Injury and recovery of Escherichia coli after sublethal acidification.

Over 99% of the viable cells of Escherichia coli K-12 were injured after a 60-min exposure to 0.3 M sodium acetate buffer at pH 4.2. Injured cells were those able to grow on Trypticase soy agar but unable to grow on violet red bile agar. The extent of both death and injury of acid-treated cells increased with decreasing pH or increasing concentration of acid. Injured cells were able to recover their colony-forming ability on violet red bile agar by incubation in Trypticase soy broth or potassium phosphate buffer before plating on the agar media. Direct neutralization of the injury medium did not allow recovery and, in fact, was lethal to the population. The addition of metabolic inhibitors to the Trypticase soy recovery broth was used to study the repair process. It was not affected by the presence of inhibitors of protein, cell wall, deoxyribonucleic acid, or ribonucleic acid syntheses. The addition of 2,4-dinitrophenol to the recovery medium also did not inhibit recovery. Actinomycin D and N,N'-dicyclohexylcarbodiimide were lethal to a proportion of the acidified cells but allowed another fraction of the population to recover. There were no detectable amounts of 260- or 280-nm-absorbing materials leaked during the course of acid injury.     

Effect of aeration on minimal medium recovery of heated Salmonella typhimurium.

The effect of presence or absence of air on minimal medium recovery of heated Salmonella typhimurium was investigated. It was determined that the expression of minimal medium recovery is not only dependent on heat and a nutritionally complex medium but also on air. Unlike in the presence of air, in the presence of nitrogen, cells were able to recover their ability to grow on Trypticase soy agar enriched with 0.5% yeast extract (TSY) when incubated in TSY broth. It was established that in the presence of nitrogen the number of heat-TSY- induced, single-straneded breaks in deoxyribonucleic acid (DNA) were less than in the presence of air. Furthermore, the DNA breaks in nitrogen were repaired, whereas DNA breaks in air were not. The ability of cells to grow on TSY agar corresponded well with their ability to repair damage to DNA.     

Repair and Enumeration of Injured Coliforms in Frozen Foods

Two strains of Escherichia coli manifested death and repairable injury after being frozen in water or sterile foods at -20 C. The injured survivors were inhibited from forming colonies on violet red bile agar (VRBA) or deoxycholate lactose agar; this inhibition was greater when enumeration was done by the pour plate method instead of the surface or surface-overlay method. Injured cells repaired rapidly in Trypticase soy broth (TSB), and the repair was about maximum after 1 h at 25 C. When the injured cells were added to different foods and incubated at 25 C, repair also occurred; however, recovery was better and more uniform when the samples were mixed with TSB and incubated 1 h at 25 C. Cell multiplication was not evident until after 90 to 120 min at 25 C. The enumeration of coliforms from commercially frozen foods was increased when the thawed samples were mixed with TSB and the cells were allowed to repair 1 h at 25 C. In some samples, the repair permitted at least a 20-fold increase in the coliform count. The associated flora in the commercially frozen foods gave no evidence of impairing the repair of coliforms, nor did they start multiplication prior to 90 min after being incubated in TSB at 25 C. Generally, the plating gave more reproducible recovery of coliforms than did the most probable number method. Also, a higher number of coliforms were obtained by the surface-overlay method of plating using VRBA.     

Enumeration of Escherichia coli in Frozen Samples After Recovery from Injury

More than 90% of the surviving cells of Escherichia coli NCSM were injured after freezing in water at -78 C. Injury was determined by the ability of cells to form colonies on Trypticase soy agar with yeast extract but not on violet red-bile agar and deoxycholate-lactose agar. Exposure of the injured cells to Brilliant Green-bile broth and lauryl sulfate broth prevented subsequent colony formation on Trypticase soy agar with yeast extract. The freeze-injury could be repaired rapidly in a medium such as Trypticase soy broth with yeast extract (TSYB). The repaired cells formed colonies on violet red-bile agar and deoxycholate-lactose agar and were not inhibited by Brilliant Green-bile broth and lauryl sulfate broth. At least 90% of the cells repaired in TSYB within 30 min at 20 to 45 C and began multiplication within 2 h at 25 C. When the cells were frozen in different foods, 60 to 90% of the survivors were injured. Repair of the injured cells occurred in foods during 1 h at 25 C, but generally repair was greater and more reproducible when the foods were incubated in TSYB. The study indicated that the repair of freeze-injured coliform bacteria should be accomplished before such cells are exposed to selective media for their enumeration.     

