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The survival of Campylobacter jejuni in red meats stored at different temperatures
Internanonal Journal of Food Mwrobtologv. 1 (1984) 187-196
187
Elsevier JFM 00020
The survival of Campylobacterjejuni in red meats stored at different temperatures R.A.E. B a r r e l l Publw Health Laboratory. Withington Hospttal. Manchester M20 8LR, U.K (Received 30 March 1984; accepted 6 September 1984)
The survival of 4 strains of Campylobacter.tejuni was studied in raw minced beef and raw pork sausage mixture stored in plastic stomacher bags at freezer temperatures ( - 19°C) for up to 10 weeks, refrigerator temperatures ( < 10°C) for 6 days and at 22°C for 24 h. At each of the 3 storage temperatures survival was better in minced beef. Similarly, there was less variation in percentage survival between the 4 strains in minced beef than in sausage mixture after storage at each temperature. Detailed studies were carried out with one strain of C. jejuni. Viable counts were relatively unchanged in minced beef at refrigerator temperatures and 22°C. but showed a decrease in corresponding samples of sausage mixture. At freezer temperatures decreases in count of approximately 1 log unit were observed during the first week for both meats followed by a more gradual decrease. The effect of desiccation by exposure was studied in minced beef and lamb outer carcass meat (breast) at refrigerator temperatures ( < 10°C). Decreases in viable count were observed in lamb carcass meat after 32 h although large variations were sometimes observed between duplicate samples for the same strain of C. je)uni. Counts were unchanged in exposed minced beef after storage for 48 h. Key words: Survival: Campylobacter)ejuni; Red meats
Introduction Thermotolerant Campylobacter spp. ( C jejuni and C. coli) have become widely recognised as agents of diarrhoeal disease in recent years. Isolations from humans in England, Wales and Ireland exceeded 12000 per annum in 1981 and 1982 (Anon., 1984). Red meats are recognised as a vehicle of salmonellosis but to date, there is little evidence to implicate a widespread association between red meats and Camp.vlobacter infection. A higher proportion of poultry carcasses are contaminated with campylobacters than red meats. Poultry have been implicated in more outbreaks of Campylobacter infection (Skirrow, 1982). Laboratory studies have been carried out on the survival of campylobacters in unpreserved red meats by H~inninen (1981), Gill and Harris (1981), Christopher et al. (1982). Bolton et al. (1982) examined carcasses in abattoirs and in butchers' shops. Campylobacters were isolated from 32% of beef, 70% of sheep and 56% of pig carcasses when sampled at abattoirs but not from any carcasses examined in butchers' shops. 0168-1605/84/$03.00 © 1984 Elsevier Science Publishers B.V.
l~
The object of the present study was to compare the survival of C. jejuni in an unpreserved raw meat (minced beef) with that in a preserved raw meat (pork sausage mixture) at freezer ( - 19°C) and refrigerator ( < 10°C) temperatures and at 22°C. The effect of exposure at refrigerator temperatures was also studied using a meat with a non porous consistency (lamb carcass breast) and one with a porous consistency (minced beef).
Materials and Methods
Two series of experiments were carried out. In the first (study 1) survival of campylobacters was studied in raw minced beef and pork sausage mixture stored in plastic stomacher bags (A.J. Seward Ltd., London). In the second (study 2) survival was compared in exposed minced beef and lamb outer carcass meat (breast) stored in plastic petri dishes. Sausage mixture ready for cooking was used in this study. Details of its composition were kindly supplied by the manufacturer and were as follows: total meat content (including fat), 65%; soya protein, 2%; sausage rusk (manufactured from wheat flour), 10.5%; salt, 2% and whey powder, 1%. The total carbohydrate level was 13.8% and the moisture content, 49%. The product also contained monosodium glutamate (approx. 250 ppm), colouring agent red 2G (approximately 40 p.p.m.),antioxidant (a blend of ascorbyl palmitate and a-tocopherol) equivalent to 200 p p m in the finished product and sodium metabisulphite to give a level of approximately 300 p p m of sulphur dioxide. Herbs and spices were incorporated as extracts into a pre-blended seasoning. The exact composition and addition rates were not known but were considered to be low in the finished product. The storage times studied were the maximum storage periods likely to be used in domestic circumstances.
Test strains of campylobacter Freshly isolated strains were used and details of their source and biotype are shown in Table I. The strains were identified on the basis of hippurate hydrolysis, production of hydrogen sulphide and sensitivity to nalidixic acid (Skirrow and Benjamin, 1980). The cultures were maintained on blood agar incubated in a micro-aerophilic atmosphere. This was achieved by evacuating an anaerobic jar without a catalyst and filling with a mixture of 10% carbon dioxide and 90% nitrogen. Cultures were subcultured at intervals of 1-3 days.
