemically generated exogenous flux of superoxide radicals (O2.-). The death of thermally stressed cells was linear with time. Addition of superoxide dismutase or catalase to the O2.- generating system resulted in protection of thermally stressed and nonstressed cells, with the protective effect being greater for thermally stressed cells. Incorporation of O2-, hydroxyl radical, or singlet oxygen scavengers or antioxidants to tryptic soy agar containing 7.5% NaCl did not increase the enumeration of thermally stressed cells.     

Effect of salinity on the immune response of tiger shrimp Penaeus monodon and its susceptibility to Photobacterium damselae subsp. damselae

Addition of NaCl at 2.5% to tryptic soy broth (TSB) significantly increased the growth of Photobacterium damselae subsp. damselae. Tiger shrimp Penaeus monodon held in 25 per thousand seawater were injected with P. damsela subsp. damselae grown in TSB containing NaCl at 0.5%, 1.5%, 2.5% and 3.5% at a dose of 8.48 x 10(4)colony-forming units (cfu)shrimp(-1). Over 24-96 h, the cumulative mortality was significantly higher for the shrimp challenged with P. damselae subsp. damselae grown in 2.5% NaCl than those grown in 0.5%, 1.5% and 3.5% NaCl. In another experiment, P. monodon held in 25 per thousand were injected with TSB-grown P. damselae subsp. damselae (8.48 x 10(4)cfushrimp(-1)), and then transferred to 5 per thousand, 15 per thousand, 25 per thousand (control) and 35 per thousand. After 96 h, the mortality was highest for the P. damselae subsp. damselae-injected shrimp held in 5 per thousand, and the lowest for the P. damselae subsp. damselae-injected shrimp held in 25 per thousand. In a separate experiment, P. monodon held in 25 per thousand and then transferred to 5 per thousand, 15 per thousand, 25 per thousand (control) and 35 per thousand were examined for immune parameters, and phagocytic activity and clearance efficiency of P. damselae subsp. damselae after 12-96 h. The THC, hyaline cell, phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency decreased significantly for the shrimp held in 5 per thousand, 15 per thousand and 35 per thousand after 12h. It is concluded that tiger shrimp P. monodon transferred from 25 per thousand to low salinity levels (5 per thousand and 15 per thousand) and high salinity (35 per thousand) had reduced immune ability and decreased resistance against P. damselae subsp. damselae infection.     

Suitability of some enrichment broths and diluents for enumerating cold- and heat-stressed Vibrio parahaemolyticus.

Vibrio parahaemolyticus cells were injured by chilling and heating, and their recovery was tested in glucose-salt-Teepol broth (GSTB), tryptic soy broth containing 7% NaCl (TSBS), Horie - arabinose - ethyl violet broth (HAEB), and water blue - alizarin yellow broth (WBAY). Exponential phase cells were more sensitive to cold shock than were stationary phase cells. Exposure of chill-injured V. parahaemolyticus to GSTB and TSBS resulted in 70 to 80% death; about 70% lethality was noted for heat-injured cells inoculated into TSBS. Neither HAEB nor WBAY enrichment media were lethal to stressed cells, although rates of growth were retarded. The 3% NaCl in 0.1 M potassium phosphate (pH 7.0) diluent proved to be most suitable for protecting against inactivation of cold- and heat-injured cells.     

Effect of environmental conditions during heating on commercial spore strip performance.

Commercial biological indicator spore strips in glassine envelopes, produced by three manufacturers, were evaluated by fraction-negative procedures after being heated at 121.0 +/- 0.05 degrees C. Only one type of spore strip met the manufacturer's specifications. The strips of one manufacturer were further evaluated by fraction-negative and survivor curve-plate count procedures after being heated under several conditions (enclosed in glassine envelopes, in trypticase soy broth plus 0.0015% bromocresol purple, in Trypticase soy broth alone in Water for Injection, directly); Trypticase soy broth plus bromocresol purple and tryptic soy agar, respectively, were used as recovery media. The heating condition affected the D-value of the spore strip. Recovery procedures also had an effect; in all cases, the D-values obtained from the survivor curve tests were larger than those obtained from fraction-negative tests carried out under the same conditions. To determine if the differences in D-values between the two evaluation procedures were caused by the recovery media, we evaluated, by both methods, one type of spore strip heated directly and in glassine envelopes, using tryptic soy agar plus bromocresol purple and Trypticase soy broth plus 1.5% agar, respectively, as the recovery media. The survivor curve results showed that for both enclosed and unenclosed spore strips, there was a marked difference between the two recovery media; however, there was no difference when fraction-negative tests were used.     