Repair and Enumeration of Injured Coliforms by a Plating Procedure

Surface plating of coliforms on Trypticase soy agar, followed by 1 to 2 h of incubation at 25 C and subsequent overlay with violet red bile agar, was found to be a useful method for the repair and enumeration of coliforms injured by freezing.     

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.     

Evaluation of the ability of primary selective enrichment to resuscitate heat-injured and freeze-injured Listeria monocytogenes cells.

Resuscitation rates of injured Listeria monocytogenes on conventional selective Listeria enrichment broth and nonselective Trypticase soy broth containing 0.6% yeast extract were compared. Cells were heated to 60 degrees C for 5 min or frozen at -20 degrees C for 7 days. Inoculation of Trypticase soy broth-yeast extract with the stressed cells resulted in growth that was superior to that in Listeria enrichment broth. Injured cells were fully recovered at 6 to 8 h.     

Evaluation of the ability of primary selective enrichment to resuscitate heat-injured and freeze-injured Listeria monocytogenes cells.

Resuscitation rates of injured Listeria monocytogenes on conventional selective Listeria enrichment broth and nonselective Trypticase soy broth containing 0.6% yeast extract were compared. Cells were heated to 60 degrees C for 5 min or frozen at -20 degrees C for 7 days. Inoculation of Trypticase soy broth-yeast extract with the stressed cells resulted in growth that was superior to that in Listeria enrichment broth. Injured cells were fully recovered at 6 to 8 h.     

Recovery of Spores of Bacillus stearothermophilus from Thermal Injury

Bacillus stearothermophilus grown in nutrient broth produced a product which promoted recovery from thermal injury of its spores. This phenomenon was observed with nutrient agar as the plating medium but not with a medium composed of Trypticase, Phytone, dextrose and phosphate (TPDP). Recovery of injured spores was greatest in such a medium if it contained starch or charcoal. Trypticase soy agar and dextrose tryptone agar were markedly inferior to TPDP medium.     

Thermal injury of Yersinia enterocolitica

Procedures were developed to evaluate thermal injury to three strains of Yersinia enterocolitica (serotypes 0:3, 0:8, and 0:17). Serotype 0:17 (atypical strain) was more sensitive to bile salts no. 3 (BS) and to sublethal heat treatment than the typical strains, 0:3 and 0:8. When the 0:3, 0:8, and 0:17 serotypes were thermally stressed in 0.1 M PO4 buffer, pH 7.0, at 47 degrees C for 70, 60, and 12 min, respectively, greater than 99% of the total viable cell population was injured. Injury was determined by the ability of cells to form colonies on brain heart infusion (BHI) agar, but not on Trypticase soy agar (TSA) plus 0.6% BS for serotypes 0:3 and 0:8 and TSA plus 0.16% BS for 0:17. Heat injury of serotype 0:17 cells for 15 min in 0.1 M PO4 buffer caused an approximate 1,000-fold reduction in cell numbers on selective media as compared with cells heated in pork infusion (PI), BHI broth, and 10% nonfat dry milk (NFDM). The extended lag and resuscitation period in BHI broth was 2.5 times greater for 0:17 cells injured in 0.1 M PO4 than for cells injured in BHI or PI. The rate and extent of repair of Y. enterocolitica 0:17 cells in three recovery media were directly related to the heating menstruum used for injury. The use of metabolic inhibitors demonstrated that ribonucleic acid synthesis was required for repair, whereas deoxyribonucleic, cell wall, and protein synthesis were not necessary for recovery of 0:17 cells injured in 0.1 M PO4 buffer, BHI, or PI. Inhibition of respiration by 2,4-dinitrophenol slowed repair only for 0:17 cells injured in 0.1 M PO4 buffer, not for cells injured in PI or BHI.     

Thermal injury of Yersinia enterocolitica.