Preparation of inocula The test strains were spread over the surface of blood agar plates to give a lawn of growth and incubated micr~aerophilically at 42°C for 24 h. The growth from each plate was suspended in 10 mi quarter strength Ringer solution (-~ R), centrifuged for 20 min at 3000 rpm at approximately 10°C and resuspended in 10 or 1130 ml ¼R, the volume depending on the amount of growth on the blood agar plates.
lgo
Inoculation of samples For study 1. 10 g samples of minced beef or sausage mixture were placed in stomacher bags and surface inoculated with 0.5 ml Camp.vlol'acter suspension. For study 2, 20 g samples of minced beef were placed in plastic petri dishes and surface inoculated with 0.5 ml Campyh)bacter suspension. Samples of lamb carcass meat were cut into 5 x 5 cm squares, placed in plastic petri dishes and surface inoculated with 0.1 ml of Camp.vlobacter suspension spread over the surface with a sterilized bent nichrome wire. Initial viable counts of Camp.vlobacter were in the range 10~-107 colony forming units per g or cm-" of meat.
Viable counts Frozen meats were thawed at room temperature for 1 h prior to analysis. 10 g samples of meat (study 1 ) and 20 g samples (study 2) were macerated with 90 ml and 180 ml, respectively, of ¼R in a Colworth stomacher (A.J. Seward Ltd., London). Squares of lamb carcass meat (study 2) were macerated with 100 ml ~ R. Viable counts were carried out by a modified method of that described by Miles and Misra (1938). Viable counts of Campylobacter were estimated on Skirrow's agar (Skirrow. 1977) incubated micro-aerophilically at 42°C. Optimal growth of colonies was obtained after incubation for 2 4 - 4 8 h. Viable counts were carried out on 2 × 10 g of each batch of meat used in study 1 prior to inoculation as a control. For study 2. viable counts were carried out on 2 × 20 g of minced beef and from one 5 × 5 cm square from each lamb carcass sample prior to inoculation. Viable counts of aerobic mesophilic bacteria were carried out on blood agar incubated at 22°C for up to 3 days.
TABLE I Test strains of C. jejuni. Strain
Biotype
Source
1 2 l 1 1 1
poultry pouhry human human poultry human
i 1 1 1
pig cattle human human
:Study I 82/3155 82/3343 82/8606 82/16006 83/621 83/1059
Study 2 83/3089 83/3092 83/13659 83/14334
190
Storage of meats: study 1 Minced beef and sausage mixture were inoculated with C. jejuni and stored in a refrigerator at < 10°C and a laboratory freezer room at - 19’C, a domestic incubator at 22’C. The refrigerator was fitted with an automatic defrosting device and the meats were stored in the top part of the refrigerator. The temperature was checked with a maximum/minimum thermometer and was found to fluctuate on a regular cycle mostly between - 4°C and + 7°C. A survival profile was established for strain 82/8606 (C. jejuni biotype 1) at each temperature together with changes in viable counts of aerobic mesophilic bacteria in meats inoculated with this strain. At - 19°C viable counts of Camp.~lohacter and aerobic mesophilic bacteria were carried out at intervals of 1-2 weeks during a 10 week storage period as shown in Fig. 1. At refrigerator temperatures counts were carried out at 0, 1, 2, 3 and 6 days and at 22°C at 0, 2, 4, 6 and 24 h. A comparison was made between the survival of 4 strains of Camp_vlobacter in both meats after storage at - 19°C for 27 days, refrigerator temperatures for 6 days and 22°C for 24 h. Strains 82/3155, 82/3343 and 82/16006 were used together with a repeat analysis of 82/8606 at these storage temperatures and times. Only viable counts of Campylobacter were monitored. Storage
ofmeats:
study 2
The object of this study was to investigate the effect of desiccation and oxygen toxicity on survival of C. jejuni. Inoculated samples of minced beef and lamb carcass were stored at refrigerator temperatures (I 10°C for this study) in plastic petri dishes under normal atmospheric conditions. For exposed meats, petri dish lids were removed during overnight storage at I 10°C (approximately 16 h) and replaced on the following morning to minimise hazards. The refrigerator was sealed during exposure of meats. The survival of strain 83/13659 (C. jejuni biotype 1) was studied in exposed and unexposed minced beef and lamb carcass. Viable counts of Campylobacter and aerobic mesophilic bacteria were carried out after storage for 1, 2, and 3 days. The survival of strains 83/3089, 83/3092 and 83/14334 were studied in exposed lamb carcass meat after storage for 2 days.
