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Characterisation of Bacillus cereus isolated from milk silo tanks at eight different dairy plants
ARTICLE IN PRESS
International Dairy Journal 14 (2004) 17–27
Characterisation of Bacillus cereus isolated from milk silo tanks at eight different dairy plants Birgitta Svensson*, Kerstin Ekelund, Hiroshi Ogura, Anders Christiansson Swedish Dairy Association, Research and Development Department, Scheelevagen 18, SE-223 63 Lund, Sweden . Received 24 March 2003; accepted 7 July 2003
Abstract Raw milk was collected over 1 year from silo tanks at eight different dairy plants. On the average the level of spores and the percentage of psychrotrophic isolates was higher in summer milk (median 198 spores L–1; 48% psychrotrophs) than in winter milk (median 86 spores L–1; 35% psychrotrophs). Systematic differences in the average level of psychrotrophic spores were found between some dairies. There was an over all high diversity in the Bacillus cereus flora but the percentage of unique RAPD-patterns (fingerprints) among isolates differed between the dairies (34–54%). For all dairies mesophilic isolates with identical RAPD-patterns from up to 6 sampling occasions were found. An indication of a mesophilic in house flora was found at the dairy with 34% unique RAPD-patterns and the lowest percentage of psychrotrophic isolates (27%). In some dairies clusters of RAPD-patterns from psychrotrophic isolates were found. Eight of the RAPD-patterns were found among isolates from several of the investigated dairies indicating that some strains of B. cereus are widely spread and that certain strains may be selected for in the silo tank environment. r 2003 Elsevier Ltd. All rights reserved. Keywords: Bacillus cereus; Spores; Contamination; Dairy plant; RAPD-PCR; Raw milk; Psychrotrophic; Mesophilic
1. Introduction Bacillus cereus is a Gram-positive, spore-forming bacterium that is widely spread in the environment (Labots, Hup, & Galesloot, 1965; Slaghuis, te Giffel, Beumer, & Andre! , 1997). B. cereus is a common contaminant in raw milk (Johnston & Bruce, 1982; te Giffel, Beumer, Leijendekkers, & Rombouts, 1996b; te Giffel, Beumer, Slaghuis, & Rombouts, 1995). The spores survive pasteurisation and psychrotrophic strains of B. cereus limit the keeping quality of milk stored above 6 C (Griffiths, 1992; Phillips, Griffiths, & Muir, . Lindberg, & Molin, 1993). B. cereus is 1981; Ternstrom, also a potential food poisoning organism, that can produce several enterotoxins and an emetic toxin causing diarrhea and vomiting, respectively (Granum & Lund, 1997; Kramer & Gilbert, 1989). The highest numbers of B. cereus spores in raw milk are found during the grazing season (Slaghuis et al., 1997) mainly due to contamination of the teats by soil (Christiansson, Bertilsson, & Svensson, 1999). In earlier *Corresponding author. Fax: +46-46-13-70-40. E-mail address: [email protected] (B. Svensson). 0958-6946/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0958-6946(03)00152-3
investigations it was suggested that additional contamination of the milk occurs at the dairy plant (Coghill, 1982; Donovan, 1959; S^gaard, 1975), but lack of methods to enumerate low numbers of spores in the raw milk and lack of methods to distinguish between strains limited the understanding of how contamination of milk occurs in the dairy plant. Lately, a method to determine low numbers of B. cereus spores in raw milk by filtration has been developed (Christiansson, Ekelund, & Ogura, 1997a). This method allows isolation of strains for further typing. Several different typing methods for B. cereus have been used to try to find contamination sites in the dairy plant. Lin, Schraft, Odumeru, and Griffiths (1998) used fatty acid profiles to differentiate between different strains of B. cereus and RAPD-PCR was used by Svensson, Eneroth, Brendehaug, and Christiansson (1999) and te Giffel, Beumer, Bonestroo, and Rombouts (1996a). These investigations showed that the majority of spores are already present in raw milk on arrival at the dairy plant, but confirmed that additional contamination could occur in the dairy plant. Contamination has been shown to occur along the whole processing line. Svensson et al. (1999) found indications of a prolonged contamination problem
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Table 1 Number of B. cereus isolates and RAPD-PCR analyses made for each dairy plant
Number of isolates RAPD analysis
Dairy A
Dairy B
Dairy C
Dairy D
Dairy E
Dairy F
Dairy G
Dairy H
300 162
293 181
265 171
291 180
268 157
288 180
294 182
298 171
caused by mesophilic B. cereus strains early in the production chain in one dairy plant. The pasteuriser can also be a source of contamination (Svensson, Eneroth, Brendehaug, Molin, & Christiansson, 2000; te Giffel, Beumer, Langeveld, & Rombouts, 1997). Additional contamination of milk by B. cereus has been shown to occur in the filling machine (Eneroth, Svensson, Molin, & Christiansson, 2001). Different Bacillus species, and among them B. cereus, have been found on liquid packaging boards and blanks (Pirttij.arvi, Graeffe, & Salkinoja-Salonen, 1996; V.ais.anen, Mentu, & SalkinojaSalonen, 1991) and these could thus be an additional source of contamination. In this investigation, we have examined whether contamination of milk by B. cereus in silo tanks might be a common phenomenon, considering earlier findings (Svensson et al., 1999). Milk from eight dairy plants was analysed for B. cereus spores during 1 year and the isolates were characterised with respect to their ability to grow at low temperature. Furthermore, isolates were typed by RAPD-PCR to examine the possibility of an ‘‘in-house’’—flora in silo tanks. Two of the dairies were further studied to examine how the B. cereus flora varies from day to day.
2. Materials and methods
2.2. Determination of spore content and isolation of strains After heat treatment of the milk at 75 for 5 min the spore content was analysed by filtration through a membrane filter (Sartorius 11404-47-ACN) with 0.8 mm pore size in accordance with Christiansson et al. (1997a). The filters were incubated at 20 C for 48 h on the surface of blood agar plates (Blood agar base No. 2, Oxoid, with 10 ppm polymyxin B sulphate, Sigma Chemicals, and 5% bovine defibrinated blood, National Veterinary Institute, Uppsala, Sweden). With this procedure spores of both mesophilic and psychrotrophic B. cereus are collected and counted. The detection limit was 1–10 L1 depending on the volume that was filtrated. Colonies of B. cereus were recognised on the filter by their characteristic colony morphology and zone of haemolysis in the blood agar. The B. cereus (group) identity was confirmed by phase contrast microscopy and plating on MYP-agar (mannitol–egg yolk–phenol red agar) (Mossel, Koopman, & Jongerius, 1967). When in doubt, biochemical typing using API 50 CHB/20E system (Bio Me! rieux, France) was performed. Approximately 30 strains were isolated randomly from each dairy and sampling occasion (Table 1). In total 2297 isolates were collected. The purified isolates were stored in Nutrient Broth (Oxoid) with 20% glycerol at 80 C until further used.
2.1. Sampling at the dairies 2.3. Analysis of growth at low temperature Milk from farms is stored in silo tanks at the dairy plant until processed. Processing is usually done within 10–12 h but sometimes the milk can be stored up to 24 h before processing. The volume of a silo tank is about 100,000 L. The milk is chilled to below +4 C before storage in the silo tank. Milk samples were collected from silo tanks at eight dairy plants during 1 year. These plants were geographically widely spread in Sweden. Samples were taken once a month from August to September and from May to August (summer months) and every second month from October to April (winter months). The same silo tank from each plant was sampled all the time. A 1-L sample was collected in four 250 mL portions through a sampling cock or by a syringe through a membrane. The samples were frozen at 20 C and shipped frozen to the laboratory for analysis.
The strains were grown at 30 C overnight on plate count agar. This temperature was also used earlier during purification, to allow growth of all isolates, mesophilic as well as psychrotrophic. Bacteria from one colony were transferred to the surface of two milk agar plates containing 10% sterile skim milk and 2% agar. One plate was incubated at 30 C for 24 h as a positive control for growth on milk agar. Both psychrotrophic and mesophilic isolates will grow at this temperature. The second plate was incubated at 8 C for 7–10 days. Isolates giving visible growth at 8 C within this time were considered to be psychrotrophic. Isolates growing at 30 C but not at 8 C were regarded as mesophilic. Some of the isolates were also tested by the PCRmethod developed by Francis, Mayr, von Stetten, Stewart, and Scherer (1998). This method identifies the
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cspA gene encoding the major cold shock protein present in psychrotolerant strains. There is a good agreement between the agar plate method at 8 C and the method of Francis et al. (unpublished data). 2.4. Random amplification of polymorphic DNA polymerase chain reaction RAPD-PCR was performed as described by Nilsson, Svensson, Ekelund, and Christiansson (1998). Cells from one colony was suspended in 1 mL of sterile water, centrifuged at 12,000g for 5 min and resuspended in 100 mL sterile water with a loopful of activated carbon. The samples were kept frozen at 70 C over night and were then boiled for 10 min. Cell debris was removed by centrifugation at 12,000g for 5 min and the supernatant was used as template for RAPD. The primer used had the sequence 50 -CCGAGTCCA-30 (CyberGene, Huddinge, Sweden). The RAPD-patterns were analysed with GelCompar 4.0s (Applied Maths, Belgium) using the Pearson correlation coefficients between the densitometric traces and the clustering method of Ward (1963). The PCR products with sizes between 100 and 1400 bp were used in the calculation. The software enables data from different sampling occasions to be combined in dendrograms, and allows comparison between RAPDfingerprints of B. cereus isolates sampled over long time periods. Clusters containing identical RAPD-patterns were confirmed by visual comparison of the banding patterns. 2.5. Statistical analysis SYSTAT ver 9.0 (SPSS Inc., USA) was used to perform chi-square and Mann–Whitney’s U-test.
