Lethal and inhibitory effects of sodium chloride on thermally stressed Staphylococcus aureus

Iandolo and Ordal used a double-medium technique for the study of thermal injury in Staphylococcus aureus. In their system sodium chloride (salt) was used to distinguish the salt-sensitive from the salt-insensitive portions of the viable population remaining after thermal stressing. The data reported here show that nutrient medium containing salt was lethal for cells immediately postinjury and that nutrient medium containing salt was only inhibitory to injured cells if those cells had been incubated for 30 min in nutrient medium, buffered salt, or buffer alone prior to subjection to nutrient medium containing salt. Sodium ion was implicated as the active agent since nutrient media containing potassium chloride or glycerol (osmotic equivalence) produced neither inhibition nor the lethal effect. The inhibitory effect was interpreted as defining an early stage in the thermal injury recovery process.     

Coagulase Production by Injured Staphylococcus aureus MF-31 During Recovery

Suspensions of Staphylococcus aureus MF-31 injured by heat treatment at 54 C for 15 min produced coagulase during recovery in Trypticase Soy Broth. Coagulase also was produced by injured cells during recovery in a medium that did not support growth. Coagulase synthesis during recovery was independent of the molar strength of the buffer in which the cells were injured, the age of the cells, and the degree of injury. Return of salt tolerance and coagulase production required glucose, amino acids, and phosphate in the recovery medium. Vitamins stimulated coagulase production, but did not affect recovery. Although coagulase production was not necessary for repair of thermal injury to S. aureus MF-31, its detection was interpreted as an indicator of protein synthesis.     

Thermal Injury and Recovery of Bacillus subtilis

Exposure of Bacillus subtilis NCTC 8236 to sublethal temperatures produced a change in the sensitivity of the organism to salt and polymyxin. After 30 min at 47 C, 90% of the population was unable to grow on a modified sulfite polymyxin sulfadiazine agar containing an added 1% NaCl, 1% glucose, and 1% asparagine. The data presented demonstrate that thermal injury results in degradation of both 16S and 23S ribonucleic acid (RNA) and in damage to the cell membrane, suggested by leakage into the heating mestruum of material absorbing at 260 nm. When the cells were placed in a recovery medium (Trypticase soy broth), complete recovery, indicated by a returned tolerance to salt and polymyxin, occurred within 2 hr. The presence of a protein inhibitor (chloramphenicol) and cell wall inhibitors (vancomycin and penicillin) during recovery had no effect, whereas the presence of an RNA inhibitor (actinomycin D) effectively inhibited recovery. Further data demonstrated that the injured cells were able to resynthesize both species of ribosomal RNA during recovery by using the fragments which resulted from the injury process. Also, precursor 16S and precursor 23S particles accumulated during recovery. The maturation of the precursor particles during recovery was not affected by the presence of chloramphenicol in the recovery medium.     

Stability of ribosomes of Staphylococcus aureus S6 sublethally heated in different buffers.

Cells of Staphylococcus aureus heated at 52 degrees C in magnesium-chelating buffers [pH 7.2, 50 mM potassium phosphate or 50 mM tris(hydroxymethyl)-aminomethane containing 1 mM ethylenediaminetetraacetic acid] leaked 260-nm absorbing material, shown to be RNA, and suffered destruction of their ribosomes. These cells did not regain their salt tolerance when repair was carried out in the presence of actinomycin D (5 microgram/ml). Cells similarly heated in magnesium-conserving buffers [pH 7.2, 50 mM tris(hydroxymethyl)aminomethane containing 10 mM MgCl2 or piperazine buffer] did not leak RNA, suffered no ribosomal damage when heated for 15 min, and recovered, at least partially, in the presence of actinomycin D. Ribosomal damage, is therefore, a consequence of Mg2+ loss and is not an effect of heat per se. Cells suspended in either Mg2+-chelating or Mg2+-conserving buffers lost salt tolerance to about the same extent during heating at 52 degrees C. Therefore, sublethal heat injury can not be attributed to ribosomal damage.     