Results Study I Survival at freezer tempfratures ( - 19OC) Viable counts for strain 82/8606 and Fig. 1. Survival of C. jejuni was better in both meats a 1 log unit decrease in counts week of storage. Further investigation of
aerobic mesophilic bacteria are shown in minced beef than in sausage mixture. For of C. jejuni was observed during the first this initial decrease was carried out with
lql strains 8 3 / 6 2 1 and 8 3 / 1 0 5 9 . A repeat analysis was carried out for 8 3 / 1 0 5 9 . F o r each of the 3 analyses, p e r c e n t a g e survival was similar in both meats a l t h o u g h differences in the time during which a 1 log unit decrease o c c u r r e d were observed ra n g i n g from 4 h for 8 3 / 1 0 5 9 (repeat) to 48 h for 8 3 / 1 0 5 9 (first study). A f t e r 7 days the counts had decreased by 2 log units. Percentage survival of 4 strains of Campylobacter is shown in T a b l e II. This includes the initial study with strain 8 2 / 8 6 0 6 as described a b o v e and the repeat
TABLE II Percentage survival of 4 strains of C. jejuni at different temperatures. Meat
Temp. (°C)
Time(days)
Strain 82/3155
82/3343
82/8606
82/8606 (repeat)
Minced beef
- 19 < 10 " 22
27 6 1
0.02 26.4 17.6
1.3 35.7 14.3
1.7 14.3 54.0
0.8 ,~.6 57.7
Pork sausage mixture
- 19 < 10 22
27 6 1
< 0.01 14.4 12.7
1.2 5.8 0.3
0.4 4.8 0.5
< 0.01 0.5 1.1
82/16006 0.5 84.5 85.7 0.09 2.1 1.0
a R e f r i g e r a t o r temperatures.
8-
m
6-
"~-o~O~
5o 4-
~n
°~o
n
3-
2"
Storgge time (Weeks)
Fig. 1. Survival of C. jejuni biotype 1 (strain 82/g606) and viable counts of aerobic mesophilic bacteria at 22°C in minced beef (o@) and sausage meat (~.A) stored at - 1 9 ° C . Open symbols, viable counts of C. jejunt; closed symbols, viable counts of aerobic mesophilic bacteria at 22°C.
192
i
i
0
3 Storage time
6 (Days)
Fig. 2. Survival of C. jejuni biotype 1 (strain 82/8606) and viable counts of aerobic mesophilic bacteria at 22°C in minced beef (00) and sausage meat (AA.) stored at refrigerator temperatures ( < 10°C). Open symbols, viable counts of C. jejuni; closed symbols, viable counts of aerobic mesophilic bacteria at 22OC. 10
9
A/A-A8
o-o-.-~
.,:& -
o-~-o-_o_
-
OIT--------o
A\A
'A ‘A-A \ ,’
, 0
2
4
If
6
Storage time
A
_(
24
(Hours)
Fig. 3. Survival of C. jejunr biotype 1 (strain 82/8606) and viable counts of aerobic mesophilic bacteria at 22°C in minced beef (00) and sausage meat (AA) stored at 22°C. Open symbols, viable counts of C. jejuni; closed symbols. viable counts of aerobic mesophilic bacteria at 22°C.
193 analysis. Survival was better in minced beef than in sausage meat for all strains. A greater range of percentage survival was obtained in sausage mixture.
Survival at refrigerator temperatures ( < ;O°C) and 22°C Viable counts' for strain 82/8606 and aerobic mesophilic bacteria are shown in Figs. 2 and 3. Percentage survival for 4 strains of C. jejuni is shown in Table II. At both temperatures survival was better in minced beef than in sausage mixture. The range of percentage survival was greater in sausage mixture than in minced beef~ Repeat analyses of strain 8 2 / 8 6 0 6 Differences in percentage survival were obtained for repeat analyses of this strain at each of the 3 storage temperatures (Table II). The differences were greater in sausage mixture. Stud r 2
Survival of C jejuni in exposed and unexposed minced beef and lamb carcass meat at refrigerator temperatures ( < I O°C) Viable counts of strain 83/13659 are shown in Fig. 4. During the 3 day storage period the meats were exposed for 48 h. Little change in viable counts of C jejum
fir C
0
N
2
Storage t:me [Days)
Fig. 4. Survival of C. jejuni biotype 1 (( Strain 8 3 / t 3659) in exposed and unexposed minced beef (AA) and lamb outer carcass breast meat ( o 0 ) stored at refrigerator temperatures ( ~ 10°C). Open symbols.
exposed meats: closed symbols, unexposed meats.1
194
was observed in exposed and unexposed minced beef during the 3 day period. Viable counts of aerobic mesophilic bacteria increased from 4 X lO’/g to 1 X lO’/g in both exposed and unexposed minced beef. A rapid decrease in viable counts of C. jejuni was observed in exposed lamb carcass meat and no viable organisms were recovered after 2 days (32 h exposure). A less marked decrease was observed in unexposed lamb carcass. Viable counts of aerobic mesophilic bacteria increased after 2 days by approximately 1 log unit in exposed lamb carcass and approximately 2 log units in unexposed meat. Viable counts for C. jejuni and aerobic mesophilic bacteria were not carried out on day 3 for exposed lamb carcass. Viable counts were carried out for strains 83/3089, 83/3092 and 83/14334 after 0 and 2 days on exposed lamb carcass meat at refrigerator temperatures. Starting with counts of 3-8 X lo5 C. jejuni per cm’ of meat, variations between duplicate counts were observed after 2 days for strains 83/3089 (1 x 10’ and < 1 x 10’ per cm*) and 83/14334 (1 X lo4 and 1 x lo2 per cm’). No viable organisms were recovered from meats inoculated with 83/3092.