3. Results
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due to the large fluctuation in spore concentration within the summer and winter periods for the other dairies (Fig. 1). The highest spore concentrations for all dairies were found during the summer months. Some dairies had a very high spore content, up to 1300 L1, in the silo tank milk at some sampling occasions. Occasionally low levels, i.e. less than 100 L1, were found during the summer months. There was a difference between years as well, cf. August data for year 1 and 2. At Dairy G systematically higher spore counts (80–250 L1) were found during winter than for the other dairies. Occasional elevated spore levels (>100 L1) were found in winter for several dairies. During the 1-year study the samples were taken once a month or once every second month. This gives a limited view of the B. cereus flora in the silo tank milk, taking into account the possibility of a transient colonisation by an in-house flora. Therefore, Dairies F and G were sampled during 4 days a row for 2 weeks during winter and 2 weeks during the autumn (summer) to study the variation in RAPD-patterns from day to day. These dairies were chosen because they had very different patterns of occurrence of B. cereus in silo milk (Fig. 1). The study was performed 2 years after the 1year study. The spore content during the 2-week winter sampling period was very low in milk from both dairies (Fig. 2). Milk from Dairy F contained between 3 and 11 spores L1 (median 5), while milk from Dairy G varied between 1 and 71 spores L1 (median 10). All spore concentrations, except for one day on Dairy G, were lower than those observed during winter in the 1-year study. The spore level of the silo tank milk varied much more from day to day at Dairy G than at Dairy F during winter (Fig. 2). In the autumn (summer) sampling in Dairy F there was a steady decrease of spores in the silo tank milk from 270 to 105 L1 (median 173) (Fig. 2). Milk from Dairy G contained between 89 and 1200 spores L1 (median 302) with large fluctuations from day to day.
3.1. Spore concentration in silo tank milk 3.2. Occurrence of psychrotrophic strains The spore concentration in the silo tank milk varied at all the dairies during the year, with the lowest numbers generally occurring during the winter months (Fig. 1). The spore content during winter varied between 29 and 308 spores L1 (median 86 spores L1). During summer the spore content was between 25 and 1355 spores L1 (median 198 spores L1). The proportion of silo milk samples with more than 100 spores per litre was significantly higher during summer than during winter (chi-square, po0.001), taking into account all data from all dairies. However, when the spore content during the summer and winter months was compared for each dairy separately, only Dairy D and F showed a significant difference in spore content between summer and winter (Mann–Whitney’s U-test; po0.05). This was
The average proportion of psychrotrophic strains was different among the dairies (Fig. 1). At Dairy F and H more than half of the analysed strains were psychrotrophic, i.e. 58% and 60%, respectively. In Dairy G only 27% psychrotrophic strains were found and for the other dairies intermediate figures were found: Dairy A 34%; Dairy B 49%; Dairy C 39%; Dairy D 43% and Dairy E 37% psychrotrophs. The proportion of psychrotrophic strains was not constant over the year (Fig. 1). During the grazing season (summer) the proportion of psychrotrophic strains was higher than during the indoor season (chi-square; po0.001), taking data from all dairies. This was also true for five of the individual dairies (A, D, E, F, and G; chi-square;
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Sampling occasion 1 2 3 4 5
Aug Year1 Sep Year1 Oct Year1 Dec Year1 Feb Year2
6 Apr Year2 7 May Year2 8 Jun Year2 9 Jul Year2 10 Aug Year2
Fig. 1. Concentration of Bacillus cereus spores and the percentage of mesophilic and psychrotrophic isolates in milk from each dairy plant (A–H) at 10 sampling occasions during 1-year. Sampling occasions 3–6 are referred to as ‘‘winter’’ in the text and the remaining occasions as ‘‘summer’’.
po0.05). For Dairy C and H there was no statistically significant difference between summer and winter. Dairy B had a significantly higher proportion of psychrotrophic strains in milk during the winter. However, the fluctuation in the proportion of psychrotrophic strains in the silo tank milk was large for each dairy during both the summer and winter months. The day-to-day study confirmed that milk from Dairy F had, on the average, a higher percentage of
psychrotrophic strains than Dairy G (Fig. 2). The average proportion of psychrotrophic strains at Dairy F was 32% during the winter weeks and 92% during the autumn (summer) weeks and at Dairy G 8% during winter and 61% during autumn (summer) weeks. During the autumn weeks the proportion of psychrotrophic strains varied a lot between different days at Dairy G (Fig. 2). Interestingly, one of the days when the spore content was high (800 spores L1, September 6)
ARTICLE IN PRESS B. Svensson et al. / International Dairy Journal 14 (2004) 17–27
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Sampling day 1 2 3 4 5
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6 Apr Year2 7 May Year2 8 Jun Year2 9 Jul Year2 10 Aug Year2
Fig. 2. Results from a day-to-day study during two winter and two autumn (summer) weeks at dairies F and G. For comparison data from the 1-year study are included.
the proportion of mesophilic strains was 100%. At Dairy F no days with extremely high spore contents, i.e. >500 spores L1, were seen, but here 100% of the isolates were psychrotrophic during some days (Fig. 2). 3.3. RAPD-PCR typing of isolates from the 1-year study About two thirds of the isolated strains from each dairy plant were typed by RAPD-PCR; in total 1384 (Table 1). Depending on the dairy, between 34% and
54% of the analysed isolates were unique strains, i.e. the same RAPD banding pattern was not found for two isolates from the same dairy plant. The proportion of unique RAPD-patterns for the different dairies was: Dairy A 54%; Dairy B 34%; Dairy C 38%; Dairy D 50%; Dairy E 41%; Dairy F 53%; Dairy G 34% and Dairy H 47%. Milk from Dairy A, D, F and H had significantly higher proportions of unique RAPDpatterns than the other dairies (chi-square, po0.05). Five of the dairies (A, C, F, G and H) had significantly
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B. Svensson et al. / International Dairy Journal 14 (2004) 17–27
higher proportion of unique strains during the summer than during winter (chi-square, po0.05), while for Dairy B, D and E there was no difference in the proportion of unique strains between summer and winter (data not shown). The RAPD-patterns that were not unique were found in clusters in the dendrograms. Fig. 3 shows a dendrogram where selected RAPD-patterns of mesophilic strains isolated at Dairy G form two large clusters, and isolates from different sampling occasions are represented. Examples of unique RAPD-patterns from every sampling occasion are also shown. At all the dairies several small clusters with identical RAPDpatterns (2–4 isolates) were found (Table 2). These originated from the same, and less frequently from different sampling occasions. Isolates in clusters with more than 4 members more frequently originated from several sampling days than from only 1 day (data not shown). Large clusters (>8 members) with mesophilic isolates were found at all dairies (Table 2). The isolates came from up to six different sampling occasions at the individual dairies. The largest cluster was found in Dairy E (36 members), but very large clusters (>20 members) were also found at Dairy A, B, F and G. At dairies (E, F, G, H) large clusters (>8 members) with identical
RAPD-patterns from psychrotrophic strains isolated from up to four different sampling occasions were found (Table 2). At Dairy B a very large cluster with 21 RAPD-patterns from psychrotrophic strains isolated from one single sampling occasion was found. 3.4. RAPD-PCR typing of isolates from the day to day study The occurrence of different RAPD-patterns was studied in detail for Dairies F and G during the 2-week periods in summer and winter in year 4. The day to day study confirmed that the average proportion of isolates with unique RAPD-patterns was larger at Dairy F than at Dairy G, 64% and 36%, respectively. The spore content of the milk samples collected from Dairy F during the winter was so low that only a few strains could be isolated. All of these isolates had unique RAPD-patterns. Of the winter isolates from Dairy G only 22% had unique RAPD-patterns. Among the isolates from the autumn (summer) milk samples 60% from Dairy F and 45% from Dairy G had unique RAPD-patterns. Table 3 summarises the distribution of different RAPD-patterns of isolates from the day-to-day study. From Dairy F only psychrotrophic isolates were present in clusters. The mesophilic isolates from Dairy F were few and all of them were unique. The number of isolates in the clusters were larger at Dairy G. One of the clusters from Dairy G contained 60 isolates. All clusters with more than 10 isolates, except one, included isolates from more than one sampling day. In the largest clusters from Dairy G isolates from both winter and autumn sampling were present. For comparison, a summary of the distribution of RAPD-patterns from Dairy F and G from the 1-year study is shown in Table 4. More psychrotrophic RAPDpatterns were found among isolates from milk from Dairy F than from Dairy G, which could be due to the higher number of psychrotrophic isolates from Dairy F. For both dairies a similar distribution of small clusters was found and these usually included RAPD-patterns from isolates from the same sampling occasion. The larger clusters, with more than 10 isolates, usually comprised RAPD-patterns of mesophilic isolates from different sampling occasions. The differences between Dairy F and Dairy G were thus quite stable over several years, both regarding the proportion of psychrophic isolates and the distribution of RAPD-patterns. 3.5. Comparison of RAPD-patterns from the two studies at Dairy F and Dairy G
Fig. 3. Dendrogram of RAPD-patterns of isolates from Dairy G. Note clusters of identical mesophilic (M) isolates marked by a thick line. P: psychrotrophic isolates.
