Survival and Growth of Campylobacter fetus subsp. jejuni on Meat and in Cooked Foods

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 192, p. 259-263 99-224/2/259-5$2./ Vol. 44, No. 2 Survival and Growth of Campylobacter fetus subsp. jejuni on Meat and in Cooked Foods C.. GILL* AND LYNDA M. HARRIS Meat Industry Research Institute of New Zealand Inc., Hamilton, New Zealand Received 23 February 192/Accepted 4 May 192 Twelve strains of Campylobacter fetus subsp. jejuni isolated from humans and animals grew at temperatures ranging from 34 to 45 C and ph minima between 5.7 and 5.9. Only one strain grew at ph 5. with lactic acid present at a concentration similar to that in meat. All strains had decimal reduction times of less than 1 min at 6 C. Further examination of a typical strain showed that it grew at 37 C on highph meat but not at 37 C on normal-ph meat. Bacterial numbers on both high (6.4)-pH and normal (5.)-pH inoculated meat declined at a similar rate when the meat was stored at 25 C. At -1PC, the rate of die-off was somewhat slower on normal-ph meat but was very much slower on high-ph meat. The initial fall in bacterial numbers that occurred when meat was froen was also greater for normal-ph meat than for high-ph meat. The organism exhibited a long lag phase (1 to 2 days) when grown in cooked-meat medium at 37 C and died in meat pies stored at 37 or 43 C. Evaluation of the risk of Campylobacter contamination of red-meat carcasses to human health must take into account the limited potential of the organism to grow or even survive on fresh meats and in warm prepared foods. It is now well established that Campylobacter fetus subsp. jejuni is a common cause of human enteric disease. However, the epidemiology of the disease remains uncertain, largely because an effective system of strain differentiation has yet to be developed. The organism has been isolated from most common domestic animal species, so it has been inferred that direct transmission of the disease to humans might occur via consumption of animal products (2). In particular, milk and poultry meat have been implicated (11). The evidence linking some outbreaks of campylobacter enteritis with the consumption of unpasteuried milk is very strong (9). The evidence regarding poultry meat is far less conclusive, although there is no doubt about the high incidence of poultry carcass contamination by C. fetus subsp. jejuni (6). More recently, it has been shown that carcasses of most other animals providing meat can be contaminated by C. fetus subsp. jejuni (12, 13). The incidence varies with animal species, but generally, the degree of contamination of carcasses seems to be low (4). Whether such contamination is of concern for public health depends in part upon the survival of the organisms during storage and their ability to grow on raw meats or in any prepared dishes to which they may be inadvertently transferred. This paper reports on the behavior on stored meat and in cooked food of C. fetus subsp. jejuni isolates from human and animal sources. MATERIALS AND METHODS Organisms. Six C. fetus subsp. jejuni strains were isolated from sheep and calf feces as previously described (4). Six strains isolated from humans with enteritis were obtained from a medical laboratory. All strains grew at 43 C but not at 25 C and showed typical Campylobacter morphology and movement when examined microscopically (2). The organisms were grown and maintained on Campylobacter agar, which is composed of brucella medium base (CM169; Oxoid Ltd., Basingstoke, England) supplemented with 5% whole human blood and Campylobacter growth supplement (SR4; Oxoid), under an atmosphere of 5% 2-1% C2-5% N2. All strains were subcultured weekly. Liquid cultures were grown in a broth containing 2 g of proteose peptone (Difco Laboratories, Detroit, Mich.) per liter and 1 g of yeast extract (Difco) per liter under the reducedoxygen atmosphere. Growth temperature range. Colonies of all strains were picked from maintenance plates and streaked on Campylobacter agar plates. Individual plates were then incubated at one test temperature for 7 days in an anaerobic jar containing the low-oxygen atmosphere. We maintained the incubation temperatures by immersing each jar in a water bath controlled to stay within.5 C of the required temperature. Incubation temperatures were increased from 43 C in 1 C steps and decreased similarly from 36 C. Minimum ph values. Each strain was streaked onto a separate plate of Campylobacter agar the ph of which was adjusted after steriliation and cooling by the addition of 2 N HCl. The ph values of the plates ranged from 6.2 to 5.5 (.1 increments). All plates were incubated for 7 days at 37 C under the low-oxygen 259

