3050/04/89
Journal of Applied Bacteriology 1990,68, 157-162
Growth of Listeria monocytogenes at refrigeration temperatures S.J. W A L K E RP. , A R C H E R& J.G. B A N K SCampden Food and Drink Research Association, Chipping Campden, Glos. GL55 6LD, U K Accepted 14 June 1989
W A L K E R .S . J.. A R C H E R . P. & B A N K S , J.G. 1990, Growth of Listeria monocytogenes at 'refrigeration temperatures. Journal of Applied Bacteriology 68, 157-162. The growth of three strains of Listeria rnonocytogenes at refrigeration temperatures (-0.5 to 9.3"C) in chicken broth and/or UHT milk was determined using a rocking temperature gradient incubator. Minimum growth temperatures ranged from -0.1 to -0.4"C for the three strains. Lag times of 1-3 d and 3 to > 34 d were observed with incubation at 5 and 0°C respectively. Corresponding generation times ranged from 13-24 h at 5°C and 62-131 h at 0°C. The type of culture medium had an influence on both the rate and extent of growth. Incubation of cultures at 4°C before inoculation caused a marked reduction in the lag time when compared with cultures which had been previously incubated at 30°C.
Human listeriosis may cause a serious illness in those individuals whose immune defence system is compromised and the rate of mortality may be high (Marth 1988). Listeria monocytogenes has been implicated as an agent of foodborne disease in several outbreaks; foods involved include coleslaw (Schlech et al. 1983), pasteurized milk (Fleming et al. 1985) and cheese (James et al. 1985; Bannister 1987; Azadian et al. 1989). The ability of L. monocytogenes to grow at refrigeration temperatures may be of concern, particularly with chilled foods. It has been postulated that in two outbreaks of listeriosis (Schlech et al. 1983; Fleming et al. 1985), refrigerated storage may have allowed the micro-organisms to multiply in the food before consumption. Although the growth of L. monocytogenes is well characterized at 4°C (Donnelly & Briggs 1986; Rosenow & Marth 1987), little is known about the ability of strains to grow at lower temperatures. The aim of this study was to investigate the growth of L. monocytogenes at a range of temperatures typical of refrigerated storage.
Materials and Methods M A I N TEN A N CE OF S U B - 0 P TI M A L TEMPERATURES
Sub-optimal temperature incubation of bacterial cultures was done in a rocking temperature gradient incubator (TGI) described previously by Alcock et al. (1984). The TGI was placed in a refrigerated room set at the mean temperature of the TGI, to optimize the linearity of the temperature gradient along the instrument. A range of - 1 . 5 to 1O"C was used with a minimum of 15 intermediate settings. The temperature of the medium in the culture tubes was monitored hourly at five equidistant points along the block with platinum resistance detectors connected to a Comark 6600 Microprocessor Datalogger (Comark Electronics, Rustington, W. Sussex). The average temperature at each point was used to produce a calibration graph of the tube position with temperature. A maximum variation of +0.25"C was noted at any detection point during the incubation period of up to 50 d.
S . J . Walker et al.
158
0
.,
I
0
1
I
1
1
I
10
20
30
40
50
Time (days)
Fig. 1. Growth of Listeria monocytogenes CRA 433 in chicken broth when incubated at 8.7, 3.5, 1.5 and 0.8”C 0and 0 respectively).