The RAPD-patterns of the isolates from Dairy F and G from the day-to-day study were compared to the
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Table 2 Number of clusters with more than one isolate from the different dairies. Distribution of isolates of B. cereus with respect to RAPD-patterns and number of isolates in clusters of different sizes in milk from silo tanks Number of isolates in each cluster
Dairy Aa
Dairy Ba
Dairy Ca
Dairy Da
Dairy Ea
Dairy Fa
Dairy Ga
Dairy Ha
2 3 4 5 6 7 8 9 10 11 14 15 16 17 19 20 21 30 31 36
4 2 1 2 1
10 3 3 2
8 (4)
10 4 2 4
3 (0) 2 (1)
7 (7) 2 (1) 1 (1)
6 (1) 2 (0) 3 (0)
8 5 1 3 2
a
(2) (2) (1) (0) (0)
(3) (1) (3) (0)
4 (1) 3 (2) 1 (0)
(2) (2) (0) (1)
1 (0) 1 (0)
2 (1)
2 1 1 1
(1) (1) (0) (1)
1 (0) 1 (0)
2 (1)
2 (2)
1 (0)
(5) (2) (1) (1) (2)
2 (0) 1 (1)
1 (0) 1 (0)
2 (1)
1 (0) 1 (1) 1 (0) 1 (0) 1 (0) 1 (0)
2 (1) 1 (0) 1 (0) 1 (0)
Number of clusters with psychrotrophic isolates in parenthesis.
Table 3 Number of clusters with more than one isolate from the day-to-day study. Distribution of RAPD-patterns into clusters for isolates from the day-today study in year 4 Number of isolates in each cluster
2 3 5 6 10 11 14 15 17 20 44 60 a
Dairy F
Dairy G a
Number of clusters
Description
Number of clusters
Descriptiona
4 4 1 1
PDA; 3 PSaA; PDA; 3 PSaA PSaA PSaA
5 1
PDWA; MDWA; PDA; MSaW; MPSaA PSaA
1
PDWA
1 1 1
PDA PDA PDA
2 1 1 1 1
MDW; PSaA MPDA MPDWA MDWA MDWA
P=psychrotrophic; M=mesophilic; D=from different sampling occasions; Sa=from the same sampling occasion; A=autumn; W=winter.
patterns in the 1-year study (data not shown). From Dairy F only two clusters were found that included patterns from both studies. One cluster contained a unique summer isolate from the 1-year study and 15 isolates from the day-to-day study (Table 3). In the other cluster a unique isolate from the autumn sampling of the day-to-day study and 6 mesophilic isolates from both winter and summer samplings in the 1-year study were found (Table 4). From Dairy G four clusters with
isolates from both studies were found. Here 31 mesophilic isolates from both summer and winter sampling in the 1-year study (Table 4) clustered together with 60 mesophilic isolates from winter and autumn sampling in the day-to-day study (Table 3)! A second cluster included 21 isolates where 17 were from two different sampling dates in the autumn sampling from day-to-day study (Table 3) and 4 were mesophilic isolates from summer and winter samples in the 1-year
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Table 4 Number of clusters with more than one isolate from the 1-year study. Distribution of RAPD-patterns into clusters for isolates from the 1-year study at dairy F and G Number of isolates in each cluster
Dairy F
2 3 4 6 9 10 11 20 30 31 a
Dairy G a
Number of clusters
Description
7 2 1 2 2
3 PSaW; 4 PSaSu PSaSu; MSaSu PSaW PSaSu; MDWSu PSaW; PDWSu
1 1
MDWSu MDWSu
Number of clusters
Descriptiona
6 2 3
MSaW; 4 MSaSu; PSaW MDWSu; MSaW MSaSu; 2 MDWSu
2 1
MDW; MDWSu PDWSu
1 1
MDWSu MDWSu
P=psyhrotrophic; M=mesophilic; D=from different sampling occasions; Sa=from the same sampling occasion; Su=summer; W=winter.
Table 5 Number of isolates with identical RAPD-patterns at the different dairies RAPD-patterna
Dairy A
Dairy B
Dairy C
Dairy D
Dairy E
Dairy F
Dairy G
Dairy H
Summary
1 2 3 4 5 6 7 8
21 19 5
21 2 12 1
13 19 22 4
10 15 13 4
7 14 40
11 20 9 6
31 30 2 3
11
2 1 5
2 4
2 1 6
14 8 4 5 3 1 2 12
128 127 107 23 8 22 8 34
M M M/P M M M P P a
5 4 1
10
M=mesophilic; P=psychrotrophic.
study. One unique isolate from summer in the 1-year study clustered with 15 mesophilic isolates from winter samples in the day-to-day study (Table 3). Another cluster contained 3 mesophilic isolates from winter and summer sampling in the 1-year study (Table 4) and 2 isolates from winter and autumn sampling in the day to day study (Table 3). The large number of strains with identical RAPD-patterns indicates existence of an inhouse flora of B. cereus at Dairy G. 3.6. Widely distributed strains of B. cereus The silo tank of Dairy G seemed to house an endemic flora of strains of B. cereus. Could these strains be found elsewhere? RAPD-patterns from the two largest clusters in Table 4, with 30 and 31 strains, respectively, were compared with the RAPD-patterns of isolates from the other 7 dairies in the 1-year study by searching the whole database of RAPD-patterns. These two RAPD-patterns were represented among the isolates from all the studied dairies (Table 5). The cluster with 31 isolates from Dairy G is found in RAPD-pattern 1 in Table 5, and the cluster with 30 isolates is included in RAPD-pattern 2.
As strains with identical RAPD-patterns could be found at all the dairies, a systematic comparison of RAPDpatterns from clusters with more than 4 isolates from different sampling occasions was made among RAPDpatterns of all isolates from every individual dairy. This resulted in eight RAPD-patterns that were present among the isolates from more than one dairy (Table 5). The members of these eight patterns represent 34% of all isolates that were RAPD-typed. The isolates in the eight groups originated in most cases from clusters, within each dairy, with representatives from several sampling occasions. Most of these groups were mesophilic isolates, but two of the groups were made up by psychrotrophic isolates. In one of the groups it was difficult to determine whether the isolates could grow on plates at low temperature or not. When tested with the PCR-method by Francis et al. (1998) these isolates were mesophilic. Some of the isolates from this group were grown in PC-broth at 7.6 C without shaking. After a lag-phase of about 5 days they grew with a generation time of 27 h (results not shown). These isolates can adapt to low temperature without the major cold shock protein encoded by the cspA gene.
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4. Discussion A problem with studies of B. cereus in raw milk has been the low level of spores and the need for enrichment. When using enrichment procedures in liquid medium, incubation at any given temperature will favour the fastest germinating spores and the strains that grow best at the incubation temperature. Thus isolates collected after enrichment will only represent part of the flora present. In contrast to enrichment procedures, the use of filtration to collect B. cereus spores from a raw milk sample does not result in selection of certain types of strains. This gives a unique possibility to study the entire flora of B. cereus spores that is present in a silo tank at the dairy. This investigation is the first to thoroughly examine the B. cereus flora in silo tank milk at the dairy silo level. The finding, that the B. cereus spore concentration and the proportion of psychrotrophic isolates in raw milk was higher in the summer than in winter, is in agreement with the results of earlier investigations (Larsen & J^rgensen, 1997; Phillips & Griffiths, 1986; te Giffel, Beumer, Slaghuis, & Rombouts, 1995). This is also in agreement with the fact that elevated levels of B. cereus are found in pasteurised milk during the grazing period. But the fluctuation in spore content between different sampling occasions at the same dairy during each season made it difficult to demonstrate a significant difference in spore content between summer and winter samples at most of the individual dairies. There are probably several reasons for the fluctuations. During the grazing period (summer) the major source of contamination of milk is dirty, soil contaminated, teats. The level of spores in milk was shown to be dependent on the water content of the soil (Christiansson et al., 1999). Thus low levels may be found during dry weather, whereas rainfall generally coincides with an increased spore level in milk. Weather dependent factors can therefore affect the spore level in silo milk in the summer. In winter the contamination of raw milk is much lower than in summer, although some farm housing systems have a higher risk for growth of B. cereus in bedding material (Christiansson, Magnusson, Nilsson, Ekelund, & Samuelsson, 1997b). In addition, contamination in the silo tank, if present, might increase the spore concentration in milk during winter-time. The finding that there were more psychrotrophic isolates in silo milk during the summer, when the cows were grazing, indicates that the psychrotrophs originate from the outdoor environment at the farm. Similar conclusions have been drawn by others (te Giffel et al., 1995). This is also supported by the fact that the number of unique isolates (RAPD-patterns) was larger during summer than in winter. This indicates that the B. cereus flora in soil is highly diverse.