Discussion Viable counts of C. jejuni biotype 1 showed decreases of less than 1 log unit during storage in minced beef in plastic bags at refrigerator temperatures ( < 1O’C) for 6 days and at 22°C for 1 day. Similar observations were made in sterile beef (Christopher et al., 1982) and in chicken (Blankenship and Craven, 1982) stored at similar temperatures. At freezer temperatures (- 19°C) the viable counts decreased by 1-2 log units during the first week of storage and this was followed by a more gradual decrease. Similar results were obtained by Hanninen (1981) with frozen raw ground beef whereas Christopher et al. (1982) observed a relatively uniform rate of decrease in frozen sterile beef. Oosterom et al. (1983) studied the effect of drying on C. jejuni. Viable counts on the surfaces of pig carcasses showed a more rapid decrease at a relative humidity of 60-7058 with ventilation than at a relative humidity of 95% without ventilation. Tiles of different materials were inoculated and stored in open and closed petri dishes at room temperature. Survival was better in the closed dishes and viable organisms were not recovered from tiles in open or closed dishes when the surfaces were visibly dry. Similarly, in the present study, survival was better on non-porous meat surfaces (lamb outer carcass breast) stored in closed petri dishes at refrigerator temperatures (Fig. 4). However, viable counts remained unchanged in minced beef exposed for the same period despite the very dry appearance of the meat after exposure. The inoculum was absorbed into the minced beef which had a protective effect against desiccation, oxygen toxicity or both these factors. Repeat analyses for strain 82/8606 showed differences in percentage survival of less than 1 log unit in minced beef at each temperature and in sausage mixture at 22°C but were greater in sausage mixture at freezer and refrigerator temperatures (Table II). The differences in percentage survival for repeat studies with this strain were similar to the overall range of survival shown by the 4 strains of C. jejuni in
195
each meat at each temperature. Thus, the differences in survival found in this study could be due to intrinsic factors in different batches of meat rather than to actual differences in survival potential between the different strains. No obvious association was found between" the initial viable count of aerobic mesophilic bacteria and the survival of C. jejuni 1 in minced beef and pork sausage mixture at each storage temperature. The survival of C. jejuni 1 was better in minced beef than in pork sausage mixture at each of the 3 storage temperatures and there was less variation in percentage survival between the 4 strains in minced beef. In a survey carried out in England and Wales (Turnbuli and Rose, 1982) campylobacters were found in a higher proportion of minced beef samples (1.0%) than in sausage mixture (0.1%). This is probably due to the presence of salt and sulphur dioxide in the sausage mixture. To conclude, it can be seen that C. jejuni is unlikely to multiply on meats at or below temperatures prevailing in temperate climates and the organism will be inactivated on exposed surfaces of carcasses as a result of desiccation and. possibly, oxygen sensitivity. This could explain the low incidence of reported Camp.vlobacter infection associated with red meats. The higher incidence associated with poultry could be due to enhanced survival on unexposed parts of the carcass such as the gut area and under the wings.
Acknowledgements The author would like to thank Dr J.D. Abbott for biotyping the cultures of C.
jejuni.
References Anon. (1984). W.H.O. surveillance programme for the control of foodborne infections and intoxications in Europe. Newsletter N u m b e r 3. January, 1984. Blankenship. C.C. and S.E. Craven, (1982). Camp.vlobacter)e/uni survival in chicken meat as a function of temperature. Appl. Environ. Microbiol. 44. 88-92. Bohon. F.J., H.C. Dawkins and L. Robertson (1982). Cump.vlobac'ter]ejuni/coli in abattoirs and butchers" shops. J. Infect. 4. 243-245. Christopher, F.M., G.C. Smith and C. Vanderzant. (1982). Effect of temperature and pH on the survival of Campvlobacterfetus. J. Food Protect. 45, 253-259. Gill. C.C. and L.M. Harris. (1982). Survival and growth of Compvlobacterfetus subsp.je/uni on meat and • in cooked foods. Appl. Environ. Microbiol. 44. 259-263. H~inninen. M.L.. (1981). Survival of Camp.vlobacterjejuni/coli in ground refrigerated and Jn ground frozen beef liver and in frozen broiler carcasses. Acta Veterin. Scand. 22, 566-577. Miles. A.A. and S.S. Misra, (1938). The estimation of the bactericidal power of the blood. J. Hyg. (Cambridge) 38, 732-749. Oosterom. J.. G.J.A. De Wilde. E. De Boer. L.H. De Blaaw and H. Karman. {1983). Survival of CampvlolTacterjejuni during poultry processing and pig slaughtering. J. Food Protect. 46. 702-706. Skirrow, M.B.. (1977). Campvlobacterenteritis: a "new" disease. Br. Med. J. 2. 9-11. Skirrow. M.B. and J. Benjamin, 0980). "1001 Campylobactcrs'. Cultural characteristics of intestinal campylobacters from m a n and animals. J. Hyg. (Cambridge) 85, 427-442.
