Interactive Effect of Temperature, Atmosphere and Storage Time on the Probability of Colony Formation on Blood Agar by Four Listeria Species

Transcription

1 JOURNAL OF FOOD PROTECTION, VOL. 55, FEBRUARY 99 Journal of Food Protection, Vol. 55. No., Pages -9 (February 99) Copyright, International Association of Milk, Food and Environmental Sanitarians Interactive Effect of Temperature, Atmosphere and Storage Time on the Probability of Colony Formation on Blood Agar by Four Listeria Species VADOOD RAZAVILAR and CONSTANTIN GENIGEORGIS* Department of Epidemiology and Preventive Medicine, School of Veterinary Medicine, University of California, Davis, California 95 (Received for publication July, 99) ABSTRACT The probability (P) of one Listeria cell and the cells needed to initiate colony formation on sheep blood agar plates as affected by atmospheric conditions (AC), storage temperature (T), time (t), and Listeria species was evaluated. The factorial design experiments included Listeria monocytogenes ( strains), Listeria seeligeri, Listeria ivanovii, and Listeria innocua, as test organisms, storage of the plates at,, 0, and 0 C under air (A), modified atmosphere (MA) of 5% O, + 0% C0 + 5% N 00% C0 (C0 ), vacuum (V), and candle jar (CJ) for 7,,,, and 5 d. Statistical analysis indicated the significant effect of AC (p<0.000), T (p<0.000), t (p<0.000) but not of species (p>0.7). None of the interactions with atmospheric conditions were significant (AC x species, AC x t, AC x T, all with p>0.). Pairwise comparison of the P's for each of the AC's indicated that 00% C0 was significantly more inhibitory to growth initiation than any other AC (p<0.00). No difference among the other AC's was shown. The effect of C0 on delaying growth of Listeria was enhanced with decreasing storage T. Thus, under C0, < cells of L. monocytogenes formed a colony within 7 d at 0 C and d at C. L. ivanovii was the most sensitive to C0 and required.7 x 0 cells to form a colony after d at C. C0 (00%) extended the lag phase at < C and decreased the rate of growth of the test organisms at C but not at higher temperatures. In recent years major foodborne outbreaks of listeriosis (5,5,,) and sporadic cases (,,0,0) have been reported, while the epidemiology of many sporadic cases remains unclear (). Of the various species of Listeria, Listeria monocytogenes is the most important to human and animal health. Rare human cases due to Listeria ivanovii and Listeria seeligeri have been reported (9,9). L. monocytogenes is widely distributed in meat, poultry, fish, and raw agricultural products (,,5,,7). According to Seeliger (5), Listeria thrives best at reduced oxygen tension. He also stated that growth was excellent after replacement of oxygen by C0, while anaerobic growth in the absence of C0 was scanty or absent. Many freshly isolated cultures and all laboratory strains grew profusely under aerobic cultural conditions. It seems that primary isolations from human and animal sources were facilitated by the use of reduced oxygen pressure (5). Today Listeria is considered a facultative anaerobe (). L. monocytogenes can grow at temperatures of -0. to 5 C (,7) and generation times at C have been described (). In recent years there has been a great expansion in the use of modified atmospheres (vacuum, gas blends) to extend the shelf life of raw and cooked foods at low temperatures (7). Considering the psychrotrophic nature of Listeria, and the extended product shelf life under modified atmospheres, selective cold enrichment becomes a possibility which may lead to human listeriosis (0). The behavior of Listeria in foods stored under modified atmospheres has not been studied extensively. Growth has been recorded in raw meat under vacuum at 0 to C (9,0,,), fresh vegetables under controlled atmosphere (0, C0, N ) at > C (,), and cooked chicken under