Thermal Injury and Recovery of Streptococcus faecalis

Exposure of Streptococcus faecalis R57 to sublethal heating produced a temporary change in the salt tolerance and growth of the organism. After sublethal heat treatment at 60 C for 15 min, greater than 99.0% of the viable population was unable to reproduce on media containing 6% NaCl. In addition, the heated cells displayed a sensitivity to incubation temperature, pH, and 0.01% methylene blue. When the injured cells were placed in a synthetic medium, recovery occurred at a much slower rate than in a complex medium. However, both media supported comparable growth of the uninjured organism. Various media used for the enrichment of streptococci also provided a suitable environment for the recovery of the injured cells. Generally, as more selective agents were present in the medium, the rates of recovery decreased. Metabolic inhibitor studies with chloramphenicol, penicillin, and actinomycin D substantiated the fact that the process involved was recovery and not growth, and that this recovery was linked to ribonucleic acid synthesis.     

Physiological Studies on the Recovery of Salt Tolerance by Staphylococcus aureus After Sublethal Heating

Cultures of S. aureus in 100 mM potassium phosphate buffer heated at 52 C for 15 min lost their tolerance to 7.5% NaCl. After incubation in a complex growth medium or in a diluted dialyzed medium in which unheated cells were unable to grow, salt tolerance was regained. Heat injury caused 30% loss of lipid. During recovery, the concentration of C(15) and C(17) fatty acids returned to normal, and there appeared to be an oversynthesis of C(16) and C(18) unsaturated acids. Penicillin abolished the latter reaction without affecting recovery; chloramphenicol did not affect fatty acid oversynthesis but reduced recovery. The K/Na ratio was 12.6 in control cells and 3.4 in injured cells, where it remained during the recovery of salt tolerance. Aspartate uptake was about 10% of the control level after injury and about 35% at recovery. Control cells grew without a lag on subculture, but injured cells which had regained their salt tolerance needed about 2 more h of incubation. Cells recovering with penicillin needed 6 more h, and cells recovering with chloramphenicol did not grow without a prolonged lag. Cells of S. aureus, therefore, may recover their salt tolerance while various membrane functions are still damaged.     

Destruction of Microbacterium lacticum, Escherichia coli and Staphylococcus aureus in milk by spray-drying. I. Selective count of surviving bacteria]

In this paper a method which allows the measure of microbial death rate during spray-drying by means of a streptomycin-resistant mutant that can be grown on a streptomycin-containing agar is described. Plate counts of Microbacterium lacticum, Escherichia coli, and Staphylococcus aureus recovered from skim milk powders were done on plate count agar in the presence and absence of streptomycin and on various selective media. The powders were produced from evaporated milk previously inoculated with those organisms. Our results showed that the proposed method allows the recovery of 78% of M. lacticum, 61% of E. coli, and 100% of S. aureus that survived spray-drying. Recoveries of surviving E. coli on violet bile agar and brilliant green bile 2% were 34% and 29% respectively. Baird-Parker and mannitol salt agar media allow the recovery of all surviving S. aureus, thus showing that S. aureus cells did not lose their ability to grow in media containig 7.5% NaCl. Our results show that physiological injury of the cells during spray-drying differs from injury due to heating only.     

Function of cell wall teichoic acid in thermally injured Staphylococcus aureus.

Thermally injured cells of Staphylococcus aureus lack the ability to grow on tryptic soy agar containing 7.5% NaCl. This injury phenomenon was examined in three strains of S. aureus: MF-31; H (Str); and, isolated from H (Str), 52A5, a mutant which lacks teichoic acid in the cell wall. Temperatures for sublethal heat treatment were selected to produce maximum injury with minimum death for each strain. Examination of isolated cell walls showed that magnesium was lost from the wall during heating, and that the degree of cell injury was accentuated when magnesium ions were either removed from or made unavailable to the cell. S. aureus 52A5 was more heat sensitive than its parent strain. Cells containing higher levels of wall teichoic acid generally showed less injury than normal cells. Cells with the weaker cation-binding polymer, teichuronic acid, in the cell wall generally showed greater injury. These data suggest that cell wall teichoic acid of S. aureus aids in the survival of the cell by the maintenance of an accessible surface pool of magnesium.     

