Radiation Survival of Food Pathogens in

Transcription

1 APPLIED MICROBIOLOGY, Jan., 1966 Vol. 14, No. 1 Copyright 1966 American Society for Microbiology Printed in U.S.A. Radiation Survival of Food Pathogens in Complex Media JOHN K. DYER, A. W. ANDERSON, AND PISAWAT DUTIYABODHI Department of Microbiology, School of Science, Oregon State University, Corvallis, Oregon Received for publication 17 August 1965 ABSTRACT DYER, JOHN K. (Oregon State University, Corvallis), A. W. ANDERSON, AND PISAWAT DUTIYABODHI. Radiation survival of food pathogens in complex media. Appl. Microbiol. 14: When 15 bacterial species representing genera associated with food-borne diseases were irradiated individually, all except Escherichia coli and Streptococcus faecalis showed typical linear dose-survival curves in Hartsell's The minimal lethal dose (MLD) for the organisms tested ranged from 3.0 X 105 to 6.0 X 105 rad. Salmonella paratyphi B, S. wichita, S. typhi, E. coli, and S. faecalis were found to be the least sensitive to radiation. In commercially canned crabmeat the survival curves of S. typhi, S. paratyphi B, and S. wichita exhibited to varying degrees an initial linear death decline with increasing radiation doses, followed by a distinct tailing effect caused by survival of low numbers at the higher doses. The above species of Salmonella were further individually subjected to y-radiation in various dilutions of crabmeat. The "tailing effect" gradually disappeared, with the dose-survival curve tending to become linear as the concentration of the crabmeat decreased. An important aspect of the future applications of radiation to the preservation of foods is that of insuring that food commodities made available for public consumption be free of any pathogenic microorganisms. A "working" dose sufficient to free any food commodity from pathogens should be considered, keeping in mind the factors which may influence the radiosensitivity of a microorganism. Among the more important of these is the irradiating medium. Previous studies have shown microorganisms to be more resistant when irradiated in a medium such as nutrient broth, as compared with buffer or distilled water (6). Further work has shown that, as the chemical complexity of the growth environment increases, there is a concomitant increase in the resistance of the microorganism (5). Thus, it is possible that a microorganism when irradiated in a new complex medium would exhibit a new and entirely different type of dose-survival curve. Such a curve would possibly necessitate changes in "working" doses. The present report is intended (i) to compare the relative resistance and to observe resulting survival patterns in Hartsell's broth of 15 bacterial species representing genera associated with food-borne diseases after individual exposure to 7y-radiation, (ii) to similarly examine the survival patterns in commercially canned crabmeat of Salmonella wichita, S. paratyphi B, and S. typhi, and (iii) to observe the effects of added water in influencing the "tailing" phenomenon of the radiation inactivation patterns found in crabmeat for the above Salmonella species. MATERIALS AND METHODS Organisms. The microorganisms selected in this study represented either species known to be pathogenic or nonpathogenic members of various genera containing pathogenic species. S. paratyphi and S. wichita were obtained from the culture collection maintained at the Communicable Disease Center in Atlanta, Ga. The following species were obtained from the culture collection maintained in the Department of Microbiology, Oregon State University, Corvallis: S. typhi, S. paratyphi A, S. choleraesuis, S. enteritidis, S. pullorum, Shigella dysenteriae, S. paradysenteriae, S. sonnei, Escherichia coli, Streptococcus faecalis, Proteus vulgaris, Neisseria catarrhalis, and Mycobacterium smegmatis. Culture media. Hartsell's broth of the following composition was used: yeast extract, 5.0 g; tryptone, 5.0 g; Proteose Peptone (Difco), 5.0 g; NaCl, 5.0 g; veal infusion, 100 ml; and distilled water to make 1 liter. The ph was adjusted so that after autoclaving at 121 C for 20 min it would be 7.2. Hartsell's agar was prepared by adding 20.0 g of agar to 1 liter of 92