25
The differences in the proportion of psychrotrophic strains between dairy plants have not been described before. It is possible that a large part of the differences reflects the B. cereus flora in the environment at the farms were the milk was produced. von Stetten, Mayr, and Scherer (1999) found that climate could influence the proportion of psychrotrophic strains in soil. But in our case the dairies with the highest proportion of psychrotrophic strains are located in different climate zones in the country. There were more psychrotrophs among the isolates from Dairy F than from Dairy G both in the 1-year study and in the day-to-day investigations 2 years later, indicating that the B. cereus flora in the silo tank or environment at the farm is rather stable. If this is the case, dairies in areas with a high proportion of psychrotrophic strains in the environment may have larger problems with the keeping quality of pasteurised milk than dairies in areas with low proportion of psychrotrophic strains; even if the level of spores in the silo milk is lower. According to the dairies participating in the study this may well be the case. One-third to half of the isolates from each dairy had unique RAPD-patterns, indicating a large diversity among B. cereus. In addition, the results indicate that the RAPD-PCR method has a high degree of resolution. Among isolates from milk at all the dairies many small clusters with isolates from the same sampling occasion were found. Small clusters with isolates from different sampling occasions were also found. The significance of these clusters is not clear. However, larger clusters with isolates from the same sampling occasion might indicate problems at a farm or a temporary cleaning problem of the silo tank at the dairy plant. Some of the dairies had larger clusters of psychrotrophic isolates with strains that were present at several sampling occasions. Such contamination obviously may affect the keeping quality of the pasteurised milk. However, large clusters of mesophilic RAPD-patterns with isolates from several sampling occasions were found in milk from all the investigated dairies. There was no connection between the average percentage of mesophilic isolates in milk and the occurrence of these clusters. This indicates that an in-house flora of mesophilic B. cereus is common in silo tanks and that selection for mesophilic strains may take place in the silo environment. A possible reason could be that mesophilic spores are more heat-resistant than psychrotrophic spores (Dufrenne, Soentoro, Tatini, Day, & Notermans, 1994) and therefore survive the cleaning of the silo tanks better. Possibly, the in-house flora might have a better ability to adhere to the surface of the tanks than other strains. But the presence of mesophilic strains could also be explained by the fact that the content of mesophilic B. cereus in the pasteurised milk is not monitored and therefore the
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dairy manufacturer is not aware of their presence in the silo tank. During winter the level of spores was generally lower, and a higher proportion of mesophilic strains was found. Under these circumstances any house flora would be more easily detected than during summer. This is reflected in the results from Dairy G, where the level of spores was higher than average, the proportion of mesophilic spores was extremely high and a majority of the isolates belonged to a few RAPD-patterns. The large fluctuation in spore levels during the winter weeks in Dairy G as compared to Dairy F could be caused by a biofilm of B. cereus in the silo tank, part of which is released at irregular intervals into the silo milk. Considering the great diversity of RAPD-patterns among the isolates, it is surprising to find that certain RAPD-patterns were widely present in all or several silo tanks of the dairies. The properties of these strains and the mechanisms that enabled them to be selected in the silo environment merit further studies. In addition, these strains must be evaluated for the presence of virulence factors such as ability to produce toxins, in order to assess any possible health risk associated with them. Psychrotrophic strains limit the keeping quality of pasteurised milk but both mesophilic and psychrotrophic strains have been shown to produce toxins and the mesophilic strains are important when milk powder is produced (Becker, Schaller, von Wiese, & Terplan, 1994). The presence of an in-house flora of mesophilic strains indicates that the cleaning system of the silo tanks may not be satisfactory. The use of RAPD-PCR for strain typing seems to be a useful tool to identify the presence of in-house strains of B. cereus in milk silo tanks at dairy plants.
Acknowledgements We like to thank the staff at the studied dairies for their help with collection of the samples. This work was financed by the Swedish dairy industry.
References Becker, H., Schaller, G., von Wiese, W., & Terplan, G. (1994). Bacillus cereus in infant foods and dried milk products. International Journal of Food Microbiology, 23, 1–15. Christiansson, A., Bertilsson, J., & Svensson, B. (1999). Bacillus cereus spores in raw milk: Factors affecting the contamination of milk during the grazing period. Journal of Dairy Science, 82, 305–314. Christiansson, A., Ekelund, K., & Ogura, H. (1997a). Membrane filtration method for enumeration and isolation of spores of Bacillus cereus from milk. International Dairy Journal, 7, 743–748. Christiansson, A., Magnusson, M., Nilsson, J., Ekelund, K., & Samuelsson, O. (1997b). The occurrence of Bacillus cereus-spores in bulk tank milk during the indoor winter season. Rep. No. 114. SLU-JBT, Alnarp, Sweden.
Coghill, D. (1982). Studies on the thermoduric psychrotrophic bacteria in South East Queensland dairy products. The Australian Journal of Dairy Technology, 37, 147–148. Donovan, K. O. (1959). The occurrence of Bacillus cereus in milk and on dairy equipment. Journal of Applied Bacteriology, 22, 131–137. Dufrenne, J., Soentoro, P., Tatini, S., Day, T., & Notermans, S. (1994). Characteristics of Bacillus cereus related to safe food production. International Journal of Food Microbiology, 23, 99–109. ( Svensson, B., Molin, G., & Christiansson, A. (2001). Eneroth, A., Contamination of pasteurised milk by Bacillus cereus in the filling machine. Journal of Dairy Research, 68, 189–196. Francis, K. P., Mayr, R., von Stetten, F., Stewart, G. S. A. B., & Scherer, S. (1998). Discrimination of psychrotrophic and mesophilic strains of the Bacillus cereus group by PCR targeting of major cold shock protein genes. Applied and Environmental Microbiology, 64, 3525–3529. Granum, P. E., & Lund, T. (1997). Bacillus cereus and its food poisoning toxins. FEMS Microbiology Letters, 157, 223–228. Griffiths, M. W. (1992). Bacillus cereus in liquid milk and other milk products. Bulletin of the International Dairy Federation, 275, 36–39. Johnston, D. W., & Bruce, J. (1982). Incidence of thermoduric psychrotrophs in milk produced in the west of Scotland. Journal of Applied Bacteriology, 52, 333–337. Kramer, J. M., & Gilbert, R. J. (1989). Bacillus cereus and other Bacillus species. In M. P. Doyle (Ed.), Foodborne bacterial pathogens (pp. 21–70). New York, Basel: Marcel Dekker, Inc. Labots, H., Hup, G., & Galesloot, T. E. (1965). Bacillus cereus in raw and pasteurised milk. III. The contamination of raw milk with B. cereus spores during its production. Netherlands Milk and Dairy Journal, 19, 191–221. Larsen, H. D., & J^rgensen, K. (1997). The occurrence of Bacillus cereus in Danish pasteurised milk. International Journal of Food Microbiology, 34, 179–186. Lin, S., Schraft, H., Odumeru, J. A., & Griffiths, M. W. (1998). Identification of contamination sources of Bacillus cereus in pasteurised milk. International Journal of Food Microbiology, 43, 159–171. Mossel, D. A. A., Koopman, M. J., & Jongerius, E. (1967). Enumeration of Bacillus cereus in food. Applied Microbiology, 15, 650–653. Nilsson, J., Svensson, B., Ekelund, K., & Christiansson, A. (1998). A RAPD-PCR method for large-scale typing of Bacillus cereus. Letters in Applied Microbiology, 27, 168–172. Phillips, J. D., & Griffiths, M. W. (1986). Factors contributing to the seasonal variation of Bacillus spp. in pasteurised dairy products. Journal of Applied Bacteriology, 61, 275–285. Phillips, J. D., Griffiths, M. W., & Muir, D. D. (1981). Growth and associated enzymic activity of spoilage bacteria in pasteurised double cream. Journal of the Society of Dairy Technology, 34, 113–118. Pirttij.arvi, T. S. M., Graeffe, T. H., & Salkinoja-Salonen, M. S. (1996). Bacterial contaminants in liquid packaging boards: Assessment of potential for food spoilage. Journal of Applied Bacteriology, 81, 445–458. Slaghuis, B. A., te Giffel, M. C., Beumer, R. R., & Andr!e, G. (1997). Effect of pasturing on the incidence of Bacillus cereus spores in raw milk. International Dairy Journal, 7, 201–205. ( Bredehaug, J., & Christiansson, A. (1999). Svensson, B., Eneroth, A., Investigation of Bacillus cereus contamination sites in a dairy plant with RAPD-PCR. International Dairy Journal, 9, 903–912. ( Brendehaug, J., Molin, G., & ChristiansSvensson, B., Eneroth, A., son, A. (2000). Involvment of a pasteuriser in the contamination of milk by Bacillus cereus in a commercial dairy plant. Journal of Dairy Research, 67, 455–460.