Enumeration of sublethally heated staphylococci in some dried foods.

The effect of 45 substances to restore the salt tolerance of sublethally heat-injured Staphylococcus aureus was tested. Sodium pyruvate, yeast extract, L-histine, casitone (Difco), adenosine triphosphate, and acetylphosphate were effective. For enumeration a repair medium was first used, containing sodium pyruvate and penicillin in 1% skim milk. This step was followed by counting on Baird-Parker agar with penicillinase. This method was selective; fewer than 100 staphylococci/g food could be enumerated and it gave counts about 8 times higher than the method of Giolitti and Cantoni used as a five-tube most probable number technique. Heat injury sensitized S. aureus to polymyxin.     

Injury to Staphylococcus aureus during sausage fermentation.

Staphylococcus aureus 196E added to a beef sausage containing starter culture and 0.5 to 2.0% glucose and incubated at 35 degrees C was unable to grow when plated on tryptic soy agar (TSA) containing 7.5% NaCl. The injury, presumed to be due to the lactic acid produced during fermentation, was more pronounced at the lower concentrations of glucose (and lower acid levels). In the absence of glucose and/or starter culture, no injury was observed. When sausages containing S. aureus injured by fermentation at 35 degrees C were incubated at 5 degrees C, the counts on TSA (measures both injured and uninjured cells) and TSA containing 7.5% NaCl (measures uninjured cells only) remained constant; however, upon reincubation of the cold-stored sausage at 35 degrees C, the staphylococcus counts on TSA and TSA containing 7.5% NaCl and were similar to the counts of S. aureus present in fermenting sausages that had never been subjected to 5 degrees C. The demonstration of acid injury indicated that the injury phenomenon must be considered when determining numbers of viable S. aureus in fermented sausages.     

Injury to Staphylococcus aureus during sausage fermentation.

Staphylococcus aureus 196E added to a beef sausage containing starter culture and 0.5 to 2.0% glucose and incubated at 35 degrees C was unable to grow when plated on tryptic soy agar (TSA) containing 7.5% NaCl. The injury, presumed to be due to the lactic acid produced during fermentation, was more pronounced at the lower concentrations of glucose (and lower acid levels). In the absence of glucose and/or starter culture, no injury was observed. When sausages containing S. aureus injured by fermentation at 35 degrees C were incubated at 5 degrees C, the counts on TSA (measures both injured and uninjured cells) and TSA containing 7.5% NaCl (measures uninjured cells only) remained constant; however, upon reincubation of the cold-stored sausage at 35 degrees C, the staphylococcus counts on TSA and TSA containing 7.5% NaCl and were similar to the counts of S. aureus present in fermenting sausages that had never been subjected to 5 degrees C. The demonstration of acid injury indicated that the injury phenomenon must be considered when determining numbers of viable S. aureus in fermented sausages.     

Loss of D-alanine during sublethal heating of Staphylococcus aureus S6 and magnesium binding during repair.

Staphylococcus aureus S6 sublethally heated at 52 degrees C for 15 min to 0-1 M-potassium phosphate buffer pH 7-2, lost neither the ribitol teichoic acid of the wall nor the glycerol teichoic acid of the membrane. Hurst et al. (1974) showed that this heating caused 40% loss of the cellular Mg, and we now report the loss of 65% of the ester-bound D-alanine of teichoic acid. Repair from sublethal heat injury, measured by the return of salt tolerance, occurs in a simple no-growth medium provided that the cell concentration is less than 5 x 10(8)/ml. During repair, D-alanine is rapidly synthesized. Fully-repaired cells contain four times more D-alanine than do freshly-injured cells. Magnesium is present in the medium at only 3 x 10(-6) M, yet the cellular Mg concentration returns to normal within 1 h of incubation, even in the presence of EDTA. The results suggest that repair occurs in two stages. Soon after injury, in the absence of the competitive effect of D-alanine, Mg is strongly bound to teichoic acid. In repaired or uninjured cells Mg is less strongly bound. The implications of these findings are discussed in relation to the cation-binding function of teichoic acid.     