196 Skirrow. M.B.. (1982). Campdobacrer enteritis - the first 5 years. J. Hyg. (Cambridge) 89. 175-184. Svedhem. A., B. Kaijser and E. Sjiigren. (1981). The occurrence of Camp_vlobacrerjejuni in fresh food and survival under different conditions. J. Hyg. (Cambridge) 87. 421-425. Turnbull, P.C.B. and P. Rose, (1982). Camp_vlobacrer jejuni and .Sa/mone//u in raw red meats. J. Hyg. (Cambridge) 88. 29-37.
187
Elsevier JFM 00020
The survival of Campylobacterjejuni in red meats stored at different temperatures R.A.E. B a r r e l l Publw Health Laboratory. Withington Hospttal. Manchester M20 8LR, U.K (Received 30 March 1984; accepted 6 September 1984)
The survival of 4 strains of Campylobacter.tejuni was studied in raw minced beef and raw pork sausage mixture stored in plastic stomacher bags at freezer temperatures ( - 19°C) for up to 10 weeks, refrigerator temperatures ( < 10°C) for 6 days and at 22°C for 24 h. At each of the 3 storage temperatures survival was better in minced beef. Similarly, there was less variation in percentage survival between the 4 strains in minced beef than in sausage mixture after storage at each temperature. Detailed studies were carried out with one strain of C. jejuni. Viable counts were relatively unchanged in minced beef at refrigerator temperatures and 22°C. but showed a decrease in corresponding samples of sausage mixture. At freezer temperatures decreases in count of approximately 1 log unit were observed during the first week for both meats followed by a more gradual decrease. The effect of desiccation by exposure was studied in minced beef and lamb outer carcass meat (breast) at refrigerator temperatures ( < 10°C). Decreases in viable count were observed in lamb carcass meat after 32 h although large variations were sometimes observed between duplicate samples for the same strain of C. je)uni. Counts were unchanged in exposed minced beef after storage for 48 h. Key words: Survival: Campylobacter)ejuni; Red meats
Introduction Thermotolerant Campylobacter spp. ( C jejuni and C. coli) have become widely recognised as agents of diarrhoeal disease in recent years. Isolations from humans in England, Wales and Ireland exceeded 12000 per annum in 1981 and 1982 (Anon., 1984). Red meats are recognised as a vehicle of salmonellosis but to date, there is little evidence to implicate a widespread association between red meats and Camp.vlobacter infection. A higher proportion of poultry carcasses are contaminated with campylobacters than red meats. Poultry have been implicated in more outbreaks of Campylobacter infection (Skirrow, 1982). Laboratory studies have been carried out on the survival of campylobacters in unpreserved red meats by H~inninen (1981), Gill and Harris (1981), Christopher et al. (1982). Bolton et al. (1982) examined carcasses in abattoirs and in butchers' shops. Campylobacters were isolated from 32% of beef, 70% of sheep and 56% of pig carcasses when sampled at abattoirs but not from any carcasses examined in butchers' shops. 0168-1605/84/$03.00 © 1984 Elsevier Science Publishers B.V.
l~
The object of the present study was to compare the survival of C. jejuni in an unpreserved raw meat (minced beef) with that in a preserved raw meat (pork sausage mixture) at freezer ( - 19°C) and refrigerator ( < 10°C) temperatures and at 22°C. The effect of exposure at refrigerator temperatures was also studied using a meat with a non porous consistency (lamb carcass breast) and one with a porous consistency (minced beef).
Materials and Methods
Two series of experiments were carried out. In the first (study 1) survival of campylobacters was studied in raw minced beef and pork sausage mixture stored in plastic stomacher bags (A.J. Seward Ltd., London). In the second (study 2) survival was compared in exposed minced beef and lamb outer carcass meat (breast) stored in plastic petri dishes. Sausage mixture ready for cooking was used in this study. Details of its composition were kindly supplied by the manufacturer and were as follows: total meat content (including fat), 65%; soya protein, 2%; sausage rusk (manufactured from wheat flour), 10.5%; salt, 2% and whey powder, 1%. The total carbohydrate level was 13.8% and the moisture content, 49%. The product also contained monosodium glutamate (approx. 250 ppm), colouring agent red 2G (approximately 40 p.p.m.),antioxidant (a blend of ascorbyl palmitate and a-tocopherol) equivalent to 200 p p m in the finished product and sodium metabisulphite to give a level of approximately 300 p p m of sulphur dioxide. Herbs and spices were incorporated as extracts into a pre-blended seasoning. The exact composition and addition rates were not known but were considered to be low in the finished product. The storage times studied were the maximum storage periods likely to be used in domestic circumstances.