2 VOL. 14, 1966 RADIATION SURVIVAL OF FOOD PATHOGENS 93 Preparation of suspension. To maintain a similar population for each of the cultures used for inoculation, an inoculum from the pure culture was transferred to 15 ml of Hartsell's broth and incubated for 24 hr at 35 C. From the above culture, 1 ml was inoculated into 99 ml and again incubated as above. Two flasks were inoculated in a proportion similar to that above with cells from the latter culture. The resulting cultures were transferred similarly into four flasks, providing 400 ml of suspended 18-hr-old cells, which were pooled. The population of the pooled cells was determined by measuring the optical density with a Bausch & Lomb Spectronic-20 colorimeter at 550 miu. The desired population was obtained by dilution and by varying the amount added to each sample. Type of seafood. The crabmeat was obtained from a commercial firm as sterile dungeness crabmeat (Cancer magister), packaged in no. ½2 flat tins in quantities of 150 g each, along with water, salt, and citric acid. Radiation source. The inoculated samples were irradiated in a Co60 irradiator located at Oregon State University, Corvallis. The source is composed of 12 CO60 rods, containing radioactivity equal to approximately 3,600 c. The dosimetry in the high flux chamber was determined by the Fricke ferrous sulfate method and was found to be 8.13 X 105 i 0.36 rad/hr on 29 January Corrections for decay were made after each exposure. Preparation of broth samples. Quantities of 2 and 5 ml from the final cell concentration, 107 cells per milliliter, were transferred into screw-cap tubes, 1.0 by 5.0 cm. The tubes were packed and sealed into no. 2 cans and maintained at a low temperature (3 to 5 C) prior to irradiation. All samples of the same species were replicated from three to eight times. Unirradiated inoculated control samples were treated similarly in each experiment. Pre- and postirradation treatment of solid crabmeat. The crabmeat was broken into small pieces and distributed in equal amounts (17 g) among sterile glass vials 3.8 by 6.3 cm. Each sample was inoculated with approximately 106 cells per gram of crabmeat. The microorganisms were mixed thoroughly throughout the crabmeat. The inoculated samples were positioned in a specially designed, opened, cylindrical container for the high flux chamber of the irradiator. Inoculated samples in groups of three, selected randomly from the numbers irradiated at the same dose, were pooled and blended with 70 ml of M phosphate buffer (ph 7.0). The meat-cell-buffer mixture was blended at a slow speed in a Waring Blendor. The resulting homogenate was transferred to a flask. The blending container was washed with 80 ml of sterile cold phosphate buffer, which was added to the homogenate. During the entire procedure, the samples and homogenates were kept at a low temperature (3 to 5 C). Six unirradiated inoculated control samples were included in each experiment. These were treated in a manner identical to that referred to above for the test specimens. Pre- and postirradiation treatment of diluted crabmeat. A 50-g amount of crabmeat mixed with 50 ml 100I FIG. 1. Radiation inactivation patterns of Salmonella in Hartsell's C 500 FIG. 2. Radiation inactivation patterns of Shigella in Hartsell's

3 94 DYER, ANDERSON, AND DUTIYABODHI APPL. MICROBIOL. of sterile distilled water was blended for 3 min. This homogenate was diluted as desired and inoculated with 106 cells per gram. The inoculated homogenate was transferred to sterile vials (3.8 by 6.3 cm) in quantities of 17 g per vial. I T T DOSE (KS FIG. 3. Radiation inactivation patterns of Neisseria, Mycobacterium, Streptococcus, Escherichia, and Proteus in Hartsell's QY D (U) H 0.1 z LUJ ) 0.01 LUJ 0- s J FIG. 5. Radiation inactivation pattern of Salmonella paratyphi B in solid crabmeat as compared with Hartsell's Fio. 6 Radiation inactivation pattern of Salmonella wichita in solid crabmeat as compared with Hartsell's FIG. 4. Radiation inactivation pattern of Salmonella typhi in solid crabmeat as compared with Hartsell's Two inoculated control samples each containing 50 g of crabmeat diluted in a manner similar to that described above were included in each experiment. Microbial examination. Amounts of 1 ml of the irradiated and unirradiated samples of the crabmeat homogenates and the Hartsell's broth cultures were diluted to a suitable concentration with M phosphate buffer and plated in Hartsell's agar. The plates