Control of the rootknot nematode meloidogyne javanica by Bacillus cereus

Exposure of Meloidogyne javanica second‐stage juveniles to the bacterium Bacillus cereus in soil inhibited the penetration of the juvenile nematodes into tomato roots. Culture filtrate of the bacterium grown on nutrient broth and tryptic soy broth revealed nematocidal activity on M. javanica juveniles and eggs. Loss of the nematocidal activity of the media by lowering pH, boiling or dialysis raised the possibility that the active ingredient in the culture filtrate was ammonia, released during the breakdown process of peptides in the media by bacterial activity. Free ammonia (NH₃) concentrations in the nutrient broth and tryptic soy broth culture filtrates measured after 48 h were 140 and 190 µg ml^?1 respectively. Exposure of second‐stage juveniles to 9.3 µg ml^?1 ammonia for 40 h in vitro was lethal to 95% of the nematode population. In a nitrate medium, nitrite accumulated up to 250 µg ml^?1 during the growth of the bacterium, and its culture filtrate revealed nematocidal activity. The nematocidal activity of the bacterium increased when the bacterium was applied with various proteinaceous supplements to soil. Soil treated with the bacteria and peptone showed an earlier nematocidal activity than either the bacteria or peptone applied alone, and also had a higher level of ammonia than the individual treatments. However, the level of ammonia was lower than the lethal level for second‐stage juveniles recorded in vitro. The nematocidal activity exhibited by the bacterium‐proteinaceous amendment combination is not fully understood; the ammonia released during protein degradation by the bacterium may contribute significantly to the recorded nematocidal activity.     

The effect of culture conditions on hydrophobic properties of Pseudomonas aeruginosa

The cell surface hydrophobicity (CSH) plays an important role in a adhesion of bacteria on solid surfaces. CSH of 62 Pseudomonas aeruginosa strains isolated from humans and different animals was assessed using the ammonium sulfate salt aggregation test. Bacteria were grown for 24 h and 48 h at a room temperature (22 degrees C) and 37 degrees C on enrichment broth and agar (Biomed) and tryptic soy agar (Difco). The hydrophobic properties of the Pseudomonas aeruginosa strains were depended on the temperature, time of the culture of bacteria and the kind of media. CSH properties were most frequently expressed when the analyzed strains were cultured in enrichment broth. In a such conditions Pseudomonas aeruginosa strains were more hydrophobic when grown at 22 degrees C (94% after 24 h and 87% after 48%) than those at 37 degrees C (72% after 24 h and 71% after 48 h). Among strains cultured in tryptic soy agar at 37 degrees C, 48% after 24 h and 75% after 48 h were autoaggregating, representing very strong hydrophobic properties.     

Relationships between heat resistance and phospholipid fatty acid composition of Vibrio parahaemolyticus.

Vibrio parahaemolyticus was grown in tryptic soy broth (TSB) containing NaCl levels of 0.5, 3.0, and 7.5% (wt/vol). Cultures incubated at 21, 29, and 37 C were harvested in late exponential phases and thermal death times at 47 C (D47 c; time at 47 C required to reduce the viable population by 90%) were determined in phosphate buffer containing 0.5, 3.0, and 7.5% NaCl. At a given NaCl concentration in the growth medium, D47 c values increased with elevated incubation temperatures and with elevated levels of NaCl in the heating menstrua. Differences in thermal resistance of cells cultured at a particular temperature were greater between those grown in TSB containing 0.5 and 3.0% NaCl than between those grown in TSB containing 3.0 and 7.5% NaCl. D47c values ranged from 0.8 min (grown at 21 C in TSB with 0.5% NaCl) to 6.5 min (grown at 37 C in TSB with 7.5%, heated in 7.5% NaCl buffer). Methyl esters of major phospholipid fatty acids extracted from cells were quantitated. The ratio of saturated to unsaturated fatty acids in cells grown at a given NaCl concentration increased with elevated incubation temperature. At a particular growth temperature, however, saturated to unsaturated fatty acids ratios were lowest for cells grown in TSB containing 3.0% NaCl.     

Influence of media and temperature on bacteriocin production by<Emphasis Type="Italic"> Bacillus cereus</Emphasis> 8A during batch cultivation

Cerein 8A is a bacteriocin produced by the soil bacterium Bacillus cereus 8A, isolated from native woodlands of Brazil. The influence of temperature and media on the growth of B. cereus 8A and the production of this bacteriocin was studied during batch cultivation. Maximum activity was detected by cultivation in brain/heart infusion broth, reaching 3200 activity units ml^–1. Bacteriocin was also produced in peptone, MRS, Mueller–Hinton and nutrient broth, while no activity was observed during cultivation in thioglycollate or tryptic soy broth. Temperature had a strong influence on bacteriocin production, which was higher at 30 °C than at 25 °C. An important decrease in bacteriocin activity was observed at 37 °C. The relationship between growth and specific production rates, as a function of the temperature, showed different kinetics of production and there were several peaks in the specific production rates during growth. Bacteriocin was produced at the stationary phase, indicating it is synthesized as a secondary metabolite.     

Listeriolysin O secretion by Listeria monocytogenes in the presence of cysteine and sorbate

The influence of cysteine or cysteine-HCl and their combination with potassium sorbate on growth of Listeria monocytogenes and listeriolysin O (LLO) secretion, and on activation of LLO in the haemolysin assay system was studied. Both cysteine and cysteine-HCl (10 and 20 mmol 1^-1) enhanced LLO secretion in tryptic soy broth supplemented with yeast extract during 24 h incubation at 35°C. While sorbate did not affect growth, it suppressed both LLO secretion and LLO activation by cysteine in the haemolysin activity assay. These findings provide further evidence that sulphydryl-containing enzymes are implicated in the mechanism of microbial inhibition of sorbate. Addition of sorbate to foods has the potential of inactivating listeriolysin and other thiol-dependent toxins.