Test strains of campylobacter Freshly isolated strains were used and details of their source and biotype are shown in Table I. The strains were identified on the basis of hippurate hydrolysis, production of hydrogen sulphide and sensitivity to nalidixic acid (Skirrow and Benjamin, 1980). The cultures were maintained on blood agar incubated in a micro-aerophilic atmosphere. This was achieved by evacuating an anaerobic jar without a catalyst and filling with a mixture of 10% carbon dioxide and 90% nitrogen. Cultures were subcultured at intervals of 1-3 days.
Preparation of inocula The test strains were spread over the surface of blood agar plates to give a lawn of growth and incubated micr~aerophilically at 42°C for 24 h. The growth from each plate was suspended in 10 mi quarter strength Ringer solution (-~ R), centrifuged for 20 min at 3000 rpm at approximately 10°C and resuspended in 10 or 1130 ml ¼R, the volume depending on the amount of growth on the blood agar plates.
lgo
Inoculation of samples For study 1. 10 g samples of minced beef or sausage mixture were placed in stomacher bags and surface inoculated with 0.5 ml Camp.vlol'acter suspension. For study 2, 20 g samples of minced beef were placed in plastic petri dishes and surface inoculated with 0.5 ml Campyh)bacter suspension. Samples of lamb carcass meat were cut into 5 x 5 cm squares, placed in plastic petri dishes and surface inoculated with 0.1 ml of Camp.vlobacter suspension spread over the surface with a sterilized bent nichrome wire. Initial viable counts of Camp.vlobacter were in the range 10~-107 colony forming units per g or cm-" of meat.
Viable counts Frozen meats were thawed at room temperature for 1 h prior to analysis. 10 g samples of meat (study 1 ) and 20 g samples (study 2) were macerated with 90 ml and 180 ml, respectively, of ¼R in a Colworth stomacher (A.J. Seward Ltd., London). Squares of lamb carcass meat (study 2) were macerated with 100 ml ~ R. Viable counts were carried out by a modified method of that described by Miles and Misra (1938). Viable counts of Campylobacter were estimated on Skirrow's agar (Skirrow. 1977) incubated micro-aerophilically at 42°C. Optimal growth of colonies was obtained after incubation for 2 4 - 4 8 h. Viable counts were carried out on 2 × 10 g of each batch of meat used in study 1 prior to inoculation as a control. For study 2. viable counts were carried out on 2 × 20 g of minced beef and from one 5 × 5 cm square from each lamb carcass sample prior to inoculation. Viable counts of aerobic mesophilic bacteria were carried out on blood agar incubated at 22°C for up to 3 days.
TABLE I Test strains of C. jejuni. Strain
Biotype
Source
1 2 l 1 1 1
poultry pouhry human human poultry human
i 1 1 1
pig cattle human human
:Study I 82/3155 82/3343 82/8606 82/16006 83/621 83/1059
Study 2 83/3089 83/3092 83/13659 83/14334
190
Storage of meats: study 1 Minced beef and sausage mixture were inoculated with C. jejuni and stored in a refrigerator at < 10°C and a laboratory freezer room at - 19’C, a domestic incubator at 22’C. The refrigerator was fitted with an automatic defrosting device and the meats were stored in the top part of the refrigerator. The temperature was checked with a maximum/minimum thermometer and was found to fluctuate on a regular cycle mostly between - 4°C and + 7°C. A survival profile was established for strain 82/8606 (C. jejuni biotype 1) at each temperature together with changes in viable counts of aerobic mesophilic bacteria in meats inoculated with this strain. At - 19°C viable counts of Camp.~lohacter and aerobic mesophilic bacteria were carried out at intervals of 1-2 weeks during a 10 week storage period as shown in Fig. 1. At refrigerator temperatures counts were carried out at 0, 1, 2, 3 and 6 days and at 22°C at 0, 2, 4, 6 and 24 h. A comparison was made between the survival of 4 strains of Camp_vlobacter in both meats after storage at - 19°C for 27 days, refrigerator temperatures for 6 days and 22°C for 24 h. Strains 82/3155, 82/3343 and 82/16006 were used together with a repeat analysis of 82/8606 at these storage temperatures and times. Only viable counts of Campylobacter were monitored. Storage
ofmeats:
study 2
The object of this study was to investigate the effect of desiccation and oxygen toxicity on survival of C. jejuni. Inoculated samples of minced beef and lamb carcass were stored at refrigerator temperatures (I 10°C for this study) in plastic petri dishes under normal atmospheric conditions. For exposed meats, petri dish lids were removed during overnight storage at I 10°C (approximately 16 h) and replaced on the following morning to minimise hazards. The refrigerator was sealed during exposure of meats. The survival of strain 83/13659 (C. jejuni biotype 1) was studied in exposed and unexposed minced beef and lamb carcass. Viable counts of Campylobacter and aerobic mesophilic bacteria were carried out after storage for 1, 2, and 3 days. The survival of strains 83/3089, 83/3092 and 83/14334 were studied in exposed lamb carcass meat after storage for 2 days.