ARTICLE IN PRESS B. Svensson et al. / International Dairy Journal 14 (2004) 17–27 S^gaard, H. (1975). Market milk products, infected by Bacillus cereus in the dairy. Nordeuropeisk Mejeri-Tidsskrift, 7, 248–253. te Giffel, M. C., Beumer, R. R., Bonestroo, M. H., & Rombouts, F. M. (1996a). Incidence and characterization of Bacillus cereus in two dairy processing plants. Netherlands Milk and Dairy Journal, 50, 479–492. te Giffel, M. C., Beumer, R. R., Langeveld, L. P. M., & Rombouts, F. M. (1997). The role of heat exchangers in the contamination of milk with Bacillus cereus in dairy processing plants. International Journal of Dairy Technology, 50, 43–47. te Giffel, M. C., Beumer, R. R., Leijendekkers, S., & Rombouts, F. M. (1996b). Incidence of Bacillus cereus and Bacillus subtilis in foods in the Netherlands. Food Microbiology, 13, 53–58. te Giffel, M. C., Beumer, R. R., Slaghuis, B. A., & Rombouts, F. M. (1995). Occurrence and characterization of (psychrotrophic)
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Bacillus cereus on farms in the Netherlands. Netherlands Milk and Dairy Journal, 49, 125–138. . A., Lindberg, A.-M., & Molin, G. (1993). Classification of Ternstrom, the spoilage flora of raw and pasteurised bovine milk, with special reference to Pseudomonas and Bacillus. Journal of Applied Bacteriology, 75, 25–34. V.ais.anen, O. M., Mentu, J., & Salkinoja-Salonen, M. S. (1991). Bacteria in food packaging paper and board. Journal of Applied Bacteriology, 71, 130–133. von Stetten, F., Mayr, R., & Scherer, S. (1999). Climate influence on mesophilic Bacillus cereus and psychrotolerant Bacillus weihenstephanensis populations in tropical, temperate and alpine soil. Environmental Microbiology, 1, 503–515. Ward, J. H. (1963). Hierarchial grouping to optimise an objective function. Journal of the American Statistical Association, 58, 236–244.
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Characterisation of Bacillus cereus isolated from milk silo tanks at eight different dairy plants Birgitta Svensson*, Kerstin Ekelund, Hiroshi Ogura, Anders Christiansson Swedish Dairy Association, Research and Development Department, Scheelevagen 18, SE-223 63 Lund, Sweden . Received 24 March 2003; accepted 7 July 2003
Abstract Raw milk was collected over 1 year from silo tanks at eight different dairy plants. On the average the level of spores and the percentage of psychrotrophic isolates was higher in summer milk (median 198 spores L–1; 48% psychrotrophs) than in winter milk (median 86 spores L–1; 35% psychrotrophs). Systematic differences in the average level of psychrotrophic spores were found between some dairies. There was an over all high diversity in the Bacillus cereus flora but the percentage of unique RAPD-patterns (fingerprints) among isolates differed between the dairies (34–54%). For all dairies mesophilic isolates with identical RAPD-patterns from up to 6 sampling occasions were found. An indication of a mesophilic in house flora was found at the dairy with 34% unique RAPD-patterns and the lowest percentage of psychrotrophic isolates (27%). In some dairies clusters of RAPD-patterns from psychrotrophic isolates were found. Eight of the RAPD-patterns were found among isolates from several of the investigated dairies indicating that some strains of B. cereus are widely spread and that certain strains may be selected for in the silo tank environment. r 2003 Elsevier Ltd. All rights reserved. Keywords: Bacillus cereus; Spores; Contamination; Dairy plant; RAPD-PCR; Raw milk; Psychrotrophic; Mesophilic
1. Introduction Bacillus cereus is a Gram-positive, spore-forming bacterium that is widely spread in the environment (Labots, Hup, & Galesloot, 1965; Slaghuis, te Giffel, Beumer, & Andre! , 1997). B. cereus is a common contaminant in raw milk (Johnston & Bruce, 1982; te Giffel, Beumer, Leijendekkers, & Rombouts, 1996b; te Giffel, Beumer, Slaghuis, & Rombouts, 1995). The spores survive pasteurisation and psychrotrophic strains of B. cereus limit the keeping quality of milk stored above 6 C (Griffiths, 1992; Phillips, Griffiths, & Muir, . Lindberg, & Molin, 1993). B. cereus is 1981; Ternstrom, also a potential food poisoning organism, that can produce several enterotoxins and an emetic toxin causing diarrhea and vomiting, respectively (Granum & Lund, 1997; Kramer & Gilbert, 1989). The highest numbers of B. cereus spores in raw milk are found during the grazing season (Slaghuis et al., 1997) mainly due to contamination of the teats by soil (Christiansson, Bertilsson, & Svensson, 1999). In earlier *Corresponding author. Fax: +46-46-13-70-40. E-mail address: [email protected] (B. Svensson). 0958-6946/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0958-6946(03)00152-3
investigations it was suggested that additional contamination of the milk occurs at the dairy plant (Coghill, 1982; Donovan, 1959; S^gaard, 1975), but lack of methods to enumerate low numbers of spores in the raw milk and lack of methods to distinguish between strains limited the understanding of how contamination of milk occurs in the dairy plant. Lately, a method to determine low numbers of B. cereus spores in raw milk by filtration has been developed (Christiansson, Ekelund, & Ogura, 1997a). This method allows isolation of strains for further typing. Several different typing methods for B. cereus have been used to try to find contamination sites in the dairy plant. Lin, Schraft, Odumeru, and Griffiths (1998) used fatty acid profiles to differentiate between different strains of B. cereus and RAPD-PCR was used by Svensson, Eneroth, Brendehaug, and Christiansson (1999) and te Giffel, Beumer, Bonestroo, and Rombouts (1996a). These investigations showed that the majority of spores are already present in raw milk on arrival at the dairy plant, but confirmed that additional contamination could occur in the dairy plant. Contamination has been shown to occur along the whole processing line. Svensson et al. (1999) found indications of a prolonged contamination problem
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Table 1 Number of B. cereus isolates and RAPD-PCR analyses made for each dairy plant
Number of isolates RAPD analysis
Dairy A
Dairy B
Dairy C
Dairy D
Dairy E
Dairy F
Dairy G
Dairy H
300 162
293 181
265 171
291 180
268 157
288 180
294 182
298 171
caused by mesophilic B. cereus strains early in the production chain in one dairy plant. The pasteuriser can also be a source of contamination (Svensson, Eneroth, Brendehaug, Molin, & Christiansson, 2000; te Giffel, Beumer, Langeveld, & Rombouts, 1997). Additional contamination of milk by B. cereus has been shown to occur in the filling machine (Eneroth, Svensson, Molin, & Christiansson, 2001). Different Bacillus species, and among them B. cereus, have been found on liquid packaging boards and blanks (Pirttij.arvi, Graeffe, & Salkinoja-Salonen, 1996; V.ais.anen, Mentu, & SalkinojaSalonen, 1991) and these could thus be an additional source of contamination. In this investigation, we have examined whether contamination of milk by B. cereus in silo tanks might be a common phenomenon, considering earlier findings (Svensson et al., 1999). Milk from eight dairy plants was analysed for B. cereus spores during 1 year and the isolates were characterised with respect to their ability to grow at low temperature. Furthermore, isolates were typed by RAPD-PCR to examine the possibility of an ‘‘in-house’’—flora in silo tanks. Two of the dairies were further studied to examine how the B. cereus flora varies from day to day.