(0,
Tubes containing sterile growth media were allowed to equilibrate in the TGI for at least 24 h before inoculation. MICRO-ORGANISMS A N D I N O C U L A T I O N
Three strains of L. monocytogenes (serotype 4b) were used; L. monocytogenes CRA 197 and CRA 198 were isolated from a patient and food implicated in the milkborne outbreak (Fleming et al. 1985) and L. monocytogenes CRA 433 (NCTC 11994) was isolated from soft cheese implicated in a foodborne case of meningitis (Bannister 1987). Reference cultures were freeze-dried for storage. Routine culture and maintenance was on Nutrient Agar (Oxoid CM3) slopes stored at 4°C. In general, the bacterial strains were inoculated into Nutrient Broth (Oxoid CM1) and incubated at 30°C for 48 h. Further dilution in the same medium was used to inoculate sterile growth media to achieve final populations of 104-105 cfu/ml. In addition to incubation at 30°C for 48 h, L. monocytogenes CRA 433 was also incubated at 4°C for 6 d before inoculation. Both regimes resulted in approximately the same level of inoculum and phase (mid-late logarithmic) of growth. G R O W T H MEDIA
Chicken broth (pH ca 6.4) was prepared in bulk (approx 3 1) (Alcock 1984), dispensed in 7 ml
volumes into TGI culture tubes and autoclaved. UHT milk (pH ca 6.6) was also used and dispensed (7 ml volumes) into sterile culture tubes. The sterility of both media was tested by subculture on Plate Count Agar (Oxoid CM325) incubated at 30°C for 48 h. These were chosen as growth media as chickens (Pini & Gilbert 1988) and milk (Garayzabal et al. 1986; Lovett et al. 1987) often contain L. monocytogenes.
ASSESSMENT O F G R O W T H
A Gilson PlOO micropipette (Anachem, Luton, Bedfordshire) fitted with slim tips (Universal Extended Finntip, Jencons Scientific Ltd, Leighton Buzzard, Bedfordshire) was used to sample each culture tube at appropriate time intervals of 1-7 d. Samples were diluted in Maximum Recovery Diluent (Oxoid CM733) and enumerated in duplicate by a pour plate method on Plate Count Agar. Incubation was at 30°C for 48 h. Results The lowest temperatures that permitted growth of L. monocytogenes CRA 197, 198 and 433 in chicken broth were -0.1, -0.4 and -0.2”C respectively. At lower temperatures,, the media froze and could not be sampled. The minimum growth temperature ( - 0.2”C) was unaffected by the culture medium for L. monocytogenes CRA 433. Listeria monocytogenes CRA 433 was
Growth of L. monocytogenes unable to grow at -0.2"C in chicken broth after pre-incubation at 30°C and under these circumstances the minimum growth temperature was 0.5"C. As the incubation temperature was lowered both the lag time before growth and the generation time during the logarithmic phase increased and in general the maximum population size was reduced (Fig. 1). Similar growth curves were obtained for all the strains of L. monocytogenes. At the lowest incubation temperatures, the lag phase could exceed 30 d and the generation time 100 h (Table 1). A reduction in the temperature of incubation had an increasingly marked effect on both the lag and generation time.
159
Pre-incubation of L. monocytogenes CRA 433 at 4°C before inoculation of culture media greatly reduced the lag time particularly at low ( < 2°C) incubation temperatures (Fig. 2). At higher ( > 7°C) incubation temperatures, this effect was not evident. The pre-incubation temperature did not markedly affect the generation time or the maximum population size observed with the culture media. Similar trends were noted with both chicken broth and UHT milk, although the effect was less dramatic in the latter medium. Growth of L. monocytogenes CRA 433 was also influenced by the culture medium per se (Fig. 3). With culture in UHT milk, the lag phase was greatly reduced, particularly as the
9
8
E
\
2 7 0
m
-
6
5 0
20
10
30
50
40
Time (days)
Fig. 2. Effect of previous incubation at 30°C (open symbols) and 4°C (closed symbols) on the growth of Listeria monocytogenes CRA 433 in chicken broth when growth at 84-8.7"C (0,B)and 1.5-1.8"C (0, 0).
I
0
10
20
30
40
50
Time (days)
Fig. 3. Growth of Listeria monocytogenes CRA 433 when cultured in chicken broth (open symbols) and UHT milk (closed symbols) incubated at 8.7"Cand 1.5"C(0,and 0, respectively).
160
S . J . Walker et al.
micro-organisms approached their minimum temperature for growth (Table 1). The growth medium had a marked effect on the generation time only at temperatures less than 2"C, with more rapid growth observed in UHT milk. At higher ( > 7°C) incubation temperatures no constant effects were noted. Irrespective of preincubation treatment or incubation temperature the maximum population achieved in chicken broth was greater than in UHT milk.