modified atmosphere (C0, 0 ) at > C (), but not in raw chicken breast stored at C under 0-00% C0 (). The present study was undertaken in order to quantify and compare the effect of five atmospheric conditions on the probability (P) of growth initiation of four species Listeria by one cell, at to 0 C on a model blood agar system. The study was designed in a factorial fashion, to allow appropriate statistical analysis. MATERIALS AND METHODS Test organisms and preparation of inocula Listeria monocytogenes strains VPH- (a clinical isolate, serotype b), and VPH- (isolated from cheese, serotype / b), L. ivanovii, L. seeligeri, and L. innocua (all obtained from Dr. Hailu Kinde, California, Veterinary Diagnostic Laboratory Services), were used as test organisms. Culture stocks lyophilized on porcelain beads were kept at C. Working cultures were maintained on tryptose-soy-agar with 5% sheep blood agar (TSBA) (PML Microbiologicals, Sacramento, CA). Listeria inocula were prepared by transferring cells from working cultures to brain heart infusion broth (Difco Laboratories, Detroit, MI). After h incubation at 5 C, a second subculture was prepared which was incubated for h at 5 C and then used as inoculum. The

2 COLONY FORMATION ON BLOOD AGAR BY LISTERIA SPP. 9 number of cells in the h culture was estimated by plating appropriate dilutions in duplicate on TSBA and incubating at 5 C for to h. Effect of atmosphere on Listeria growth initiation To compare the effect of five atmospheric conditions (AC), the ability of decreasing concentrations of Listeria inoculum to form colonies on TSBA stored under each condition was evaluated. An h Listeria culture grown in brain heart infusion broth at 5 C was diluted 0-fold in 0.% peptone water from W to W. From each dilution, 0.00 ml was spotted in triplicate on the surface of a TSBA plate (x spots per plate) using a standard platinum loop. After the spotted inocula dried, the 0 plates (two for each atmospheric condition and temperature time combination) were stored under a particular atmospheric condition. This included air (A), vacuum (V), 00% C0 (C0 ), modified atmosphere (MA) composed of 5% 0 0% C0 and 5% N,, and a candle jar (CJ) atmosphere ( to % 0 and 5 to 0% C0 ). Plates to be incubated aerobically were wrapped with gas permeable cellophane with 0 permeability of cc/m / h at 5 C (Strech'n Cling Wrap, Safeway Stores, Inc., Oakland, CA). For MA, V, and C0 atmospheres, the plates were placed in anaerobic jars (Vacu- Quick, Almore International Inc., Portland, OR). Commercial gas cylinders (Liquid Carbonic Industrial Medical Corporation, Chicago, IL) were used to fill C0 and MA in the jars. The V jar was vacuumed after three flushings with C0. To avoid dryness of the plates, wet paper towels were placed inside the jars. Plates representing all AC were stored at,, 0, and 0 C. The number of spots resulting in colony formation on each plate was checked after 7,,,, and 5 d, and identified as Listeria. From the number of spots showing growth out of on each plate and x most probable number (MPN) tables (,,), the fraction of the inoculum which was inhibited by each AC was estimated from the formula log 0 I/G, where I = the number of inoculated cells, in the highest cell concentration spot estimated by plating and G = MPN of cells in the highest cell concentration spot on the agar stored under a particular AC (,,). The probability (P, %) of any given cell initiating growth under each AC was defined as P (%) = 00/antilog (log 0 I-log 0 G). Based on MPN scaling (), when no growth took place on the agar then G was set at 0.7 cells. The number of cells in each spotted inoculum needed for colony formation was estimated from 00/P (%). The use of multiple inocula and P to compare the five AC was considered more appropriate than the use of one inoculum level and direct plating (). Statistical analysis A complete factorial design (5x5xx5) was employed to assess the effects of atmosphere (A, MA, C0, V, and CJ), types of Listeria (five strains, four species), temperatures (0, 0,, and C), and time of storage (7,,,, and 5 d). One and two way analysis of variance were applied using BMDP (BMDP Statistical Software Inc., Los Angeles, CA) and SAS (SAS Institute Inc., Cary, NC) computer programs to determine the effect of atmosphere, temperature, and time on the growth of Listeria. Log P% of one cell initiating growth was chosen as the dependent variable. Pairwise comparisons were made to evaluate the significance of the various differences found in the levels of atmosphere, types of Listeria, temperatures, and times of storage. For each of the main comparison, an overall significance level of a = 0.05 was chosen, with the Bonferoni adjustment for the multiple comparisons that were to be made. Thus, if m multiple comparisons were to be made, at an overall a level, each pairwise comparison was made at a/m (e.g., 0.05/0 = 0.005). RESULTS AND DISCUSSION The observed log 0 P% for one Listeria cell forming a colony on the TSBA plate as affected by the independent variables, strain, temperature, AC, and time is presented in Fig. and. Since there was no remarkable difference between 0 and 0 C, only P curves relating to,, and 0 C incubation were plotted. As the temperature of storage decreased from 0 C to and C, the AC containing 00% C0 became more inhibitory to Listeria growth initiation. The effect of AC and temperature on P was affected by species and strains with L. ivanovii being the most sensitive to C0 at C, and L. monocytogenes strain VPH- the most resistant. This is an interesting observation since strain VPH- is a clinical serotype b strain, one which is most frequently incriminated in human disease (7,9). The difference among species disappeared as the temperature increased to >0 C. L.MONOCYTOGENES VPH- LMONOCYTOGENES VPH- Time(days) Figure l. Comparison of air (A), modified atmosphere (MA), consisting of 5% 0 + 0% C0 + 5% N, 00% C0 (C0 ), vacuum (V), and microaerophilic atmosphere of candle jar (CJ) on the probability of colony formation by one L. monocytogenes cell on TSBA agar incubated at temperatures of to 0 C for up to 5 d. L.SEELGER L.INNOCUA L.IVANOVII Time(days) Figure. Comparison of effect of air (A), modified atmosphere (MA) consisting of 5% 0 + 0% C0 + 5% N, 00% C0 (CO ), vacuum (V), and microaerophilic atmosphere of candle jar (CJ) on the probability of colony formation by one cell of L. seeligeri, L. innocua, and L. ivanovii on TSBA agar incubated at temperatures of 5 to 0 C for up to 5 d. JOURNAL OF FOOD PROTECTION, VOL. 55, FEBRUARY 99

3 90 RAZAVILAR AND GENIGEORGIS A more refined way to observe any difference on the effect of the independent variables on the P of L. monocytogenes (two strains) is presented in Table, where the observed log 0 P% is replaced by the calculated number of cells needed to form a colony on the agar plate. The trends apparent in this table are similar to those of Fig.. Under all AC conditions, even - cells of L. monocytogenes could form a colony within 7 d at >0 C. At C, formation of a colony within 7 d required. x 0 5, 0,, 9, and cells of strain VPH- exposed to C0, CJ, V, MA, and A atmospheres, respectively. The corresponding numbers of cells for the C incubation (within 7 d), were 7. x 0 5 for all atmospheres. After d incubation at C, the corresponding numbers were.7 x 0, 7, 7, 9, and for C0, MA, A, CJ, and V, respectively. After d storage at C, even - cells were able to form a colony under all AC for both L. monocytogenes strains. The growth profile of L. seeligeri, L. innocua, and L. ivanovii at to 0 C under all atmospheric conditions was very similar (Fig. ). At C, under 00% C0, L. ivanovii was the most sensitive and L. monocytogenes VPH- was the most resistant. Analysis of variance indicated that the log 0 P% was affected significantly by three main [AC (p<0.000), temperature (p<0.000), and time of storage (p<0.000)] but not by the species or strains of Listeria (p>0.7). None of the interactions with AC were statistically significant [AC x strain (p>0.99), AC x time (p>0.5), and AC x temperature (p>0.)]