Results Study I Survival at freezer tempfratures ( - 19OC) Viable counts for strain 82/8606 and Fig. 1. Survival of C. jejuni was better in both meats a 1 log unit decrease in counts week of storage. Further investigation of
aerobic mesophilic bacteria are shown in minced beef than in sausage mixture. For of C. jejuni was observed during the first this initial decrease was carried out with
lql strains 8 3 / 6 2 1 and 8 3 / 1 0 5 9 . A repeat analysis was carried out for 8 3 / 1 0 5 9 . F o r each of the 3 analyses, p e r c e n t a g e survival was similar in both meats a l t h o u g h differences in the time during which a 1 log unit decrease o c c u r r e d were observed ra n g i n g from 4 h for 8 3 / 1 0 5 9 (repeat) to 48 h for 8 3 / 1 0 5 9 (first study). A f t e r 7 days the counts had decreased by 2 log units. Percentage survival of 4 strains of Campylobacter is shown in T a b l e II. This includes the initial study with strain 8 2 / 8 6 0 6 as described a b o v e and the repeat
TABLE II Percentage survival of 4 strains of C. jejuni at different temperatures. Meat
Temp. (°C)
Time(days)
Strain 82/3155
82/3343
82/8606
82/8606 (repeat)
Minced beef
- 19 < 10 " 22
27 6 1
0.02 26.4 17.6
1.3 35.7 14.3
1.7 14.3 54.0
0.8 ,~.6 57.7
Pork sausage mixture
- 19 < 10 22
27 6 1
< 0.01 14.4 12.7
1.2 5.8 0.3
0.4 4.8 0.5
< 0.01 0.5 1.1
82/16006 0.5 84.5 85.7 0.09 2.1 1.0
a R e f r i g e r a t o r temperatures.
8-
m
6-
"~-o~O~
5o 4-
~n
°~o
n
3-
2"
Storgge time (Weeks)
Fig. 1. Survival of C. jejuni biotype 1 (strain 82/g606) and viable counts of aerobic mesophilic bacteria at 22°C in minced beef (o@) and sausage meat (~.A) stored at - 1 9 ° C . Open symbols, viable counts of C. jejunt; closed symbols, viable counts of aerobic mesophilic bacteria at 22°C.
192
i
i
0
3 Storage time
6 (Days)
Fig. 2. Survival of C. jejuni biotype 1 (strain 82/8606) and viable counts of aerobic mesophilic bacteria at 22°C in minced beef (00) and sausage meat (AA.) stored at refrigerator temperatures ( < 10°C). Open symbols, viable counts of C. jejuni; closed symbols, viable counts of aerobic mesophilic bacteria at 22OC. 10
9
A/A-A8
o-o-.-~
.,:& -
o-~-o-_o_
-
OIT--------o
A\A
'A ‘A-A \ ,’
, 0
2
4
If
6
Storage time
A
_(
24
(Hours)
Fig. 3. Survival of C. jejunr biotype 1 (strain 82/8606) and viable counts of aerobic mesophilic bacteria at 22°C in minced beef (00) and sausage meat (AA) stored at 22°C. Open symbols, viable counts of C. jejuni; closed symbols. viable counts of aerobic mesophilic bacteria at 22°C.
193 analysis. Survival was better in minced beef than in sausage meat for all strains. A greater range of percentage survival was obtained in sausage mixture.
Survival at refrigerator temperatures ( < ;O°C) and 22°C Viable counts' for strain 82/8606 and aerobic mesophilic bacteria are shown in Figs. 2 and 3. Percentage survival for 4 strains of C. jejuni is shown in Table II. At both temperatures survival was better in minced beef than in sausage mixture. The range of percentage survival was greater in sausage mixture than in minced beef~ Repeat analyses of strain 8 2 / 8 6 0 6 Differences in percentage survival were obtained for repeat analyses of this strain at each of the 3 storage temperatures (Table II). The differences were greater in sausage mixture. Stud r 2
Survival of C jejuni in exposed and unexposed minced beef and lamb carcass meat at refrigerator temperatures ( < I O°C) Viable counts of strain 83/13659 are shown in Fig. 4. During the 3 day storage period the meats were exposed for 48 h. Little change in viable counts of C jejum
fir C
0
N
2
Storage t:me [Days)
Fig. 4. Survival of C. jejuni biotype 1 (( Strain 8 3 / t 3659) in exposed and unexposed minced beef (AA) and lamb outer carcass breast meat ( o 0 ) stored at refrigerator temperatures ( ~ 10°C). Open symbols.