Thermal Injury and Recovery of Salmonella typhimurium and Its Effect on Enumeration Procedures

Exposure of Salmonella typhimurium 7136 to sublethal heating produced a temporary change in the tolerance of the organism to a particular stress medium. After sublethal heat treatment at 48 C for 30 min, greater than 90% of the viable population was unable to reproduce on Levine Eosin Methylene Blue Agar containing 2% NaCl. This sensitivity was dependent on the pH of the heating menstruum. In addition, the heated cells displayed a sensitivity to Brilliant Green Agar, Levine Eosin Methylene Blue Agar, Salmonella-Shigella Agar, and Desoxycholate Citrate Agar. Unheated cells displayed a sensitivity to Brilliant Green Agar, Salmonella-Shigella Agar, and Desoxycholate Citrate Agar. When the injured cells were placed in a suitable medium (Trypticase Soy Broth), they recovered and grew at a rate equal to that of normal cells. Recovery was also possible in Nutrient Broth, Lactose Broth, and Lauryl Tryptose Broth. Although recovery of the injured cell occurred in Tetrathionate Broth and Selenite F Broth, they were less than ideal growth media for the organism.     

Absence or presence of glucose in growth medium and its effect on heat injury inStaphylococcus aureus

Summary Staphylococcus aureus 196E, when grown in a glucose (0.25% wt./vol.)-containing medium, produced cells that would undergo injury when subjected to sublethal heat conditions (45 min at 50°C); however, if glucose was omitted from the growth medium, the extent of injury was greatly reduced. Media containing glucose sterilized by filtration or by separate autoclaving produced cells equal in injury susceptibility to medium in which glucose was autoclaved as part of the medium components. Injury also occurred when other sugars such as fructose, mannose, maltose, or lactose were substituted for glucose. Sugar-containing media that producedStaphylococcus aureus of maximal susceptibility to heat injury reached a pH of approximately 6 or lower during growth of the cells. Incubation of staphylococci in growth medium acidified with acetic or lactic acids or HCl did not lead to cells that would undergo injury under the stated conditions. The stimulatory effect of glucose on injury appears to be related to the metabolism of the sugar byStaphylococcus aureus.Agricultural Research Service, U.S. Department of Agriculture. Reference to brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.     

The Effect of Recovery Medium on the Isolation of Staphylococcus aureus after Heat Treatment and after the Storage of Frozen or Dried Cells

The recovery of heated or dried cells of Staphylococcus aureus was best on media containing blood or sodium pyruvate. It is suggested that the catalase of S. aureus may be destroyed or its activity reduced by heating or drying and that one of the main reasons for the better recoveries on these media is that they are able to destroy peroxide. All media tested adequately supported the recovery of frozen cells.     

一株广谱抑菌活性乳酸菌的筛选及特性研究

【目的】从贵州剑河采集的传统自然发酵豆酱中分离筛选具有广谱抑菌效果的乳酸菌,并进行肠道益生特性的研究。【方法】通过抑菌试验分离筛选得到菌株DJ-04,对其进行人工胃肠液耐受性、胆盐耐受性和渗透压耐受性的研究,并对其进行生理生化鉴定和16S r RNA鉴定。【结果】菌株DJ-04对大肠杆菌、沙门氏菌、金黄色葡萄球菌、志贺氏菌和铜绿假单胞菌的生长有很好的抑制作用;在p H值为2.5的人工胃液中处理3 h活菌数达到107 CFU/m L以上;在人工肠液中处理3 h活菌数达到108 CFU/m L以上,对人工胃肠液表现出良好的耐受性。能耐受一定浓度的牛胆盐,在质量浓度0.2 g/100 m L的牛胆盐环境中活菌数可达到107 CFU/m L;具有较高的渗透压耐受能力,在Na Cl质量浓度为10 g/100 m L的液体MRS中培养24 h后,活菌数仍在107 CFU/m L以上。经鉴定,DJ-04为植物乳杆菌。【结论】植物乳杆菌DJ-04具有良好的人工胃肠液耐受性以及耐胆盐和耐渗透压能力,具有肠道益生菌的潜能。     