Procedures were developed to evaluate thermal injury to three strains of Yersinia enterocolitica (serotypes 0:3, 0:8, and 0:17). Serotype 0:17 (atypical strain) was more sensitive to bile salts no. 3 (BS) and to sublethal heat treatment than the typical strains, 0:3 and 0:8. When the 0:3, 0:8, and 0:17 serotypes were thermally stressed in 0.1 M PO4 buffer, pH 7.0, at 47 degrees C for 70, 60, and 12 min, respectively, greater than 99% of the total viable cell population was injured. Injury was determined by the ability of cells to form colonies on brain heart infusion (BHI) agar, but not on Trypticase soy agar (TSA) plus 0.6% BS for serotypes 0:3 and 0:8 and TSA plus 0.16% BS for 0:17. Heat injury of serotype 0:17 cells for 15 min in 0.1 M PO4 buffer caused an approximate 1,000-fold reduction in cell numbers on selective media as compared with cells heated in pork infusion (PI), BHI broth, and 10% nonfat dry milk (NFDM). The extended lag and resuscitation period in BHI broth was 2.5 times greater for 0:17 cells injured in 0.1 M PO4 than for cells injured in BHI or PI. The rate and extent of repair of Y. enterocolitica 0:17 cells in three recovery media were directly related to the heating menstruum used for injury. The use of metabolic inhibitors demonstrated that ribonucleic acid synthesis was required for repair, whereas deoxyribonucleic, cell wall, and protein synthesis were not necessary for recovery of 0:17 cells injured in 0.1 M PO4 buffer, BHI, or PI. Inhibition of respiration by 2,4-dinitrophenol slowed repair only for 0:17 cells injured in 0.1 M PO4 buffer, not for cells injured in PI or BHI.     

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.     

Repair detection procedure for enumeration of fecal coliforms and enterococci from seafoods and marine environments.

The repair detection procedure of Speck et al. (Appl. Microbiol. 29:549-550, 1975) was adapted for the enumeration of coliforms, fecal coliforms, and enterococci in seafood and environmental samples. Samples were pour plated with Trypticase soy agar, followed by a 1- to 2-h incubation to effect repair; the plates were then overlaid with the selective medium and incubated. Violet red bile agar and an incubation temperature of 45 degrees C were used as the selective conditions for fecal coliforms, and KF streptococcal agar was used for the enumeration of enterococci. The method was more efficient than the standard most-probable-number method for fecal coliform enumeration and also allowed enumeration of the injured cells, which might have remained undetected when selective medium in the most-probable-number method was used. The repair detection method effectively recovered the injured portion of the population of enterococci capable of growing on KF streptococcal agar. The repair enumeration method was not suitable for coliforms in marine samples because associative marine bacteria mimicked coliforms in violet red bile agar plates incubated at 35 degrees C. The marine bacteria did not grow at 45 degrees C and therefore did not interfere with fecal coliform enumeration.     

Plating procedure for the enumeration of coliforms from dairy products.

A "repair-detection" procedure consisting of pour plating of food samples with Trypticase soy agar, followed by 1-h repair incubation at room temperature and subsequent overlay with violet red bile agar, was found to be an effective method for the detection of injuried and uninjuried coliforms from dairy products. This method was relatively less effective for the detection of coliforms in many semipreserved foods as compared with dairy products, but more effective than the most-probable-number method.     

Plating procedure for the enumeration of coliforms from dairy products.

A "repair-detection" procedure consisting of pour plating of food samples with Trypticase soy agar, followed by 1-h repair incubation at room temperature and subsequent overlay with violet red bile agar, was found to be an effective method for the detection of injuried and uninjuried coliforms from dairy products. This method was relatively less effective for the detection of coliforms in many semipreserved foods as compared with dairy products, but more effective than the most-probable-number method.     

Identification of nutritional components in trypticase responsible for recovery of Escherichia coli injured by freezing

Moss, C. Wayne (North Carolina State University, Raleigh), and M. L. Speck. Identification of nutritional components in Trypticase responsible for recovery of Escherichia coli injured by freezing. J. Bacteriol. 91:1098-1104. 1966.-Freezing and storage of Escherichia coli at -20 C resulted in nonlethal or "metabolic" injury to a proportion of the surviving population. The injury was manifested as an increased nutritional requirement after freezing. Injured cells could not grow on a minimal agar medium, but could develop on Trypticase Soy Agar. The percentage of injured survivors varied among strains, but was little affected by altering the freezing menstruum. Trypticase was found to be the component in Trypticase Soy Agar responsible for the recovery of injured cells, and contained five closely related peptides that possessed most of the biological activity. Isolation of the peptides was accomplished by Sephadex gel chromatography, paper chromatography, and high-voltage paper electrophoresis. Hydrolysis of the peptides destroyed the ability to restore injured cells.     