26 GILL AND HARRIS atmosphere. After incubation, the ph values of the plates were rechecked with a glass electrode. Tolerance of lactic acid. Campylobacter agar was supplemented with lactic acid at geometrically increasil.g concentrations of 1 to 16 mg/ml, and the final ph was adjusted to 5. in all cases with 2N HCl. Agar plates were inoculated and incubated as described above. Thermal inactivation. Test tubes (16-mm diameter) equal in length to the depth of sockets in a heating block were heated in the block to the required temperature before the addition of.2-ml portions of C. fetus subsp. jejuni broth cultures containing approximately 1 cells per ml. At 2-s intervals between and 3 min, 1.-ml portions of ice-cold broth were added to the tubes, which were immediately placed in an ice bath. Samples suitably diluted in broth at room temperature (2 C) were plated on Campylobacter agar for survivor enumeration. Growth and survival on fresh meat. Slices of meat (5 by 5 by 1 cm) were aseptically cut (5) from three beef strip loins of ph 6., 6.4, or 5.. Each slice was placed in its own sterile petri dish and inoculated with a suspension of C. fetus subsp. jejuni prepared by emulsification of colonies from Campylobacter agar plates in.1% peptone, which gave an initial cell density of about 14/cm2. Slices from each strip were incubated at 37 C in air or under the reduced-oxygen atmosphere. Plates stored in air were sealed in plastic bags to prevent dehydration of the meat slices. At daily intervals, a slice from each strip incubated under each atmosphere was removed for microbiological examination. We macerated slices with 4 ml of.1% peptone, using a Colworth Stomacher, and spread suitable dilutions on Campylobacter agar for enumeration. Another group of unsterile meat slices of ph 6.4 was similarly placed in petri dishes, inoculated, and examined for C. fetus subsp. jejuni growth after incubation in air at 37 C. Macerates from this group were spread on nutrient or Campylobacter agar plates which contained the antibiotic supplement (SR69; Oxoid) recommended by Skirrow (11) for Campylobacter isolation. Survival of C. fetus subsp. jejuni on stored meat. Sterile meat slices from beef strip loins of ph 6.4 or 5. were inoculated with C. fetus subsp. jejuni, packed as described above, and then stored in air at 25 C for 2 weeks and at -1 or -1'C for 6 weeks. Growth in cooked-meat medium. Culture tubes of cooked-meat medium, six for each strain, were inoculated with stationary-phase broth cultures. The tubes were incubated without shaking at 37'C. At daily intervals, we removed a tube of each strain and vigorously agitated it to ensure thorough mixing of the contents, and then we determined the cell density by plating suitably diluted samples. For comparison of growth rates of Campylobacter strains with those of other pathogens, stationary-phase cultures of Salmonella typhimurium, Clostridium perfringens, and Staphylococcus aureus, all of which had been isolated from meat, were similarly inoculated into cooked-meat medium. Tubes of these cultures were incubated in the same manner used for Campylobacter strains except that samples were examined at intervals of 4 to h. Growth of C. fetus subsp. jejuni in meat pies. Four groups of six meat pies (2 g) each were prepared as APPL. ENVIRON. MICROBIOL. follows: (i) no inoculation (control), (ii) inoculation by injection through the crust of.1 ml of a suspension containing 14 C. fetus subsp. jejuni cells per ml, (iii) inoculation by injection of.1 ml of a suspension containing 1 C. fetus subsp. jejuni cells per ml, or (iv) inoculation by insertion under the crust of a piece (5 g) of normally contaminated meat to which had been added 17 C. fetus subsp. jejuni cells. We incubated all pies at 37 C and examined them daily for growth of bacteria by macerating the pie contents with 15 ml of.1% peptone-.2% tergitol and spreading suitably diluted samples of the macerate on plates containing the following: nutrient agar, Campylobacter selective agar, violet-red bile agar (CM17; Oxoid), Shahidi- Ferguson perfringens agar (1), or Baird-Parker Medium (CM275; Oxoid) for the respective estimation of total counts, C. fetus subsp. jejuni, Enterobacteriaceae strains, C. perfringens, or S. aureus. A second group of pies was prepared and examined as above after incubation at 43 C. RESULTS Effects of temperature, ph, and lactic acid concentration on growth. The widest temperature range, 34 to 45 C, was found for only one strain. For all other strains, the minimum temperature was 35 or 36 C, and half of the strains were unable to grow at temperatures above 44 C. For most strains, the minimum ph for growth was 5.. Response to lactic acid was variable, but only one strain (human strain 1) could grow at ph 5. with lactic acid present at mg/ml (Table 1). Thermal inactivation. The organisms were rapidly inactivated when heated to 5 C or above (Table 2). All strains had a decimal reduction time (D-value) of less than 1 min at 6 C (Table 3). Growth on meat. Studies of growth and survival of Campylobacter strains on meat and in pies were performed with apparently typical animal strain 6. This organism grew on high (6.4)-pH meat in air as well as it did under the reduced oxygen atmosphere (Fig. 1), but it did not grow on normal (5.)-pH meat. The early stages of growth on high-ph meat were not inhibited by the presence of other organisms, but when the spoilage flora approached maximum density (191Cm2), Campylobacter numbers began to decrease (Fig. 2). Three human strains (2, 3, and 6) were also examined for growth on meat, and these also grew on high-ph meat incubated at 37 C in air but did not grow on normal-ph meat. Survival on stored meat. Froen storage (-1 C) resulted in a decrease in Campylobacter numbers during the first 2 weeks, but there was little subsequent change. The cell density decreased by about one order of magnitude on high-ph meat but by two orders of magnitude on normal-ph meat. Bacterial numbers on chilled (-1C) high-ph meat declined slowly through-