2. Materials and methods
2.2. Determination of spore content and isolation of strains After heat treatment of the milk at 75 for 5 min the spore content was analysed by filtration through a membrane filter (Sartorius 11404-47-ACN) with 0.8 mm pore size in accordance with Christiansson et al. (1997a). The filters were incubated at 20 C for 48 h on the surface of blood agar plates (Blood agar base No. 2, Oxoid, with 10 ppm polymyxin B sulphate, Sigma Chemicals, and 5% bovine defibrinated blood, National Veterinary Institute, Uppsala, Sweden). With this procedure spores of both mesophilic and psychrotrophic B. cereus are collected and counted. The detection limit was 1–10 L1 depending on the volume that was filtrated. Colonies of B. cereus were recognised on the filter by their characteristic colony morphology and zone of haemolysis in the blood agar. The B. cereus (group) identity was confirmed by phase contrast microscopy and plating on MYP-agar (mannitol–egg yolk–phenol red agar) (Mossel, Koopman, & Jongerius, 1967). When in doubt, biochemical typing using API 50 CHB/20E system (Bio Me! rieux, France) was performed. Approximately 30 strains were isolated randomly from each dairy and sampling occasion (Table 1). In total 2297 isolates were collected. The purified isolates were stored in Nutrient Broth (Oxoid) with 20% glycerol at 80 C until further used.
2.1. Sampling at the dairies 2.3. Analysis of growth at low temperature Milk from farms is stored in silo tanks at the dairy plant until processed. Processing is usually done within 10–12 h but sometimes the milk can be stored up to 24 h before processing. The volume of a silo tank is about 100,000 L. The milk is chilled to below +4 C before storage in the silo tank. Milk samples were collected from silo tanks at eight dairy plants during 1 year. These plants were geographically widely spread in Sweden. Samples were taken once a month from August to September and from May to August (summer months) and every second month from October to April (winter months). The same silo tank from each plant was sampled all the time. A 1-L sample was collected in four 250 mL portions through a sampling cock or by a syringe through a membrane. The samples were frozen at 20 C and shipped frozen to the laboratory for analysis.
The strains were grown at 30 C overnight on plate count agar. This temperature was also used earlier during purification, to allow growth of all isolates, mesophilic as well as psychrotrophic. Bacteria from one colony were transferred to the surface of two milk agar plates containing 10% sterile skim milk and 2% agar. One plate was incubated at 30 C for 24 h as a positive control for growth on milk agar. Both psychrotrophic and mesophilic isolates will grow at this temperature. The second plate was incubated at 8 C for 7–10 days. Isolates giving visible growth at 8 C within this time were considered to be psychrotrophic. Isolates growing at 30 C but not at 8 C were regarded as mesophilic. Some of the isolates were also tested by the PCRmethod developed by Francis, Mayr, von Stetten, Stewart, and Scherer (1998). This method identifies the
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cspA gene encoding the major cold shock protein present in psychrotolerant strains. There is a good agreement between the agar plate method at 8 C and the method of Francis et al. (unpublished data). 2.4. Random amplification of polymorphic DNA polymerase chain reaction RAPD-PCR was performed as described by Nilsson, Svensson, Ekelund, and Christiansson (1998). Cells from one colony was suspended in 1 mL of sterile water, centrifuged at 12,000g for 5 min and resuspended in 100 mL sterile water with a loopful of activated carbon. The samples were kept frozen at 70 C over night and were then boiled for 10 min. Cell debris was removed by centrifugation at 12,000g for 5 min and the supernatant was used as template for RAPD. The primer used had the sequence 50 -CCGAGTCCA-30 (CyberGene, Huddinge, Sweden). The RAPD-patterns were analysed with GelCompar 4.0s (Applied Maths, Belgium) using the Pearson correlation coefficients between the densitometric traces and the clustering method of Ward (1963). The PCR products with sizes between 100 and 1400 bp were used in the calculation. The software enables data from different sampling occasions to be combined in dendrograms, and allows comparison between RAPDfingerprints of B. cereus isolates sampled over long time periods. Clusters containing identical RAPD-patterns were confirmed by visual comparison of the banding patterns. 2.5. Statistical analysis SYSTAT ver 9.0 (SPSS Inc., USA) was used to perform chi-square and Mann–Whitney’s U-test.
3. Results
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due to the large fluctuation in spore concentration within the summer and winter periods for the other dairies (Fig. 1). The highest spore concentrations for all dairies were found during the summer months. Some dairies had a very high spore content, up to 1300 L1, in the silo tank milk at some sampling occasions. Occasionally low levels, i.e. less than 100 L1, were found during the summer months. There was a difference between years as well, cf. August data for year 1 and 2. At Dairy G systematically higher spore counts (80–250 L1) were found during winter than for the other dairies. Occasional elevated spore levels (>100 L1) were found in winter for several dairies. During the 1-year study the samples were taken once a month or once every second month. This gives a limited view of the B. cereus flora in the silo tank milk, taking into account the possibility of a transient colonisation by an in-house flora. Therefore, Dairies F and G were sampled during 4 days a row for 2 weeks during winter and 2 weeks during the autumn (summer) to study the variation in RAPD-patterns from day to day. These dairies were chosen because they had very different patterns of occurrence of B. cereus in silo milk (Fig. 1). The study was performed 2 years after the 1year study. The spore content during the 2-week winter sampling period was very low in milk from both dairies (Fig. 2). Milk from Dairy F contained between 3 and 11 spores L1 (median 5), while milk from Dairy G varied between 1 and 71 spores L1 (median 10). All spore concentrations, except for one day on Dairy G, were lower than those observed during winter in the 1-year study. The spore level of the silo tank milk varied much more from day to day at Dairy G than at Dairy F during winter (Fig. 2). In the autumn (summer) sampling in Dairy F there was a steady decrease of spores in the silo tank milk from 270 to 105 L1 (median 173) (Fig. 2). Milk from Dairy G contained between 89 and 1200 spores L1 (median 302) with large fluctuations from day to day.
3.1. Spore concentration in silo tank milk 3.2. Occurrence of psychrotrophic strains The spore concentration in the silo tank milk varied at all the dairies during the year, with the lowest numbers generally occurring during the winter months (Fig. 1). The spore content during winter varied between 29 and 308 spores L1 (median 86 spores L1). During summer the spore content was between 25 and 1355 spores L1 (median 198 spores L1). The proportion of silo milk samples with more than 100 spores per litre was significantly higher during summer than during winter (chi-square, po0.001), taking into account all data from all dairies. However, when the spore content during the summer and winter months was compared for each dairy separately, only Dairy D and F showed a significant difference in spore content between summer and winter (Mann–Whitney’s U-test; po0.05). This was
The average proportion of psychrotrophic strains was different among the dairies (Fig. 1). At Dairy F and H more than half of the analysed strains were psychrotrophic, i.e. 58% and 60%, respectively. In Dairy G only 27% psychrotrophic strains were found and for the other dairies intermediate figures were found: Dairy A 34%; Dairy B 49%; Dairy C 39%; Dairy D 43% and Dairy E 37% psychrotrophs. The proportion of psychrotrophic strains was not constant over the year (Fig. 1). During the grazing season (summer) the proportion of psychrotrophic strains was higher than during the indoor season (chi-square; po0.001), taking data from all dairies. This was also true for five of the individual dairies (A, D, E, F, and G; chi-square;
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Fig. 1. Concentration of Bacillus cereus spores and the percentage of mesophilic and psychrotrophic isolates in milk from each dairy plant (A–H) at 10 sampling occasions during 1-year. Sampling occasions 3–6 are referred to as ‘‘winter’’ in the text and the remaining occasions as ‘‘summer’’.
po0.05). For Dairy C and H there was no statistically significant difference between summer and winter. Dairy B had a significantly higher proportion of psychrotrophic strains in milk during the winter. However, the fluctuation in the proportion of psychrotrophic strains in the silo tank milk was large for each dairy during both the summer and winter months. The day-to-day study confirmed that milk from Dairy F had, on the average, a higher percentage of
psychrotrophic strains than Dairy G (Fig. 2). The average proportion of psychrotrophic strains at Dairy F was 32% during the winter weeks and 92% during the autumn (summer) weeks and at Dairy G 8% during winter and 61% during autumn (summer) weeks. During the autumn weeks the proportion of psychrotrophic strains varied a lot between different days at Dairy G (Fig. 2). Interestingly, one of the days when the spore content was high (800 spores L1, September 6)
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%
Dairy F Year
100 90 80 70 60 50 40 30 20 10 0 1
2 3
4 5
6 7
8
L-1
%
Dairy G Year
1200 100 90 1000 80 70 800 60 600 50 40 400 30 20 200 10 0 0
9 10
%
Dairy F Winter
600 400 200 0 2 3
4 5
6 7
8
9 10
Sampling occasion L-1
Dairy G Winter
% 80
60 50 40 30 20 10 0
100 90 80 70 60 50 40 30 20 10 0
7 8 9 10 14 15 16 17 Feb Feb Feb Feb Feb Feb Feb Feb
L-1 80 70 60 50 40 30 20 10
0 7 8 9 10 14 15 16 17 Feb Feb Feb Feb Feb Feb Feb Feb
Sampling day Dairy F Autumn
1200
800
1
70
%
L-1
1000
Sampling occasion 100 90 80 70 60 50 40 30 20 10 0
21
Sampling day L-1
100 90 80 70 60 50 40 30 20 10 0
% 100 1200 90 1000 80 70 800 60 50 600 40 400 30 20 200 10 0 0
28 29 30 31 4 5 6 7 Aug Aug Aug Aug Sep Sep Sep Sep
Dairy G Autumn
1200 1000 800 600 400 200 0 28 29 30 31 4 5 6 7 Aug Aug Aug Aug Sep Sep Sep Sep
Sampling day % psychrotrophic % mesophilic B.cereus L-1
L-1
Sampling day 1 2 3 4 5
Aug Year1 Sep Year1 Oct Year1 Dec Year1 Feb Year2
6 Apr Year2 7 May Year2 8 Jun Year2 9 Jul Year2 10 Aug Year2
Fig. 2. Results from a day-to-day study during two winter and two autumn (summer) weeks at dairies F and G. For comparison data from the 1-year study are included.