4 VOL. 14, 1966 RADIATION SURVIVAL OF FOOD PATHOGENS ICO FIG. 7. Radiation inactivation patterns ofsalmonella typhi in solid and diluted crabmeat. 5, > I,. J 0- z (9f- r-j 3 c 0001m 2.Oi 'J. 0( ( DI0 DILUTION 10 -C\ O 1:5 DILUTION Or 0 A 1:4 1:3 DILUTION \i \ 0 1:7 DILUTiON IL* X SOLID I I FIG. 8. Radiation inactivation patterns ofsalmonella paratyphi B in solid and diluted crabmeat. were incubated at 35 C for 72 to 96 hr. Each viable count was the average number of cells from triplicate platings. Plates containing 20 or fewer colonies were checked for Salmonella by testing each colony on S S Agar, Bismuth Sulfite Agar, and Triple Sugar Iron Agar (all Difco products). I ¾ REsuLTs AND DIscussIoN The dose-survival curves for Salmonella in Hartsell's broth are shown in Fig. 1. These results, as well as the others included in the study of microbial survival in Hartsell's broth, are the average of three determinations. In general, the curves were linear, and the species differed only in radiation resistance. S. typhi, S. paratyphi B, and S. wichita were the most resistant with a minimal lethal dose (MLD) of 5 X lo5 rad. S. pullorum was the least resistant with a MLD of 3 x 10 rad. The dose-survival curves for the species of Shigella are shown in Fig. 2. S. sonnei, the most irradiation-resistant, showed a MLD of 4 x 105 rad. S. paradysenteriae and S. dysenteriae showed a MLD of 3 X 105 rad, comparable to S. pullorum. E. coli was the most resistant of the genera shown in Fig. 3, with a MLD of 6 x 10 rad. P. vulgaris was the least resistant, with a MLD comparable to S. pullorum. The resistance of S. faecalis was similar to that found for the most resistant species of Salmonella. N. catarrhalis and M. smegmatis showed MLD values of 4 X 105 rad. The curves (Fig. 3) for S. faecalis and E. coli were sigmoidal, indicating a different order of reaction. At least part of the explanation for this could be that the microorganism has more than one "sensitive" vital site; thus, "multiple targets" must be "hit" to cause inactivation (1). The effects of y-irradiation on S. typhi, S. paratyphi B, and S. wichita suspended in solid crabmeat are shown in Fig. 4, 5, and 6, respectively. Each co-ordinate is the average number of survivors in two specimens, each from a minimum FiG. 9. Radiation inactivation patterns ofsalmonella wichita in solid and diluted crabmeat. 95