VOL. 44, 192 C. FETUS SUBSP. JEJUNI ON MEAT 261 TABLE 1. Growth temperature range, minimum ph, and lactic acid concentrations allowing growth of 12 C. fetus subsp. jejuni on Campylobacter agar' Maximum Strain no. Minimum growth Maximum Minimum tolerable lactic temp ( C) growth temp ( C) ph acid concn (mg/ml)b Animal 1 36 44 5.9 2 35 45 5. 2 3 35 44 5. 4 36 44 5. 4 5 35 45 5. 4 6 36 45 5. 4 Human 1 35 44 5. 2 35 44 5.7 2 3 36 43 5. 1 4 35 44 5. 5 35 45 5.9 6 34 45 5. 4 a Six strains were isolated from animal feces, and six were isolated from humans with enteritis. b Maximum lactic acid concentration in the geometric series 1 to 16 mg/ml at which growth occurred at ph 5.. -, No growth at 1 mg/ml. out the storage period but declined rapidly on chilled normal-ph meat until bacteria could not be detected (after days). Storage of meat at 25 C resulted in equally rapid declines in Campylobacter numbers on both normal- and highph meats (Fig. 3). Growth in cooked-meat medium. All C. fetus subsp. jejuni strains had lag phases of 1 to 3 days before they began to grow in cooked-meat medium at 37 C. Maximum cell densities of about 1/ml were attained after 2 to 3 days of growth. Growth of all Campylobacter strains was slow compared with growth of other food-borne pathogens (Fig. 4). Growth in meat pies. Uninoculated pies were spoiled in 2 to 3 days at either 37 or 43 C as the flora approached a maximum cell density of 19 cells per g of filling. The only organisms present were Bacillus species, which grew on nutrient agar plates. No colonies developed on any of the selective agars. The Campylobacter numbers of pies inoculated with C. fetus subsp. jejuni (animal strain 6) declined during storage, and even when the pies were inoculated with over 15 cells per g of TABLE 2. Decimal reduction times (D-values) for animal strain 6 in peptone-yeast extract broth Temp ( C) D-value (s) 5 132. 55 41.7 6 2.7 65 13.2 7 11.1 filling, no Campylobacter cells could be detected after 3 days of incubation at either temperature (Fig. 5). Campylobacter numbers declined similarly in pies inoculated with raw mince and C. fetus subsp. jejuni, but the final floras contained Enterobacteriaceae strains and S. aureus, both at 15 to 16/g, as well as bacilli. DISCUSSION The growth requirements of C. fetus subsp. jejuni are such that the organism could not grow on normal (5.5 to 5.)-pH meat and could grow on high-ph, dark, firm, dry meat only at temperatures of around 4 C. Thermal sensitivity TABLE 3. D-values for C. fetus subsp. jejuni in peptone-yeast extract broth at 6 C Strain no. D-value (s) Animal 1 21.7 2 23.1 3 19.3 4 15. 5 42.6 6 2.7 Human 1 21. 2 57.1 3 2.6 4 27.3 5 3.7 6 24.

262 GILL AND HARRIS APPL. ENVIRON. MICROBIOL. N, 7. o5. 2 4 6 1 t i me (h) FIG. 1. Growth of C. fetus subsp. jejuni (animal strain 6) inoculated onto sterile meat of ph 6.4 stored at 37 C in an atmosphere of 5% 2-1% C2-5% N2 () or in air (). would not allow the organism to survive even moderate cooking. Storage of meat at room or chilling temperature resulted in comparatively rapid and ultimately complete die-off, whereas freeing substantially reduced the total bacterial numbers. This would be true even for high-ph meat, on which bacterial survival during chilling and freeing was markedly better than it was on normal-ph meat. It therefore seems that any likely treatment of meat during storage and E u 4' Cu. OC. * -.---------- 1 2 3 4 FIG. 3. Survival of C. fetus subsp. jejuni (animal strain 6) inoculated onto sterile meat slices of ph 5. (open symbols) or 6.4 (solid symbols) stored at -1 C (circles), -1 C (squares), or 25 C (triangles). preparation for consumption can only reduce the initial low level of contamination (1/cm2) by C. fetus subsp. jejuni (4). The organism appears to fare little better in prepared dishes contaminated after being cooked. The failure of organisms to survive in meat pies stored at temperatures suitable for growth suggests there would be few circum- E. -W to 7 6 5 9 o o a U,e o - 1 2 3 4 5 time (h) FIG. 2. Growth of C. fetus subsp. jejuni (animal strain 6) () in competition with the normal flora () of meat of ph 6.4 stored in air at 37 C. 4 2 3 4 5 FIG. 4. Growth of C. fetus subsp. jejuni (animal strain 6) (), C. perfringens (), S. typhimurium (), and S. aureus (-) in cooked-meat medium at 37 C.

