Radiation Resistance and Injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25°C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and −30°C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at −20°C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at −20°C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at −20°C, nor did storage at −20°C alter the cell's resistance to irradiation at 25°C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36°C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36°C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5°C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36°C for 1 day than at 5°C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

Inactivation and injury of Yersinia enterocolitica by radiation and freezing.

Cell inactivation and cell injury by irradiation and freezing of the potentially enteropathogenic, food-borne gram-negative rod Yersinia enterocolitica strain WA was investigated. The radiation dose necessary to kill 90% of the initial population, i.e., one D-value, was 10.0, 14,3, and 24.0 krad when irradiation was carried out at 2 to 0, -18, and -75 degrees C, respectively. On the other hand, cell injury, i.e., inability to form colonies in agar containing 2.5% NaCl, was 32, 42 and 54% when cells were irradiated to one D-value at 2 to 0, -18, and -75 degrees C, respectively. Freezing alone (without irradiation) at -18 and -75 degrees C for 1 h resulted in 7 and 42% cell inactivation and 55 and 83% cell injury, respectively. These data show that given the same extent of cell inactivation, freezing caused substantially greater cell injury than radiation. For purposes of radiation sterilization, doses of 100 and 150 krad would be sufficient to inactivate 10 log cycles of Y. enterocolitica strain WA if irradiated at 2 to 0 and -18 degrees C, respectively. Presence of 2.5% NaCl may result in a further 50% reduction of the dose required to achieve sterility.     

Substituted Diazenes: Effect on the Growth of Enterobacteria and Possible Use as Selective Agents for Isolation of Pseudomonads

Incorporation of various diazenes into Trypticase soy media appeared selectively to permit the growth of pseudomonads while inhibiting the growth of a variety of enterobacteria. One of these diazenes, diamide (diazenedicarboxylic acid bisdimethylamide), was shown to be bactericidal for pure cultures of Escherichia coli, Proteus sp., and Salmonella enteritidis and to cause a 1- to 2-hr delay in the growth of Pseudomonas aeruginosa. When mixtures of these four organisms were inoculated into Trypticase soy broth or Trypticase soy agar (TSA) containing diamide, P. aeruginosa grew in overnight cultures. TSA containing diamide was also used successfully to isolate pseudomonads from soil, clinical urine specimens, fish, ground beef, ground pork, and ground veal.     

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.     

Method for the detection of injured Vibrio parahaemolyticus in seafoods.

The sensitivity of Vibrio parahaemolyticus cells to refrigeration and frozen storage and the development of a method for detecting injured and uninjured V. parahaemolyticus cells were studied. Cell suspensions in different kinds of seafood homogenates were either regrigerated (4 degrees C) or frozen (-20 degrees C), stored, and examined for cell survival during storage. V. parahaemolyticus cells were sensitive to both storage temperatures. Many cells died, and many survivors were sublethally injured. In general, refrigeration storage appeared to be more injurious than frozen storage. The initial recovery of the sublethally injured cells was highest in a nutritionally rich, nonselective liquid medium such as Trypticase soy broth, whereas maximum cell multiplication was observed in Trypticase soy broth containing 3% NaCl. The sublethally injured V. parahaemolyticus cells demonstrated sensitivity to the selective enrichment medium, glucose salt teepol broth. From these findings, a new method (designated as the "repair-detection" method) was developed for the isolation and enumeration of V. parahaemolyticus. Comparative studies between the recommended and the repair-detection methods showed that injured V. parahaemolyticus cells were present in commercial seafoods and that the repair-detection method was definitely more effective for the detection of total numbers of V. parahaemolyticus cells.     

Factors affecting inactivation of Moraxella-Acinetobacter cells in an irradiation process.

The effect of various stages of the irradiation processing of beef on the injury and inactivation of radiation-resistant Moraxella-Acinetobactor cells was studied. Moraxella-Acinetobacter cells were more resistant to heat inactivation and injury when heated in meat with salts (0.75% NaCl and 0.375% sodium tripolyphosphate) then in meat without salts. These salts had no effect on radiation resistance. Both radiation- and heat-injured cells were unable to form colonies at 30 degrees C in plate count agar containing 0.8% NaCl. Neither unstressed nor heat-stressed cells were able to multiply in minced beef incubated at 30 degrees C for 12 h. Only after the beef was diluted 1:10 with peptone water were the heat-injured cells able to repair and eventually multiply. Heated cells were more sensitive to radiation inactivation and injury than unheated cells. After repair, the cells regained their resistance to both NaCl and irradiation. Freezing and storage at -40 degrees C for 14 days had only a slight effect on either unstressed or heat-stressed cells.     