Discussion Three strains of the most common serotype associated with cases of human listeriosis (serotype 4b, McLauchlin 1987) were chosen for investigation. Also all strains had been implicated in cases of foodborne listeriosis. Although other serotypes of L. monocytogenes were not tested in this study, Rosenow & Marth (1987) did not detect any significant differences between the growth rates of four strains (various serotypes) when cultured in milk at chill temperatures. The minimum growth temperature (MGT) for the three strains of L. monocytogenes ranged from -0.1 to -0.4"C. This is 1 to 1.5"C lower than the minimum (1°C) previously reported in the literature (Seeliger & Jones 1986). Growth at lower temperatures cannot be discounted as the model growth media froze at temperatures below the recorded minimum and precluded sampling. The MGT will depend also on the nutrient status of the culture medium (Ingram & Mackey 1976) and so may be lower in other situations. Michener & Elliott (1964) have suggested that the true MGT can be determined only when all other conditions are optimal. In the present study, sterile growth media were used as other micro-organisms may cause changes (eg pH change, depletion of nutrients) and so alter the growth characteristics of L. monocytogenes. A reduction in the incubation temperature of cultures resulted in a longer time before growth occurred and a slower rate of growth, which produce a series of growth curves with respect to temperature. As temperatures approached the MGT, the rate of increase in both the lag and generation times became greater. The lag and generation times observed were shorter than those reported by Rosenow & Marth (1987) for four strains of L. monocytogenes
grown in sterilized milk (i.e. lag times of 5-10 d and generation times of 29 h 44 min-45 h 33 min when incubated at 4°C). This may be due to differences between the strains used and/or growth media. In the present study strain differences were generally more pronounced as temperatures approached the minimum for growth. A reduction in the incubation temperature generally resulted in a decrease in the maximum population size of the culture. Rosenow & Marth (1987) reported that maximum stationary phase populations were obtained under optimal incubation temperature conditions. In most investigations into the growth of L. monocytogenes at low temperatures strains were grown at the optimum growth temperature before inoculation of the growth medium. Thus the population experiences a dramatic and sudden decrease in temperature which may induce 'cold shock' (Ingram & Mackey 1976). The degree of 'cold shock' is related to both the temperature difference and the rate of cooling (Ingram & Mackey 1976). In the present study L. monocytogenes CRA 433 was cultured at two different temperatures although in both instances the population was in the same growth phase and with approximately the same concentration of micro-organisms. Previous incubation of the culture at a low (4°C) temperature generally resulted in a decrease in the lag time which would indicate that the 'cold shock' phenomenon was less pronounced as compared with cultures which has been previously incubated at higher temperatures (30°C). As L. monocytogenes is common in the food processing environment (Anon 1987a; Anon 1987b; Cox et al. 1989) the microorganism may be adapted to growth at low temperatures before contamination of the food occurs. Consequently, the safety margins for food storage at low temperatures may be considerably shorter than those indicated by studies which have not considered the potential for cells to adapt to growth at low temperatures. The type of growth medium during chilled incubation also affected the lag time, with a more rapid initiation of growth in milk cf. chicken broth. Although pH does have an effect on the growth of L. monocytogenes (George et al. 1988), the growth media used had similar pH values. The differences observed most likely reflect the differing nutritional and/or inhibitory properties of the media. As the MGT was
30 30 30 4 30 4
30 30 30 4 30 4
NT, not tested; NG, No growth observed.