Sublethal heat stress of Vibrio parahaemolyticus.

When Vibrio parahaemolyticsu ATCC 17802 was heated at 41 degrees C for 30 min in 100 mM phosphate-3% NaCl buffer (pH 7.0), the plate counts obtained when using Trypticase soy agar containing 0.25% added NaCl (0.25 TSAS) were nearly 99.9% higher than plate counts using Trypticase soy agar containing 5.5% added NaCl (5.5 TSAS). A similar result was obtained when cells of V. parahaemolyticus were grown in a glucose salts medium (GSM) and heated at 45 degrees C. The injured cells recovered salt tolerance within 3 h when placed in either 2.5 TSBS or GSM at 30 degrees C. The addition of chloramphenicol, actinomycin D, or nalidixic acid to 2.5 TSBS during recovery of cells grown in 2.5 TSBS indicated that recovery was dependent upon protein, ribonucleic acid (RNA, and deoxyribonucleic acid (DNA) synthesis. Penicillin did not inhibit the recovery process. Heat-injured, GSM-grown cells required RNA synthesis but not DNA synthesis during recovery in GSM. Chemical analyses showed that total cellular RNA decreased and total cellular DNA remained constant during heat injury. The addition of [6-3H]uracil, L-[U-14C]leucine, and [methyl-3H]thymidine to the recovery media confirmed the results of the antibiotic experiments.     

Antimicrobial properties of diacetyl.

Diacetyl preparations from three commercial sources were found to be essentially similar when tested primarily against a set of 40 cultures, including 10 of lactic acid bacteria, 4 of yeasts, 12 of gram-positive non-lactic acid bacteria, and 14 of gram-negative bacteria. The compound was effective at pH less than or equal to 7.0 and progressively ineffective at pH greater than 7.0. The lactic acid bacteria were essentially unaffected by concentrations between 100 and 350 micrograms/ml over the pH range of 5.0 to 7.0. Of the 12 gram-positive non-lactic acid bacteria, 11 were inhibited by 300 micrograms/ml at pH less than or equal to 7.0. The three yeasts and the 13 gram-negative bacteria that grew at pH 5.5 were inhibited by 200 micrograms/ml. Diacetyl was ineffective against four clostridia under anaerobic conditions. It was lethal for gram-negative bacteria and generally inhibitory for gram-positive bacteria. Nongrowing cells were not affected. The effectiveness of diacetyl was considerably less in brain heart infusion broth, Trypticase soy agar, and cooked-meat medium than in nutrient broth or plate count agar. The antimicrobial activity was antagonized by glucose, acetate, and Tween 80 but not by gluconic acid. As an antimicrobial agent, diacetyl was clearly more effective against gram-negative bacteria, yeasts, and molds than against gram-positive bacteria.     

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.     

Enhanced recovery of injured Escherichia coli by compounds that degrade hydrogen peroxide or block its formation

Escherichia coli LSUFS was injured either by freezing at -10 degrees C or by heating at 57 degrees C for 12 min. Surviving cells were recovered on nonselective tryptone-glucose extract agar and selective violet red bile agar supplemented with compounds that degrade hydrogen peroxide or block its formation. Various concentrations of the following compounds were tested: sodium pyruvate, 3,3'-thiodipropionic acid, catalase, ascorbic acid, potassium permanganate, sodium thioglycolate, dimethylsulfoxide, ethoxyquin, n-propyl gallate, alpha-tocopherol sodium metabisulfite, and ferrous sulfate. Sodium pyruvate and 3,3'-thiodipropionic acid, when added to either medium, significantly (P greater than 0.01) increased recovery of injured cells. More than 90% of the heat-injured cells and 40 to 90% of the freeze-injured cells failed to grow on unsupplemented tryptone-glucose extract agar. Supplementation of violet red bile agar increased recovery, but the counts remained considerably lower than the tryptone-glucose extract agar counts. The repair detection procedure of Speck et al. (M. Speck, B. Ray, R. Read, Jr., Appl. Microbiol. 29:549-550, 1975) was greatly improved by the addition of pyruvate or 3,3'-thiodipropionic acid. However, when this improved repair detection procedure was applied to foods, pyruvate-supplemented media showed some false-positives. We therefore recommend that 3,3'-thiodipropionic acid be used to supplement media in the repair detection procedure.