Ribosome assembly during recovery of heat-injured Staphylococcus aureus cells.

The process of ribosome formation during repair of sublethal heat injury was examined in Staphylococcus aureus. Sublethal heating of this organism results in the degradation of the 30S ribosomal subunit and alteration of the 50S subunit. Cells recovering from sublethal injury were examined for changes with time in the sedimentation and electrophoretic properties of ribonucleoprotein particles and ribonucleic acid, respectively. When cells were allowed to recover in [3H]uridine, the label could be followed into ribonucleic acid species that coelectrophoresed with 23S and 16S ribonucleic acid. Three ribonucleoprotein particles (49S, 36S, and 30S) were isolated from repairing cells by sedimentation through sucrose gradients. Polyacrylamide gel electrophoresis showed that the 49S particle contained 23S ribonucleic acid, the 36S particle contained both 23S ribonucleic acid and 16S precursor and mature ribonucleic acid, and the 30S particle contained 16S and precursor 16S ribonucleic acid. Particles with similar sedimentation properties were found in unheated cells.     

Staphylococcus aureus Protoplasting Induced by d-Cycloserine

Addition of sublethal doses of d-cycloserine to growing cells of Staphylococcus aureus induces the rupture of the cell wall along an equatorial ring, thus allowing the liberation of protoplasts.     

Lack of association between glutathione content and development of thermal tolerance in human fibroblasts

Thermal tolerance is a transient state of heat resistance occurring in cells and tissues after exposure to sublethal heat or certain chemicals. Although the mechanism of such resistance is unknown, it has been recently shown that preceding its development, cellular glutathione (GSH) levels rise. We have used a glutathione synthetase-deficient [GSH(-)] human fibroblast line to study the relationship between glutathione content and thermal tolerance. The GSH(-) cells had approximately 6% as much GSH as normal fibroblasts. Normal and GSH(-) fibroblasts showed similar survival after exposure to 45 degrees C. Exposure of normal fibroblasts to heat (45 degrees C for 15 min) led to a prompt rise in cellular GSH content as well as development of transient thermal tolerance. Similar treatment of GSH(-) fibroblasts produced no change in the very low GSH levels but was associated with a degree of thermal tolerance similar to that of normal cells. Thermal tolerance decayed more rapidly in GSH(-) cells than in normal fibroblasts. We conclude that the development of thermal tolerance in human fibroblasts is independent of GSH content.     

Recovery, growth, and production of heat-stable enterotoxin by Escherichia coli after copper-induced injury

Exposure of enterotoxigenic Escherichia coli strains to a sublethal concentration (0.75 mg/liter) of copper for 3 days at 4 degrees C induced sensitivity to deoxycholate (0.1%). When placed in a complex (brain heart infusion) or a defined amino acid salt medium, the copper-injured cells recovered their tolerance to deoxycholate in 3 and 6 h, respectively, and commenced active growth. Growth and heat-stable enterotoxin production of uninjured and copper-injured cells were studied in brain heart infusion medium. A slightly altered growth curve and an initial slow rate of toxin production were observed in injured cells when compared with those corresponding uninjured controls. However, maximum heat-stable enterotoxin levels in injured cultures were comparable to those produced by uninjured cells, suggesting that the enterotoxigenic potential of copper-injured cells was fully retained.