Survival and growth characteristics of Escherichia coli associated with hemorrhagic colitis.

Escherichia coli O157:H7 in ground beef was more sensitive to heat than salmonellae, but survived for 9 months at -20 degrees C with little change in number. The organisms grew well in Trypticase soy broth (BBL Microbiology Systems) between 30 and 42 degrees C, with 37 degrees C being optimal for growth. E. coli O157:H7 grew poorly in the temperature range (44 to 45.5 degrees C) generally used for recovery of E. coli from foods.     

Relationships of Temperature and Sodium Chloride Concentration to the Survival of Vibrio parahaemolyticus in Broth and Fish Homogenate

The interaction of temperature and NaCl concentration in affecting the survival of three strains of Vibrio parahaemolyticus was studied in Trypticase soy broth and fish homogenate. Cells of V. parahaemolyticus suspended in Trypticase soy broth without NaCl were quite unstable and readily killed. The presence of NaCl appeared to be protective to the cells at 48 +/- 1 C, with the optimal concentration strain-dependent for the 3 to 12% range tested. Temperatures of 5 +/- 1, -5 +/- 1, and -18 +/- 1 C reduced the number of viable organisms per milliliter regardless of the NaCl concentration. In the presence of NaCl, viable cells, in numbers ranging up to 580 per ml, were still detected at the end of 30 days of storage. Similar results were obtained for cells suspended in fish homogenate, except that fish homogenate itself was protective as compared with Trypticase soy broth. This protection was significantly lower than that provided by NaCl in any amount tested.     

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.     

Suitability of selective plating media for recovering heat- or freeze-stressed Escherichia coli O157:H7 from tryptic soy broth and ground beef.

The efficacy of tryptic soy agar (TSA), modified sorbitol MacConkey agar (MSMA), modified eosin methylene blue (MEMB) agar, and modified SD-39 (MSD) agar in recovering a five-strain mixture of enterohemorrhagic Escherichia coli O157:H7 and five non-O157 strains of E. coli heated in tryptic soy broth at 52, 54, or 56 degrees C for 10, 20, and 30 min was determined. Nonselective TSA supported the highest recovery of heated cells. Significantly (P < or = 0.05) lower recovery of heat-stressed cells was observed on MSMA than on TSA, MEMB agar, or MSD agar. The suitability of MEMB agar or MSD agar for recovery of E. coli O157:H7 from heated or frozen (-20 degrees C) low- or high-fat ground beef was determined. Recovery of E. coli O157:H7 from heated ground beef was significantly (P < or = 0.05) higher on TSA than on MEMB agar, which in turn supported higher recovery than MSD agar did; MSMA was inferior. Recovery from frozen ground beef was also higher on MEMB and MSD agars than on MSMA. Higher populations were generally recovered from high-fat beef than from low-fat beef, but the relative performance of the recovery media was the same. The inability of MSMA to recover stressed cells of E. coli O157:H7 underscores the need to develop a better selective medium for enumerating E. coli O157:H7.     

Use of a single procedure for selective enrichment, isolation, and identification of plasmid-bearing virulent Yersinia enterocolitica of various serotypes from pork samples.

Many selective enrichment methods for the isolation of Yersinia enterocolitica from foods have been described. However, no single isolation procedure has been described for the recovery and identification of various plasmid-bearing serotypes. A single improved procedure for selective enrichment, isolation, identification, and maintenance of plasmid-bearing virulent serotypes of Y. enterocolitica from pork samples was developed. Enrichment at 12 degrees C in Trypticase soy broth containing yeast extract, bile salts, and Irgasan was found to be an efficient medium for the recovery of plasmid-bearing virulent strains of Y. enterocolitica representing O:3; O:8; O:TACOMA; O:5, O:27; and O:13 serotypes. MacConkey agar proved to be a reliable medium for the isolation of presumptive colonies, which were subsequently confirmed as plasmid-bearing virulent strains by Congo red binding and low calcium response. Further confirmation by multiplex PCR employed primers directed at the chromosomal ail and plasmid-borne virF genes, which are present only in pathogenic strains. The method was applied to pig slaughterhouse samples and was effective in isolating plasmid-bearing virulent strains of Y. enterocolitica from naturally contaminated porcine tongues. Strains isolated from ground pork and tongue expressed plasmid-associated phenotypes and mouse pathogenicity.     