exposed meats: closed symbols, unexposed meats.1
194
was observed in exposed and unexposed minced beef during the 3 day period. Viable counts of aerobic mesophilic bacteria increased from 4 X lO’/g to 1 X lO’/g in both exposed and unexposed minced beef. A rapid decrease in viable counts of C. jejuni was observed in exposed lamb carcass meat and no viable organisms were recovered after 2 days (32 h exposure). A less marked decrease was observed in unexposed lamb carcass. Viable counts of aerobic mesophilic bacteria increased after 2 days by approximately 1 log unit in exposed lamb carcass and approximately 2 log units in unexposed meat. Viable counts for C. jejuni and aerobic mesophilic bacteria were not carried out on day 3 for exposed lamb carcass. Viable counts were carried out for strains 83/3089, 83/3092 and 83/14334 after 0 and 2 days on exposed lamb carcass meat at refrigerator temperatures. Starting with counts of 3-8 X lo5 C. jejuni per cm’ of meat, variations between duplicate counts were observed after 2 days for strains 83/3089 (1 x 10’ and < 1 x 10’ per cm*) and 83/14334 (1 X lo4 and 1 x lo2 per cm’). No viable organisms were recovered from meats inoculated with 83/3092.
Discussion Viable counts of C. jejuni biotype 1 showed decreases of less than 1 log unit during storage in minced beef in plastic bags at refrigerator temperatures ( < 1O’C) for 6 days and at 22°C for 1 day. Similar observations were made in sterile beef (Christopher et al., 1982) and in chicken (Blankenship and Craven, 1982) stored at similar temperatures. At freezer temperatures (- 19°C) the viable counts decreased by 1-2 log units during the first week of storage and this was followed by a more gradual decrease. Similar results were obtained by Hanninen (1981) with frozen raw ground beef whereas Christopher et al. (1982) observed a relatively uniform rate of decrease in frozen sterile beef. Oosterom et al. (1983) studied the effect of drying on C. jejuni. Viable counts on the surfaces of pig carcasses showed a more rapid decrease at a relative humidity of 60-7058 with ventilation than at a relative humidity of 95% without ventilation. Tiles of different materials were inoculated and stored in open and closed petri dishes at room temperature. Survival was better in the closed dishes and viable organisms were not recovered from tiles in open or closed dishes when the surfaces were visibly dry. Similarly, in the present study, survival was better on non-porous meat surfaces (lamb outer carcass breast) stored in closed petri dishes at refrigerator temperatures (Fig. 4). However, viable counts remained unchanged in minced beef exposed for the same period despite the very dry appearance of the meat after exposure. The inoculum was absorbed into the minced beef which had a protective effect against desiccation, oxygen toxicity or both these factors. Repeat analyses for strain 82/8606 showed differences in percentage survival of less than 1 log unit in minced beef at each temperature and in sausage mixture at 22°C but were greater in sausage mixture at freezer and refrigerator temperatures (Table II). The differences in percentage survival for repeat studies with this strain were similar to the overall range of survival shown by the 4 strains of C. jejuni in
195
each meat at each temperature. Thus, the differences in survival found in this study could be due to intrinsic factors in different batches of meat rather than to actual differences in survival potential between the different strains. No obvious association was found between" the initial viable count of aerobic mesophilic bacteria and the survival of C. jejuni 1 in minced beef and pork sausage mixture at each storage temperature. The survival of C. jejuni 1 was better in minced beef than in pork sausage mixture at each of the 3 storage temperatures and there was less variation in percentage survival between the 4 strains in minced beef. In a survey carried out in England and Wales (Turnbuli and Rose, 1982) campylobacters were found in a higher proportion of minced beef samples (1.0%) than in sausage mixture (0.1%). This is probably due to the presence of salt and sulphur dioxide in the sausage mixture. To conclude, it can be seen that C. jejuni is unlikely to multiply on meats at or below temperatures prevailing in temperate climates and the organism will be inactivated on exposed surfaces of carcasses as a result of desiccation and. possibly, oxygen sensitivity. This could explain the low incidence of reported Camp.vlobacter infection associated with red meats. The higher incidence associated with poultry could be due to enhanced survival on unexposed parts of the carcass such as the gut area and under the wings.
Acknowledgements The author would like to thank Dr J.D. Abbott for biotyping the cultures of C.
jejuni.
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196 Skirrow. M.B.. (1982). Campdobacrer enteritis - the first 5 years. J. Hyg. (Cambridge) 89. 175-184. Svedhem. A., B. Kaijser and E. Sjiigren. (1981). The occurrence of Camp_vlobacrerjejuni in fresh food and survival under different conditions. J. Hyg. (Cambridge) 87. 421-425. Turnbull, P.C.B. and P. Rose, (1982). Camp_vlobacrer jejuni and .Sa/mone//u in raw red meats. J. Hyg. (Cambridge) 88. 29-37.