(ii) Generation time (h) L. monocytogenes 197 L. monocytogenes 198 L. monocytogenes 433
(i) Lag time (d) L. monocytogenes 197 L. monocytogenes 198 L. monocytogenes 433
Strain
Re-incubation temperature ("C)
Chicken broth Chicken broth Chicken broth Chicken broth UHT milk UHT milk
Chicken broth Chicken broth Chicken broth Chicken broth UHT milk UHT milk
Growth medium 0.3
77 98 NG 131 62 77
33 21 > 34 18 13 3 4
0.0
0.3
45 24 33
30
31 35
8 6 8 3 4 5 2
2.5
17 13 25 19 20 19
3 3 3 1-2 1-2 1
5.0 f 0.3
Temperature ("C)
NT NT 13 9 16 9.5
NT NT 2 1 1 t l
7.5 f 0.3
Table 1. Observed lag times (d) and generation times (h) for Listeria monocytogenes strains
0.3
NT NT 5 7 5.5 9
NT NT 1-2
Journal of Applied Bacteriology 1990,68, 157-162
Growth of Listeria monocytogenes at refrigeration temperatures S.J. W A L K E RP. , A R C H E R& J.G. B A N K SCampden Food and Drink Research Association, Chipping Campden, Glos. GL55 6LD, U K Accepted 14 June 1989
W A L K E R .S . J.. A R C H E R . P. & B A N K S , J.G. 1990, Growth of Listeria monocytogenes at 'refrigeration temperatures. Journal of Applied Bacteriology 68, 157-162. The growth of three strains of Listeria rnonocytogenes at refrigeration temperatures (-0.5 to 9.3"C) in chicken broth and/or UHT milk was determined using a rocking temperature gradient incubator. Minimum growth temperatures ranged from -0.1 to -0.4"C for the three strains. Lag times of 1-3 d and 3 to > 34 d were observed with incubation at 5 and 0°C respectively. Corresponding generation times ranged from 13-24 h at 5°C and 62-131 h at 0°C. The type of culture medium had an influence on both the rate and extent of growth. Incubation of cultures at 4°C before inoculation caused a marked reduction in the lag time when compared with cultures which had been previously incubated at 30°C.
Human listeriosis may cause a serious illness in those individuals whose immune defence system is compromised and the rate of mortality may be high (Marth 1988). Listeria monocytogenes has been implicated as an agent of foodborne disease in several outbreaks; foods involved include coleslaw (Schlech et al. 1983), pasteurized milk (Fleming et al. 1985) and cheese (James et al. 1985; Bannister 1987; Azadian et al. 1989). The ability of L. monocytogenes to grow at refrigeration temperatures may be of concern, particularly with chilled foods. It has been postulated that in two outbreaks of listeriosis (Schlech et al. 1983; Fleming et al. 1985), refrigerated storage may have allowed the micro-organisms to multiply in the food before consumption. Although the growth of L. monocytogenes is well characterized at 4°C (Donnelly & Briggs 1986; Rosenow & Marth 1987), little is known about the ability of strains to grow at lower temperatures. The aim of this study was to investigate the growth of L. monocytogenes at a range of temperatures typical of refrigerated storage.
Materials and Methods M A I N TEN A N CE OF S U B - 0 P TI M A L TEMPERATURES
Sub-optimal temperature incubation of bacterial cultures was done in a rocking temperature gradient incubator (TGI) described previously by Alcock et al. (1984). The TGI was placed in a refrigerated room set at the mean temperature of the TGI, to optimize the linearity of the temperature gradient along the instrument. A range of - 1 . 5 to 1O"C was used with a minimum of 15 intermediate settings. The temperature of the medium in the culture tubes was monitored hourly at five equidistant points along the block with platinum resistance detectors connected to a Comark 6600 Microprocessor Datalogger (Comark Electronics, Rustington, W. Sussex). The average temperature at each point was used to produce a calibration graph of the tube position with temperature. A maximum variation of +0.25"C was noted at any detection point during the incubation period of up to 50 d.
S . J . Walker et al.
158
0
.,
I
0
1
I
1
1
I
10
20
30
40
50
Time (days)
Fig. 1. Growth of Listeria monocytogenes CRA 433 in chicken broth when incubated at 8.7, 3.5, 1.5 and 0.8”C 0and 0 respectively).
(0,
Tubes containing sterile growth media were allowed to equilibrate in the TGI for at least 24 h before inoculation. MICRO-ORGANISMS A N D I N O C U L A T I O N
Three strains of L. monocytogenes (serotype 4b) were used; L. monocytogenes CRA 197 and CRA 198 were isolated from a patient and food implicated in the milkborne outbreak (Fleming et al. 1985) and L. monocytogenes CRA 433 (NCTC 11994) was isolated from soft cheese implicated in a foodborne case of meningitis (Bannister 1987). Reference cultures were freeze-dried for storage. Routine culture and maintenance was on Nutrient Agar (Oxoid CM3) slopes stored at 4°C. In general, the bacterial strains were inoculated into Nutrient Broth (Oxoid CM1) and incubated at 30°C for 48 h. Further dilution in the same medium was used to inoculate sterile growth media to achieve final populations of 104-105 cfu/ml. In addition to incubation at 30°C for 48 h, L. monocytogenes CRA 433 was also incubated at 4°C for 6 d before inoculation. Both regimes resulted in approximately the same level of inoculum and phase (mid-late logarithmic) of growth. G R O W T H MEDIA
Chicken broth (pH ca 6.4) was prepared in bulk (approx 3 1) (Alcock 1984), dispensed in 7 ml
volumes into TGI culture tubes and autoclaved. UHT milk (pH ca 6.6) was also used and dispensed (7 ml volumes) into sterile culture tubes. The sterility of both media was tested by subculture on Plate Count Agar (Oxoid CM325) incubated at 30°C for 48 h. These were chosen as growth media as chickens (Pini & Gilbert 1988) and milk (Garayzabal et al. 1986; Lovett et al. 1987) often contain L. monocytogenes.