. A pairwise comparison of the mean logp% of growth of Listeria spp., in each of the five AC on TSBA plates (Table ), indicated that CO, was significantly more inhibitory to growth initiation than any other AC (p<0.00). There were no statistically significant differences (p>0.) among MA, A, V, and CJ atmospheres. The significantly increasing inhibitory effect of C0 with decreasing temperature is probably due to the greater dissolution of C0 in water with decreasing temperature (,,). At C, C0 extended the lag phase (time from beginning until first change in P) of Listeria growth but did not affect the rate of growth (slope of the P curve) as compared to other AC's. At C, C0 extended the lag phase and decreased the rate of growth as compared to other AC's. It has been reported that the growth kinetics of L. monocytogenes in a broth medium stored at 5 to 7 C under aerobic and anaerobic atmospheres were similar (7,). Sterile lamb meat inoculated with L. monocytogenes and then packaged in gas permeable and gas impermeable film and stored at C showed no remarkably different effect of the type of packaging on the growth of Listeria (). Beuchat et al. () did not notice any difference in the effect of ambient air and % O, + 97% N atmosphere on the growth of L. monocytogenes strains on shredded lettuce stored at 5 and 0 C for up to 5 d. Kallander et al. () showed that a modified atmosphere containing 70% C0 + 0% N, surrounding shredded cabbage at 5 and 5 C, had no major influence on the fate of L. monocytogenes. Our findings are in disagreement with these studies since we demonstrated that only at 0 and 0 C, there was no difference among the five atmospheres tested. At < C, the atmosphere containing 00% C0 was inhibitory to all test organisms. TABLE. Estimated number of cells of L. monocytogenes needed to initiate growth and colony formation on TSBA agar under five atmospheric conditions (AC) at,, and 0 C for 7 to d. Storage time (d) AC a Temp ( C) 7 L. monocytogenes VPH- 7 L. monocytogenes VPH- A MA s 7 CO, x 0 s s.7 x x s CJ x 0 s a A = air. MA = modified atmosphere of 5% 0 + 0% CO, + 5%N, CO, = 00% CO, V = vacuum. CJ = candle jar atmosphere. JOURNAL OF FOOD PROTECTION, VOL. 55, FEBRUARY 99

4 COLONY FORMATION ON BLOOD AGAR BY LISTERIA SPP. 9 TABLE. p values for pairwise comparison of probability of colony formation of Listeria spp. on TSBA agar stored in five atmospheric conditions (AC) at to 0 C for 7 to 5 d. AC» CJ A V MA C CJ A V 0.5 a A = air. MA = modified atmosphere of 5% 0 + 0% C0 + 5% N, C0 = 00% C0. V = vacuum. CJ = candle jar atmosphere. Seeliger (5) has stated that "Listeria thrives best at reduced oxygen tension" and that "after replacement of oxygen by carbon dioxide growth is excellent". This is contradictory to our findings, which indicated an increased inhibitory effect of 00% C0 with decreasing incubation temperature, and no statistically significant difference among the other four AC's. Ingham et al. () compared the effect of air and two CO z containing modified atmospheres (C0 0% + 0 0%, and CO, 50% + O n 0%) on the growth of L. monocytogenes in sterile chicken loaf in the presence of Pseudomonas fragi at, 7, and C. After d storage at C, inoculated L. monocytogenes increased by., 0.5, and 0.7 log 0 in air, 50%, and 0% C0, atmospheres, respectively. At 7 C the corresponding log ]f) increases were.,., and. and at C they were 5., 5.59, and.77. Hart et al. () did not observe growth of L. monocytogenes on chicken