Effect of Soy Proteins on the Growth of Clostridium perfringens

Proteins that are used to fabricate imitation foods such as synthetic meats were evaluated for stimulative or inhibitory effects on the growth of Clostridium perfringens. Growth rate and extent were measured in thioglycolate medium without dextrose. This liquid medium contains Trypticase (BBL) which served as the protein control. For comparison, various soy proteins, synthetic meats, beef, turkey, sodium caseinate, and combinations of each were substituted for Trypticase. Meat loaf systems were also employed to determine the effects of protein additives to meat under actual meat loaf conditions. Growth of C. perfringens type A, strain S40, was measured in the respective media at 45 C at a pH of 7.0 and an E(h) of below -300 mv. Viable populations were enumerated by agar plate techniques on Trypticase-sulfite-yeast-citrate-agar incubated anaerobically (90% N(2)-10% CO(2)) for 18 hr at 35 C. When compared to Trypticase, some soy proteins had stimulative effects on the growth of C. perfringens, whereas sodium caseinate and some soy proteins were inhibitory. In liquid medium in which meat or soy meat was the source of protein, there was a marked stimulation by beef, chicken, and soy beef. Soy chicken supported growth at a rate less than observed with Trypticase. Under actual meat loaf conditions, the addition of soy meat or protein additives to beef did not affect the growth of C. perfringens. The addition of protein additives to turkey meat loaves significantly enhanced the rate of growth of C. perfringens. The stimulative effects of some soy proteins are significant in relation to control of foodborne disease.     

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.     

Survival of Campylobacter jejuni inoculated into ground beef.

Ground beef was inoculated with mixed cultures of Campylobacter jejuni, and the samples were subjected to various cooking and cold-storage temperatures. When samples were heated in an oven at either 190 or 218 degrees C, approximately 10(7) cells of C. jejuni per g were inactivated (less than 30 cells per g) in less than 10 min after the ground beef reached an internal temperature of 70 degrees C. When the samples were held at -15 degrees C over 14 days of storage, the numbers of C. jejuni declined by 3 log10. When inoculated samples were stored with an equal amount of Cary-Blair diluent at 4 degrees C, no changes in viability were observed over 14 days of storage. Twenty-five times as much C. jejuni was recovered from inoculated ground beef when either 10% glycerol or 10% dimethyl sulfoxide was added to an equal amount of ground beef before freezing as was recovered from peptone-diluted ground beef. Twice as much inoculated C. jejuni was recovered from ground beef plus Cary-Blair diluent as was recovered from ground beef plus peptone diluent.     

Fate of Escherichia coli O157:H7 as affected by pH or sodium chloride and in fermented, dry sausage.

The influence of pH adjusted with lactic acid or HCl or sodium chloride concentration on survival or growth of Escherichia coli O157:H7 in Trypticase soy broth (TSB) was determined. Studies also determined the fate of E. coli O157:H7 during the production and storage of fermented, dry sausage. The organism grew in TSB containing less than or equal to 6.5% NaCl or at a pH of 4.5 to 9.0, adjusted with HCl. When TSB was acidified with lactic acid, the organism grew at pH 4.6 but not at pH 4.5. A commercial sausage batter inoculated with 4.8 x 10(4) E. coli O157:H7 per g was fermented to pH 4.8 and dried until the moisture/protein ratio was less than or equal to 1.9:1. The sausage chubs were then vacuum packaged and stored at 4 degrees C for 2 months. The organism survived but did not grow during fermentation, drying, or subsequent storage at 4 degrees C and decreased by about 2 log10 CFU/g by the end of storage. These studies reveal the importance of using beef containing low populations or no E. coli O157:H7 in sausage batter, because when initially present at 10(4) CFU/g, this organism can survive fermentation, drying, and storage of fermented sausage regardless of whether an added starter culture was used.     

Repair of Injury Induced by Freezing Escherichia coli as Influenced by Recovery Medium

Freezing an aqueous suspension of Escherichia coli NCSM at -78 C for 10 min, followed by thawing in water at 8 C for 30 min, resulted in the death of approximately 50% of the cells, as determined by their inability to form colonies on Trypticase soy agar containing 0.3% yeast extract (TSYA). Among the survivors, more than 90% of the cells were injured, as they failed to form colonies on TSYA containing 0.1% deoxycholate. Microscope counts and optical density determinations at 600 nm suggested that death from freezing was not due to lysis of the cells. Death and the injury were accompanied by the loss of 260- and 280-nm absorbing materials from the intracellular pool. Injury was reversible as the injured cells repaired in many suitable media. The rate of repair was rapid and maximum in a complex nutrient medium such as Trypticase soy broth supplemented with yeast extract. However, inorganic phosphate, with or without MgSO₄, was able to facilitate repair. Repair in phosphate was dependent on the pH, the temperature, and the concentration of phosphate.     

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.     

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.     

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.