the proportion of mesophilic strains was 100%. At Dairy F no days with extremely high spore contents, i.e. >500 spores L1, were seen, but here 100% of the isolates were psychrotrophic during some days (Fig. 2). 3.3. RAPD-PCR typing of isolates from the 1-year study About two thirds of the isolated strains from each dairy plant were typed by RAPD-PCR; in total 1384 (Table 1). Depending on the dairy, between 34% and
54% of the analysed isolates were unique strains, i.e. the same RAPD banding pattern was not found for two isolates from the same dairy plant. The proportion of unique RAPD-patterns for the different dairies was: Dairy A 54%; Dairy B 34%; Dairy C 38%; Dairy D 50%; Dairy E 41%; Dairy F 53%; Dairy G 34% and Dairy H 47%. Milk from Dairy A, D, F and H had significantly higher proportions of unique RAPDpatterns than the other dairies (chi-square, po0.05). Five of the dairies (A, C, F, G and H) had significantly
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B. Svensson et al. / International Dairy Journal 14 (2004) 17–27
higher proportion of unique strains during the summer than during winter (chi-square, po0.05), while for Dairy B, D and E there was no difference in the proportion of unique strains between summer and winter (data not shown). The RAPD-patterns that were not unique were found in clusters in the dendrograms. Fig. 3 shows a dendrogram where selected RAPD-patterns of mesophilic strains isolated at Dairy G form two large clusters, and isolates from different sampling occasions are represented. Examples of unique RAPD-patterns from every sampling occasion are also shown. At all the dairies several small clusters with identical RAPDpatterns (2–4 isolates) were found (Table 2). These originated from the same, and less frequently from different sampling occasions. Isolates in clusters with more than 4 members more frequently originated from several sampling days than from only 1 day (data not shown). Large clusters (>8 members) with mesophilic isolates were found at all dairies (Table 2). The isolates came from up to six different sampling occasions at the individual dairies. The largest cluster was found in Dairy E (36 members), but very large clusters (>20 members) were also found at Dairy A, B, F and G. At dairies (E, F, G, H) large clusters (>8 members) with identical
RAPD-patterns from psychrotrophic strains isolated from up to four different sampling occasions were found (Table 2). At Dairy B a very large cluster with 21 RAPD-patterns from psychrotrophic strains isolated from one single sampling occasion was found. 3.4. RAPD-PCR typing of isolates from the day to day study The occurrence of different RAPD-patterns was studied in detail for Dairies F and G during the 2-week periods in summer and winter in year 4. The day to day study confirmed that the average proportion of isolates with unique RAPD-patterns was larger at Dairy F than at Dairy G, 64% and 36%, respectively. The spore content of the milk samples collected from Dairy F during the winter was so low that only a few strains could be isolated. All of these isolates had unique RAPD-patterns. Of the winter isolates from Dairy G only 22% had unique RAPD-patterns. Among the isolates from the autumn (summer) milk samples 60% from Dairy F and 45% from Dairy G had unique RAPD-patterns. Table 3 summarises the distribution of different RAPD-patterns of isolates from the day-to-day study. From Dairy F only psychrotrophic isolates were present in clusters. The mesophilic isolates from Dairy F were few and all of them were unique. The number of isolates in the clusters were larger at Dairy G. One of the clusters from Dairy G contained 60 isolates. All clusters with more than 10 isolates, except one, included isolates from more than one sampling day. In the largest clusters from Dairy G isolates from both winter and autumn sampling were present. For comparison, a summary of the distribution of RAPD-patterns from Dairy F and G from the 1-year study is shown in Table 4. More psychrotrophic RAPDpatterns were found among isolates from milk from Dairy F than from Dairy G, which could be due to the higher number of psychrotrophic isolates from Dairy F. For both dairies a similar distribution of small clusters was found and these usually included RAPD-patterns from isolates from the same sampling occasion. The larger clusters, with more than 10 isolates, usually comprised RAPD-patterns of mesophilic isolates from different sampling occasions. The differences between Dairy F and Dairy G were thus quite stable over several years, both regarding the proportion of psychrophic isolates and the distribution of RAPD-patterns. 3.5. Comparison of RAPD-patterns from the two studies at Dairy F and Dairy G
Fig. 3. Dendrogram of RAPD-patterns of isolates from Dairy G. Note clusters of identical mesophilic (M) isolates marked by a thick line. P: psychrotrophic isolates.
The RAPD-patterns of the isolates from Dairy F and G from the day-to-day study were compared to the
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Table 2 Number of clusters with more than one isolate from the different dairies. Distribution of isolates of B. cereus with respect to RAPD-patterns and number of isolates in clusters of different sizes in milk from silo tanks Number of isolates in each cluster
Dairy Aa
Dairy Ba
Dairy Ca
Dairy Da
Dairy Ea
Dairy Fa
Dairy Ga
Dairy Ha
2 3 4 5 6 7 8 9 10 11 14 15 16 17 19 20 21 30 31 36
4 2 1 2 1
10 3 3 2
8 (4)
10 4 2 4
3 (0) 2 (1)
7 (7) 2 (1) 1 (1)
6 (1) 2 (0) 3 (0)
8 5 1 3 2
a
(2) (2) (1) (0) (0)
(3) (1) (3) (0)
4 (1) 3 (2) 1 (0)
(2) (2) (0) (1)
1 (0) 1 (0)
2 (1)
2 1 1 1
(1) (1) (0) (1)
1 (0) 1 (0)
2 (1)
2 (2)
1 (0)
(5) (2) (1) (1) (2)
2 (0) 1 (1)
1 (0) 1 (0)
2 (1)
1 (0) 1 (1) 1 (0) 1 (0) 1 (0) 1 (0)
2 (1) 1 (0) 1 (0) 1 (0)
Number of clusters with psychrotrophic isolates in parenthesis.
Table 3 Number of clusters with more than one isolate from the day-to-day study. Distribution of RAPD-patterns into clusters for isolates from the day-today study in year 4 Number of isolates in each cluster
2 3 5 6 10 11 14 15 17 20 44 60 a
Dairy F
Dairy G a
Number of clusters
Description
Number of clusters
Descriptiona
4 4 1 1
PDA; 3 PSaA; PDA; 3 PSaA PSaA PSaA
5 1
PDWA; MDWA; PDA; MSaW; MPSaA PSaA
1
PDWA
1 1 1
PDA PDA PDA
2 1 1 1 1
MDW; PSaA MPDA MPDWA MDWA MDWA
P=psychrotrophic; M=mesophilic; D=from different sampling occasions; Sa=from the same sampling occasion; A=autumn; W=winter.
patterns in the 1-year study (data not shown). From Dairy F only two clusters were found that included patterns from both studies. One cluster contained a unique summer isolate from the 1-year study and 15 isolates from the day-to-day study (Table 3). In the other cluster a unique isolate from the autumn sampling of the day-to-day study and 6 mesophilic isolates from both winter and summer samplings in the 1-year study were found (Table 4). From Dairy G four clusters with
isolates from both studies were found. Here 31 mesophilic isolates from both summer and winter sampling in the 1-year study (Table 4) clustered together with 60 mesophilic isolates from winter and autumn sampling in the day-to-day study (Table 3)! A second cluster included 21 isolates where 17 were from two different sampling dates in the autumn sampling from day-to-day study (Table 3) and 4 were mesophilic isolates from summer and winter samples in the 1-year
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Table 4 Number of clusters with more than one isolate from the 1-year study. Distribution of RAPD-patterns into clusters for isolates from the 1-year study at dairy F and G Number of isolates in each cluster
Dairy F
2 3 4 6 9 10 11 20 30 31 a
Dairy G a
Number of clusters
Description
7 2 1 2 2
3 PSaW; 4 PSaSu PSaSu; MSaSu PSaW PSaSu; MDWSu PSaW; PDWSu
1 1
MDWSu MDWSu
Number of clusters
Descriptiona
6 2 3
MSaW; 4 MSaSu; PSaW MDWSu; MSaW MSaSu; 2 MDWSu
2 1
MDW; MDWSu PDWSu
1 1
MDWSu MDWSu
P=psyhrotrophic; M=mesophilic; D=from different sampling occasions; Sa=from the same sampling occasion; Su=summer; W=winter.