VOL. 44, 192._ (a - to. 1, 6 4 2 ID. 1 2 3 FIG. 5. Development of spoilage floras in meat pies inoculated with C. fetus subsp. jejuni (animal strain 6). Shown are total plate counts (circles) and Campylobacter counts (squares) after incubation at 37 C (open symbols) or 43 C (closed symbols). C. FETUS SUBSP. JEJUNI ON MEAT 263 stances under which bacterial numbers would increase in a warm food. Even if growth could occur, the long lag phase makes C. fetus subsp. jejuni a poor competitor with other organisms, which would normally reach unacceptable levels before Campylobacter growth commenced. Our observations on the effects of temperature on growth and survival of C. fetus subsp. jejuni are in general agreement with those of other workers (1, 3). However, the strains we examined appeared to be more sensitive to a low ph than those used by Doyle and Roman (3), who observed that their five strains grew well at ph 5.5. Strains that are relatively insensitive to a low ph might have a greater potential than our isolates had of being transmitted to humans via meat, provided that their survival on normal-ph meat is similar to that of our isolates on high-ph meat. Even so, low ph-insensitive strains would still decline in numbers during storage and preparation of meat and would be unlikely to grow in warm foods. It seems that contamination of red-meat carcasses by small numbers of C. fetus subsp. jejuni is unlikely to result in large doses of the organism being consumed. However, the consumption of small doses is a possible, if perhaps uncommon, event. Unfortunately, the order of magnitude of the infectious C. fetus subsp. jejuni dose for humans has not been established. Evidence from animals indicates that a substantial dose may be required (7, 14), but in a single case of an experimental human infection, a few hundred cells sufficed (). More data on this point will obviously be necessary for proper evaluation of the hygenic significance of contamination of red meat by C. fetus subsp. jejuni, and until such data are available, the purely qualitative demonstration of the organism on meat cannot be taken as substantial evidence that meat plays a vectorial role in human disease. LITERATURE CITED 1. Blaser, M. J., H. L. Hardesty, B. Powers, and W.-L. L. Wang. 19. Survival of Campylobacter fetus subsp. jejuni in biological milieus. J. Clin. Microbiol. 11:39-313. 2. Butler, J. P., and M. B. Skirrow. 1979. Campylobacter enteritis. Clin. Gastroenterol. :737-765. 3. Doyle, M. P., and D. J. Roman. 191. Growth and survival of Campylobacter fetus subsp. jejuni as a function of temperature and ph. J. Food Prot. 44:596-61. 4. Gill, C. O., and L. M. Harris. 192. Contamination of redmeat carcasses by Campylobacter fetus subsp. jejuni Appl. Environ. Microbiol. 43:977-9. 5. Gill, C. O., and N. Penney. 1977. Penetration of bacteria into meat. AppI. Environ. Microbiol. 33:124-126. 6. Grant, I. H., N. J. Richardson, and V. D. Bokkenheuser. 19. Broiler chickens as potential source of Campylobacter infections in humans. J. Clin. Microbiol. 11:5-51. 7. Prescott, J. F., and M. A. Karmali. 197. Attempts to transmit Campylobacter enteritis to dogs and cats. Can. Med. Assoc. J. 119:11-12.. Robinson, D. A. 191. Infective dose of Campylobacter jejuni in milk. Br. Med. J. 22:154. 9. Robinson, D. A., and D. M. Jones. 191. Milk-borne campylobacter infection. Br. Med. J. 22:1374-1376. 1. Shahidi, S. A., and A. R. Ferguson. 1971. New quantitative, qualitative and confirmatory media for rapid analysis of food for Clostridium perfringens. Appl. Microbiol. 21:5-56. 11. Skirrow, M. B. 1977. Campylobacter enteritis: a "new" disease. Br. Med. J. 2:9-11. 12. Stern, N. J. 191. Recovery rate of Campylobacter fetus ssp. jejuni on eviscerated pork, lamb and beef carcasses. J. Food Sci. 46:1291-1293. 13. Svedhem, A., and B. Kaoser. 191. Isolation of Campylobacter jejuni from domestic animals and pets: probable origin of human infection. J. Infect. 3:37-4. 14. Taylor, D. J., and P. A. Olubunmi. 191. A re-examination of the role of Campylobacter fetus suspecies coli in enteric disease of the pig. Vet. Rec. 19:112-115.