ASSESSMENT O F G R O W T H
A Gilson PlOO micropipette (Anachem, Luton, Bedfordshire) fitted with slim tips (Universal Extended Finntip, Jencons Scientific Ltd, Leighton Buzzard, Bedfordshire) was used to sample each culture tube at appropriate time intervals of 1-7 d. Samples were diluted in Maximum Recovery Diluent (Oxoid CM733) and enumerated in duplicate by a pour plate method on Plate Count Agar. Incubation was at 30°C for 48 h. Results The lowest temperatures that permitted growth of L. monocytogenes CRA 197, 198 and 433 in chicken broth were -0.1, -0.4 and -0.2”C respectively. At lower temperatures,, the media froze and could not be sampled. The minimum growth temperature ( - 0.2”C) was unaffected by the culture medium for L. monocytogenes CRA 433. Listeria monocytogenes CRA 433 was
Growth of L. monocytogenes unable to grow at -0.2"C in chicken broth after pre-incubation at 30°C and under these circumstances the minimum growth temperature was 0.5"C. As the incubation temperature was lowered both the lag time before growth and the generation time during the logarithmic phase increased and in general the maximum population size was reduced (Fig. 1). Similar growth curves were obtained for all the strains of L. monocytogenes. At the lowest incubation temperatures, the lag phase could exceed 30 d and the generation time 100 h (Table 1). A reduction in the temperature of incubation had an increasingly marked effect on both the lag and generation time.
159
Pre-incubation of L. monocytogenes CRA 433 at 4°C before inoculation of culture media greatly reduced the lag time particularly at low ( < 2°C) incubation temperatures (Fig. 2). At higher ( > 7°C) incubation temperatures, this effect was not evident. The pre-incubation temperature did not markedly affect the generation time or the maximum population size observed with the culture media. Similar trends were noted with both chicken broth and UHT milk, although the effect was less dramatic in the latter medium. Growth of L. monocytogenes CRA 433 was also influenced by the culture medium per se (Fig. 3). With culture in UHT milk, the lag phase was greatly reduced, particularly as the
9
8
E
\
2 7 0
m
-
6
5 0
20
10
30
50
40
Time (days)
Fig. 2. Effect of previous incubation at 30°C (open symbols) and 4°C (closed symbols) on the growth of Listeria monocytogenes CRA 433 in chicken broth when growth at 84-8.7"C (0,B)and 1.5-1.8"C (0, 0).
I
0
10
20
30
40
50
Time (days)
Fig. 3. Growth of Listeria monocytogenes CRA 433 when cultured in chicken broth (open symbols) and UHT milk (closed symbols) incubated at 8.7"Cand 1.5"C(0,and 0, respectively).
160
S . J . Walker et al.
micro-organisms approached their minimum temperature for growth (Table 1). The growth medium had a marked effect on the generation time only at temperatures less than 2"C, with more rapid growth observed in UHT milk. At higher ( > 7°C) incubation temperatures no constant effects were noted. Irrespective of preincubation treatment or incubation temperature the maximum population achieved in chicken broth was greater than in UHT milk.