5 96 DYER, ANDERSON, AND DUTIYABODHI APPL. MICROBIOL. TABLE 1. D values* of Salmonella typhi, S. paratyphi B, and S. wichita in solid and diluted crabmeat Organism Solid crabmeat D value in diluted crabmeat 1:3 1:4 1:5 1:7 1:10 S. typhi * S. paratyphi B S. wichita * Radiation dose (in Mrad) necessary to inactivate 90% of the initial bacterial concentration. the MLD was higher in the solid crabmeat. Further examination of the tailing effect and radiation resistance of the three species of Salmonella was conducted by irradiatifgg various dilutions of inoculated crabmeat and comparing the patterns with those in undiluted crabmeat. The radiation inactivation patterns of S. typhi, S. paratyphi B, and S. wichita in undiluted and diluted crabmeat are shown in Fig. 7, 8, and 9, respectively. Each co-ordinate is the average of viable counts of duplicate samples. The individual values agreed within 100%. The co-ordinates representing the survivors per dose in undiluted crabmeat were obtained from Fig. 4, 5, and 6. The survival curves in each of the figures showed that by diluting the crabmeat the rate of kill increased and the tailing effect tended to disappear gradually or to become less pronounced. The data obtained agree with previous observa- of three pooled samples. The values agreed to within i 10%. A higher recovery rate of Salmonella was found in solid crabmeat than in Hartsell's S. typhi irradiated in solid crabmeat showed a rapid decline in numbers of survivors with increasing dosage (Fig. 4). However, this decline became nonlinear as the radiation doses increased, showing a "tailing effect." The presence of crabmeat as the suspending medium apparently introduced this tailing effect, which resulted in a nonlinear curve. A similar phenomenon was exhibited by S. paratyphi B and S. wichita but to a lesser degree, as shown in Fig. 5 and 6, respectively. Results shown in Fig 5 indicate that S. paratyphi B is more resistant in Hartsell's However, because of the tailing effect, The D values for S. typhi, S. paratyphi B, and S. wichita irradiated in undiluted and diluted crabmeat are given in Table 1. The results show tions in that water-diluted solids show increased free radical formation and greater migration of the radicals (3). However, the increase in the MLD may also be due to the dilution of some protective substance or substances in the crabmeat. a trend in which the D values of each of the microorganisms under study decreased with increasing dilutions. In undiluted crabmeat, S. wichita appeared to be the most resistant of the three species on the basis of D values. However, in irradiationpasteurization, the primary concern is to eliminate pathogens and to increase the shelf life. In the case of Salmonella, there exists the possibility that one cell could be infectious. Thus, the objective in the present work was to determine the dose at which all cells were killed. Of the microorganisms tested, S. typhi was the most resistant, as well as the one showing the most marked tailing effect. It is apparent from the results presented that in considering the resistance of a pathogen to radiation in a food substrate, less reliance should be placed on the D value and more on an experimentally determined pasteurization dose in a particular food substrate. There does not appear to be a simple explanation to account for the tailing effect. A similar "tailing" phenomenon was obtained by Wheaton and Pratt (7) for Clostridium botulinum spores, and was observed by Brown, Venton, and Gross (2) on spores of the putrefactive anaerobe PA 3679 in cured ham and raw pork. The "tailing effect" has also been observed in various virus survival curves (4). It has been shown that pasteurization doses based on D values would be quite inadequate, since the projection is based on sigmoidal and linear inactivation curves. Also, it is obvious from this work and the work of others that the dose necessary to eliminate pathogens in buffer, broth, etc., is not sufficient for the pasteurization of a food. Thus, to insure adequate pasteurization by irradiation, it may be necessary to recommend acceptable irradiation pasteurization doses for each food based upon trials closely simulating practical operating conditions. ACKNOWLEDGMENTS We gratefully acknowledge the stimulating interest and helpful suggestions of Jong Lee, Department of

6 VoL. 14, 1966 RADIATION SURVIVAL OF FOOD PATHOGENS 97 Food Science and Technology, Oregon State University. This investigation was supported by Public Health Service grant EF from the National Institutes of Health. LITERATURE CITED 1. BACQ, Z. M., AND PETER ALEXANDER Fundamentals of radiobiology, 2nd ed. Pergamon Press, Oxford. 2. BROWN, W. L., C. VINTON, AND C. E. GROSS Radiation resistance of the natural bacterial flora of cured ham. Food Technol. 14: DESROSIBR, N. H., AND H. M. ROSENSTOCK Radiation technology in food, agriculture, and biology. Avi Publishing Co., Westport. 4. Hwrr, C. W Kinetics of the inactivation of viruses. Bacteriol. Rev. 28: NIVEN, C. F., JR Microbiological aspects of radiation preservation of food. Ann. Rev. Microbiol. 12: RAYMAN, M. M., AND A. F. BYRNE Action of ionizing radiations on microorganisms, p In Radiation preservation of food. U.S. Army Quartermaster Corps, Washington, D.C. 7. WHEATON, E., AND G. B. PRATr Radiation survival curves of Clostridium botulinum spores. J. Food Sci. 27: Downloaded from on December 23, 2018 by guest