breast meat stored at C under 0-00% C0 atmospheres, although growth occurred under aerobic conditions. According to Gill and Reichel (9), L. monocytogenes grew in vacuum-packaged beef at > C, while under atmosphere of 00% C0 growth was observed when the storage temperature was >5 C. The extensive killing effect of high C0 pressure (. atm) on L. monocytogenes in distilled water and chicken meat has been reported (). In a recent paper, Buchanan and Klawitter (9) concluded that though L. monocytogenes generally grows better under aerobic conditions, limiting oxygen appeared to enhance survival and growth when other growth factors like ph and temperature were suboptimal. In our study, only at C the inhibitory effect of air was similar to C0 for L. seeligeri but not for the other Listeria species. Overall, our study demonstrated quantitatively the increasing inhibitory effect of high concentrations of C0 on the growth of Listeria species with storage temperatures of < C. ACKNOWLEDGMENT This research was supported by the National Dairy Promotion and Research Board through a grant administrated by the California Dairy Food Research Center. Gratitude is expressed to Dr. C. Franti for his statistical support. / REFERENCES. Azadian, B. S G. T. Finnerty, and A. D. Pearson. 99. Cheeseborne Listeria meningitis in immunocompetent patient. Lancet i:-.. Bannister, B. 97. Listeria monocytogenes meningitis associated with eating soft cheese. J. Infect. 5:5-.. Berrang, M. E., R. E. Brackett, and L. R. Beuchat. 99. Growth of Listeria monocytogenes on fresh vegetables stored under controlled atmosphere. J. Food Prot. 5: Beuchat, L. R., and R. E. Brackett Survival and growth of Listeria monocytogenes on lettuce as influenced by shredding, chlorine treatment, modified atmosphere packaging and temperature. J. Food Sci. 55:755-75, Bille, J. 99. Anatomy of a foodbome listeriosis outbreak, pp. 9- : In Foodbome listeriosis. Proc. of Symp. September 7, 9. Wiesbaden, FRG. Behr's Verlag, Hamburg.. Brecr, C, and G. Breer. 9. The isolation of Listeria spp. in meat and meat products. Proc. th Int. Congr. Meat Science Technology B: Buchanan, R. L., H. G. Stahl, and R. C. Whiting. 99. Effect and interactions of temperature, sodium chloride, and sodium nitrite on the growth of Listeria monocytogenes. J. Food Prot. 5:-5.. Buchanan, R. L., and J. G. Phillips Response surface model for predicting the effects of temperature, ph, sodium chloride content, sodium nitrite concentration and atmosphere on the growth of Listeria monocytogenes. J. Food Prot. 5: Buchanan, R. L., and L. A. Klawitter Effects of temperature and oxygen on the growth of Listeria monocytogenes at ph.5. J. Food Sci. 55: Farber, J. M., A. O. Carter, P. V. Varughese, F. E. Ashton, and E. P. Ewan Listeriosis traced to the consumption of alfalfa tablets and soft cheese. N. Engl. J. Med. ;.. Farber, J. M., G. W. Sanders, and M. A. Johnson. 99. A survey of various foods for the presence of Listeria species. J. Food Prot. 5:5-5.. Fisher, R. A., and F. Yates Statistical tables for biological, agricultural and medical research, 5th ed. Oliver and Boyd, London. pp. -, 5.. Finnc, G. 9. Modified- and controlled-atmosphere storage of muscle foods. Food Technol. (): -.. Garayzabal, J. F., and C. A. Genigeorgis Quantitative evaluation of three selective enrichment broths and agars used in recovering Listeria monocytogenes. J. Food Prot. 5: Genigeorgis, A. C, D. Dutulescu, and J. F. Garayzabal. 99. Prevalence of Listeria in poultry meat at the supermarket and slaughterhouse level. J. Food Prot. 5:-.. Genigeorgis, A. C, P. Oanca, and D. Dutulescu Prevalence of Listeria spp. in turkey meat at the supermarket and slaughterhouse level. J. Food Prot. 5:-. 7. Genigeorgis, A. C. 95. Microbial and safety implications of the use of modified atmospheres to extend the storage life of fresh meat and fish. Int. J. Food Microbiol. :7-5.. Genigeorgis, C. A., S. Martin, C. E. Franti, and Hans Riemann. 