Discussion Three strains of the most common serotype associated with cases of human listeriosis (serotype 4b, McLauchlin 1987) were chosen for investigation. Also all strains had been implicated in cases of foodborne listeriosis. Although other serotypes of L. monocytogenes were not tested in this study, Rosenow & Marth (1987) did not detect any significant differences between the growth rates of four strains (various serotypes) when cultured in milk at chill temperatures. The minimum growth temperature (MGT) for the three strains of L. monocytogenes ranged from -0.1 to -0.4"C. This is 1 to 1.5"C lower than the minimum (1°C) previously reported in the literature (Seeliger & Jones 1986). Growth at lower temperatures cannot be discounted as the model growth media froze at temperatures below the recorded minimum and precluded sampling. The MGT will depend also on the nutrient status of the culture medium (Ingram & Mackey 1976) and so may be lower in other situations. Michener & Elliott (1964) have suggested that the true MGT can be determined only when all other conditions are optimal. In the present study, sterile growth media were used as other micro-organisms may cause changes (eg pH change, depletion of nutrients) and so alter the growth characteristics of L. monocytogenes. A reduction in the incubation temperature of cultures resulted in a longer time before growth occurred and a slower rate of growth, which produce a series of growth curves with respect to temperature. As temperatures approached the MGT, the rate of increase in both the lag and generation times became greater. The lag and generation times observed were shorter than those reported by Rosenow & Marth (1987) for four strains of L. monocytogenes
grown in sterilized milk (i.e. lag times of 5-10 d and generation times of 29 h 44 min-45 h 33 min when incubated at 4°C). This may be due to differences between the strains used and/or growth media. In the present study strain differences were generally more pronounced as temperatures approached the minimum for growth. A reduction in the incubation temperature generally resulted in a decrease in the maximum population size of the culture. Rosenow & Marth (1987) reported that maximum stationary phase populations were obtained under optimal incubation temperature conditions. In most investigations into the growth of L. monocytogenes at low temperatures strains were grown at the optimum growth temperature before inoculation of the growth medium. Thus the population experiences a dramatic and sudden decrease in temperature which may induce 'cold shock' (Ingram & Mackey 1976). The degree of 'cold shock' is related to both the temperature difference and the rate of cooling (Ingram & Mackey 1976). In the present study L. monocytogenes CRA 433 was cultured at two different temperatures although in both instances the population was in the same growth phase and with approximately the same concentration of micro-organisms. Previous incubation of the culture at a low (4°C) temperature generally resulted in a decrease in the lag time which would indicate that the 'cold shock' phenomenon was less pronounced as compared with cultures which has been previously incubated at higher temperatures (30°C). As L. monocytogenes is common in the food processing environment (Anon 1987a; Anon 1987b; Cox et al. 1989) the microorganism may be adapted to growth at low temperatures before contamination of the food occurs. Consequently, the safety margins for food storage at low temperatures may be considerably shorter than those indicated by studies which have not considered the potential for cells to adapt to growth at low temperatures. The type of growth medium during chilled incubation also affected the lag time, with a more rapid initiation of growth in milk cf. chicken broth. Although pH does have an effect on the growth of L. monocytogenes (George et al. 1988), the growth media used had similar pH values. The differences observed most likely reflect the differing nutritional and/or inhibitory properties of the media. As the MGT was
30 30 30 4 30 4
30 30 30 4 30 4
NT, not tested; NG, No growth observed.
(ii) Generation time (h) L. monocytogenes 197 L. monocytogenes 198 L. monocytogenes 433
(i) Lag time (d) L. monocytogenes 197 L. monocytogenes 198 L. monocytogenes 433
Strain
Re-incubation temperature ("C)
Chicken broth Chicken broth Chicken broth Chicken broth UHT milk UHT milk
Chicken broth Chicken broth Chicken broth Chicken broth UHT milk UHT milk
Growth medium 0.3
77 98 NG 131 62 77
33 21 > 34 18 13 3 4
0.0
0.3
45 24 33
30
31 35
8 6 8 3 4 5 2
2.5
17 13 25 19 20 19
3 3 3 1-2 1-2 1
5.0 f 0.3
Temperature ("C)
NT NT 13 9 16 9.5
NT NT 2 1 1 t l
7.5 f 0.3
Table 1. Observed lag times (d) and generation times (h) for Listeria monocytogenes strains
0.3
NT NT 5 7 5.5 9
NT NT 1-2