Table 5 Number of isolates with identical RAPD-patterns at the different dairies RAPD-patterna
Dairy A
Dairy B
Dairy C
Dairy D
Dairy E
Dairy F
Dairy G
Dairy H
Summary
1 2 3 4 5 6 7 8
21 19 5
21 2 12 1
13 19 22 4
10 15 13 4
7 14 40
11 20 9 6
31 30 2 3
11
2 1 5
2 4
2 1 6
14 8 4 5 3 1 2 12
128 127 107 23 8 22 8 34
M M M/P M M M P P a
5 4 1
10
M=mesophilic; P=psychrotrophic.
study. One unique isolate from summer in the 1-year study clustered with 15 mesophilic isolates from winter samples in the day-to-day study (Table 3). Another cluster contained 3 mesophilic isolates from winter and summer sampling in the 1-year study (Table 4) and 2 isolates from winter and autumn sampling in the day to day study (Table 3). The large number of strains with identical RAPD-patterns indicates existence of an inhouse flora of B. cereus at Dairy G. 3.6. Widely distributed strains of B. cereus The silo tank of Dairy G seemed to house an endemic flora of strains of B. cereus. Could these strains be found elsewhere? RAPD-patterns from the two largest clusters in Table 4, with 30 and 31 strains, respectively, were compared with the RAPD-patterns of isolates from the other 7 dairies in the 1-year study by searching the whole database of RAPD-patterns. These two RAPD-patterns were represented among the isolates from all the studied dairies (Table 5). The cluster with 31 isolates from Dairy G is found in RAPD-pattern 1 in Table 5, and the cluster with 30 isolates is included in RAPD-pattern 2.
As strains with identical RAPD-patterns could be found at all the dairies, a systematic comparison of RAPDpatterns from clusters with more than 4 isolates from different sampling occasions was made among RAPDpatterns of all isolates from every individual dairy. This resulted in eight RAPD-patterns that were present among the isolates from more than one dairy (Table 5). The members of these eight patterns represent 34% of all isolates that were RAPD-typed. The isolates in the eight groups originated in most cases from clusters, within each dairy, with representatives from several sampling occasions. Most of these groups were mesophilic isolates, but two of the groups were made up by psychrotrophic isolates. In one of the groups it was difficult to determine whether the isolates could grow on plates at low temperature or not. When tested with the PCR-method by Francis et al. (1998) these isolates were mesophilic. Some of the isolates from this group were grown in PC-broth at 7.6 C without shaking. After a lag-phase of about 5 days they grew with a generation time of 27 h (results not shown). These isolates can adapt to low temperature without the major cold shock protein encoded by the cspA gene.
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4. Discussion A problem with studies of B. cereus in raw milk has been the low level of spores and the need for enrichment. When using enrichment procedures in liquid medium, incubation at any given temperature will favour the fastest germinating spores and the strains that grow best at the incubation temperature. Thus isolates collected after enrichment will only represent part of the flora present. In contrast to enrichment procedures, the use of filtration to collect B. cereus spores from a raw milk sample does not result in selection of certain types of strains. This gives a unique possibility to study the entire flora of B. cereus spores that is present in a silo tank at the dairy. This investigation is the first to thoroughly examine the B. cereus flora in silo tank milk at the dairy silo level. The finding, that the B. cereus spore concentration and the proportion of psychrotrophic isolates in raw milk was higher in the summer than in winter, is in agreement with the results of earlier investigations (Larsen & J^rgensen, 1997; Phillips & Griffiths, 1986; te Giffel, Beumer, Slaghuis, & Rombouts, 1995). This is also in agreement with the fact that elevated levels of B. cereus are found in pasteurised milk during the grazing period. But the fluctuation in spore content between different sampling occasions at the same dairy during each season made it difficult to demonstrate a significant difference in spore content between summer and winter samples at most of the individual dairies. There are probably several reasons for the fluctuations. During the grazing period (summer) the major source of contamination of milk is dirty, soil contaminated, teats. The level of spores in milk was shown to be dependent on the water content of the soil (Christiansson et al., 1999). Thus low levels may be found during dry weather, whereas rainfall generally coincides with an increased spore level in milk. Weather dependent factors can therefore affect the spore level in silo milk in the summer. In winter the contamination of raw milk is much lower than in summer, although some farm housing systems have a higher risk for growth of B. cereus in bedding material (Christiansson, Magnusson, Nilsson, Ekelund, & Samuelsson, 1997b). In addition, contamination in the silo tank, if present, might increase the spore concentration in milk during winter-time. The finding that there were more psychrotrophic isolates in silo milk during the summer, when the cows were grazing, indicates that the psychrotrophs originate from the outdoor environment at the farm. Similar conclusions have been drawn by others (te Giffel et al., 1995). This is also supported by the fact that the number of unique isolates (RAPD-patterns) was larger during summer than in winter. This indicates that the B. cereus flora in soil is highly diverse.
25
The differences in the proportion of psychrotrophic strains between dairy plants have not been described before. It is possible that a large part of the differences reflects the B. cereus flora in the environment at the farms were the milk was produced. von Stetten, Mayr, and Scherer (1999) found that climate could influence the proportion of psychrotrophic strains in soil. But in our case the dairies with the highest proportion of psychrotrophic strains are located in different climate zones in the country. There were more psychrotrophs among the isolates from Dairy F than from Dairy G both in the 1-year study and in the day-to-day investigations 2 years later, indicating that the B. cereus flora in the silo tank or environment at the farm is rather stable. If this is the case, dairies in areas with a high proportion of psychrotrophic strains in the environment may have larger problems with the keeping quality of pasteurised milk than dairies in areas with low proportion of psychrotrophic strains; even if the level of spores in the silo milk is lower. According to the dairies participating in the study this may well be the case. One-third to half of the isolates from each dairy had unique RAPD-patterns, indicating a large diversity among B. cereus. In addition, the results indicate that the RAPD-PCR method has a high degree of resolution. Among isolates from milk at all the dairies many small clusters with isolates from the same sampling occasion were found. Small clusters with isolates from different sampling occasions were also found. The significance of these clusters is not clear. However, larger clusters with isolates from the same sampling occasion might indicate problems at a farm or a temporary cleaning problem of the silo tank at the dairy plant. Some of the dairies had larger clusters of psychrotrophic isolates with strains that were present at several sampling occasions. Such contamination obviously may affect the keeping quality of the pasteurised milk. However, large clusters of mesophilic RAPD-patterns with isolates from several sampling occasions were found in milk from all the investigated dairies. There was no connection between the average percentage of mesophilic isolates in milk and the occurrence of these clusters. This indicates that an in-house flora of mesophilic B. cereus is common in silo tanks and that selection for mesophilic strains may take place in the silo environment. A possible reason could be that mesophilic spores are more heat-resistant than psychrotrophic spores (Dufrenne, Soentoro, Tatini, Day, & Notermans, 1994) and therefore survive the cleaning of the silo tanks better. Possibly, the in-house flora might have a better ability to adhere to the surface of the tanks than other strains. But the presence of mesophilic strains could also be explained by the fact that the content of mesophilic B. cereus in the pasteurised milk is not monitored and therefore the
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dairy manufacturer is not aware of their presence in the silo tank. During winter the level of spores was generally lower, and a higher proportion of mesophilic strains was found. Under these circumstances any house flora would be more easily detected than during summer. This is reflected in the results from Dairy G, where the level of spores was higher than average, the proportion of mesophilic spores was extremely high and a majority of the isolates belonged to a few RAPD-patterns. The large fluctuation in spore levels during the winter weeks in Dairy G as compared to Dairy F could be caused by a biofilm of B. cereus in the silo tank, part of which is released at irregular intervals into the silo milk. Considering the great diversity of RAPD-patterns among the isolates, it is surprising to find that certain RAPD-patterns were widely present in all or several silo tanks of the dairies. The properties of these strains and the mechanisms that enabled them to be selected in the silo environment merit further studies. In addition, these strains must be evaluated for the presence of virulence factors such as ability to produce toxins, in order to assess any possible health risk associated with them. Psychrotrophic strains limit the keeping quality of pasteurised milk but both mesophilic and psychrotrophic strains have been shown to produce toxins and the mesophilic strains are important when milk powder is produced (Becker, Schaller, von Wiese, & Terplan, 1994). The presence of an in-house flora of mesophilic strains indicates that the cleaning system of the silo tanks may not be satisfactory. The use of RAPD-PCR for strain typing seems to be a useful tool to identify the presence of in-house strains of B. cereus in milk silo tanks at dairy plants.
Acknowledgements We like to thank the staff at the studied dairies for their help with collection of the samples. This work was financed by the Swedish dairy industry.
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