97. Initiation of staphylococcal growth in laboratory media. Appl. Microbiol. : Gill, C. O., and M. P. Reichel. 99. Growth of cold-tolerant pathogens Yersinia enterocolitica, Aeromonas hydrophila, and Listeria monocytogenes on high-ph beef packaged under vacuum or carbon dioxide. Food Microbiol. : Grau, F. H., and P. B. Vanderlinde Growth of Listeria monocytogenes on vacuum-packaged beef. J. Food Prot. 5: Hart, C. D., G. C. Mead, and A. P. Norris. 99. Effects of gaseous environment and temperature on the storage behavior of Listeria monocytogenes on chicken breast meat. J. Appl. Bacterid. 70:0-.. Ingham, S. C, J. M. Escude, and Paige McCown Comparative growth rates of Listeria monocytogenes and Pseudomonas fragi on cooked chicken loaf stored under air and two modified atmospheres. J. Food Prot. 5:9-9.. Kallander, K. D., A. D. Hitchins, G. A. Lancette, J. A. Schmieg, G. R. Garcia, H. M. Solomon, and J. N. Sofos. 99. Fate of Listeria monocytogenes in shredded cabbage stored at 5 and 5 C under a modified atmosphere. J. Food Prot. 5:0-0.. Khan, M. A., C. V. Palmas, A. Seaman, and M. Woodbine. 97. JOURNAL OF FOOD PROTECTION. VOL. 55, FEBRUARY 99

5 9 RAZAVLAR AND GENGEORGS Survival versus growth of a facultative psychrotroph. Acta Microbiol. Acad. Sci. Hung. 9: Linnan, M., L. Mascola,. D. Low, V. Goulet, S. May, C. Salminen, D, Hird, L. Yonecura, P. Hayes, R. Weaver, A. Andurier, B. D. Plikaytis, S. L. Fannin, A. Kleks, and C. V. Broome. 9. Epidemic listeriosis associated with Mexican-style cheese. N. Engl. J. Med. 9:-.. Lindroth, S. E., and C. A. Genigeorgis. 9. Probability of growth and toxin production by nonproteolytic Clostridium botulinum in rock fish stored under modified atmospheres. Int. J. Food Microbiol. : Lovett, J. 99. Listeria monocytogenes, pp.-0. In M. P. Doyle (ed.), Foodborne bacterial pathogens. Marcel Dekker, Inc., New York.. Lowry, P. D., and L. Tiong. 9. The incidence of Listeria monocytogenes in meat and meat products. Factors affecting distribution. Proc. th Int. Congr. Meat Sci. Technology. b: McLauchlin, J. 97. Listeria monocytogenes, recent advances in the taxonomy and epidemiology of listeriosis in humans. J. Appl. Bacteriol. :-. 0. Morbidity Mortality Weekly Report. 99. Listeria associated with consumption of turkey franks. Morbid. Mortal. Weekly Rep. :7-.. Ogrydziak, D. M., and W. D. Brown. 9. Temperature effects in modified-atmosphere storage of seafoods. Food Technol. (5): -9.. Rosenow, E. M., and E. H. Marth. 97. Growth of Listeria monocytogenes in skim, whole and chocolate milk, and in whipping cream during incubation at,,,, and 5 C. J. Food. Prot. 50: Schlech, W. F., P. M. Lavigne, R. A. Bortolussi, A. C. Allen, E. V. Haldane, A. J. Wort, A. W. Hightower, S. E. Johnson, S. H. King, E. S. Nicholls, and C. V. Broome. 9. Epidemic listeriosis. Evidence for transmission by food. N. Engl. J. Med. 0:0-0.. Schuchat. A., B. Swaminathan, and C. V. Broome. 99. Epidemiology of human listeriosis. Clin. Microbiol. Rev. : Seeliger, H. P. R. 9. Listeriosis. Hafner Publishing Cp., New York.. Seeliger, H. P. R., and D. Jones. 9. Genus Listeria, pp In Sneath. P. H. A., N. S. Mair, M. E. Sharpe, and J. G. Holt (ed.), Bergey's manual of systematic bacteriology, vol.. The Williams and Wilkins Co., Baltimore. 7. Walker, S. J., P. Archer, and J. G. Banks Growth of Listeria monocytogenes at refrigeration temperatures. J. Appl. Bacteriol. :57'-].. Wei, C. I., M. O. Balaban, S. Y. Fernando, and A. J. Peplow. 99. Bacterial effect of high pressure C0 treatments on foods spiked with Listeria or Salmonella. J. Food Prot. 5: World Health Organization. 9. Report of the WHO consultation on prevention and control of listeriosis. Berlin (West), pp. -. JOURNAL OF FOOD PROTECTION, VOL. 55, FEBRUARY 99