APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2003, p. 4658–4661 0099-2240/03/$08.00⫹0 DOI: 10.1128/AEM.69.8.4658–4661.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 69, No. 8
Effect of Incubation Temperature on Isolation of Campylobacter jejuni Genotypes from Foodstuffs Enriched in Preston Broth Pam Scates,1 Lynn Moran,1 and Robert H. Madden1,2* Food Microbiology Branch, Department of Agriculture and Rural Development,1 and Food Science Department, Queen’s University of Belfast,2 Belfast BT9 5PX, Northern Ireland Received 4 November 2002/Accepted 21 May 2003
Preston broth and agar incubated at either 37 or 42°C have been widely used to isolate campylobacters from foodstuffs. The consequences of using either incubation temperature were investigated. Retail packs of raw chicken (n ⴝ 24) and raw lamb liver (n ⴝ 30) were purchased. Samples were incubated in Preston broth at 37 and 42°C and then streaked onto Preston agar and incubated as before. Two Campylobacter isolates per treatment were characterized. Poultry isolates were genotyped by random amplification of polymorphic DNA (RAPD), pulsed-field gel electrophoresis (PFGE), and flagellin PCR-restriction fragment length polymorphism, and lamb isolates were genotyped by RAPD only. In total, 96% of the poultry and 73% of the lamb samples yielded campylobacters. The lamb isolates were all Campylobacter jejuni, as were 96% of the poultry isolates, with the remainder being Campylobacter lari. The incubation temperature had no significant effect on the number of positive samples or on the species isolated. However, genotyping of the C. jejuni isolates revealed profound differences in the types obtained. Overall (from poultry and lamb), the use of a single incubation temperature, 37°C, gave 56% of the total number of RAPD C. jejuni genotypes, and hence, 44% remained undetected. The effect was especially marked in the poultry samples, where incubation at 37°C gave 47% of the PFGE genotypes but 53% were exclusively recovered after incubation at 42°C. Thus, the incubation temperature of Preston media selects for certain genotypes of C. jejuni, and to detect the widest range, samples should be incubated at both 37 and 42°C. Conversely, genotyping results arising from the use of a single incubation temperature should be interpreted with caution. Campylobacter spp., especially Campylobacter jejuni and Campylobacter coli, are among the principal causes of gastroenteritis worldwide (31). Raw chicken is frequently considered to be an important source of Campylobacter spp. (1, 5), but they have also been isolated from other raw meats, such as beef, pork, and lamb, as well as cooked meats and seafood (10, 34). Procedures to isolate these organisms from foodstuffs have yet to be standardized (7), although certain media, such as Preston broth and agar (3), have been widely used (2, 19, 22, 23, 33). Incubation of these media and others has normally been conducted at 42°C to isolate the thermophilic campylobacters C. jejuni and C. coli, which were the first species found to be agents of human illness. Over recent years, there has been growing evidence that other Campylobacter spp. may also be human pathogens; however, they usually require an incubation temperature of 37°C for successful isolation and growth (8, 13). This has led to suggestions that more than one incubation temperature should be used, as well as a range of isolation media (28). It is necessary to obtain isolates of campylobacters throughout the food chain, from farms to human gastroenteritis victims, in order to determine if there is an epidemiological relationship. Campylobacter spp. isolated in epidemiological studies must then be fully characterized by the same methods in order to allow comparison.
Due to the limited biochemical activities of campylobacters, phenotyping has limited resolution; hence, genetic methods have been applied to obtain highly discriminatory typing of Campylobacter spp. (30). Pulsed-field gel electrophoresis (PFGE) has been used extensively in Campylobacter typing studies (9, 12, 27), and its discriminatory potential is well established; therefore, PFGE was used as a reference method in the study reported here. However, it is also considered to be expensive, labor-intensive, and not easily performed in routine diagnostic laboratories (26). Therefore, random amplification of polymorphic DNA (RAPD) was also used, as it is a rapid and easily performed PCR-based methodology (15) which has been successfully applied in epidemiological studies (20, 21). In addition, flagellin-based PCR-restriction fragment length polymorphism (RFLP) was used, since this technique has been widely applied to genotype campylobacters from poultry (6, 25, 27, 30). In the present study, we enriched samples of raw chicken and lamb liver in Preston media at both 37 and 42°C in order to determine if using only one incubation temperature adversely affected recoveries of Campylobacter spp., in terms of both the species and the genotypes recovered. MATERIALS AND METHODS All media used were obtained from Oxoid, Basingstoke, United Kingdom, and all chemicals were from Sigma-Aldrich, Poole, United Kingdom. Retail packs of chicken pieces were purchased in local supermarkets and transported to the laboratory, where work was initiated within 1 h of purchase. Most samples consisted of packs of legs or breasts. Legally, all packs must carry the European Community code number of the processing plant, and the packs purchased on each visit were selected to represent as wide a range of processing plants as possible. A total of 24 packs were purchased on four separate visits.
* Corresponding author. Mailing address: Food Science Division (Food Microbiology), Department of Agriculture and Rural Development, Queen’s University of Belfast, Newforge Ln., Belfast BT9 5PX, Northern Ireland. Phone: 02890 255 312. Fax: 02890 255 009. E-mail:
[email protected]. 4658
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TABLE 1. Effect of incubation temperature of Preston media on the recovery of campylobacters from raw foodstuffs Sample type
Chicken pieces (n ⫽ 24) Lamb liver (n ⫽ 30)
No. of Campylobacter-positive samples 37°C only
37 and 42°C
42°C only
2 4
19 18
2 2
Lamb liver was not as widely available and was purchased from only two local butchers, with five portions purchased on each of three visits, giving a total of 30 liver samples. In the laboratory, the samples were finely chopped with sterile scissors and thoroughly mixed, and then duplicate samples (10 g) were prepared and added to 90 ml of Preston selective broth (3). This contained (per liter) polymyxin B, 5,000 IU; rifampin, 10 mg; trimethoprim, 10 mg; and cycloheximide, 100 mg and was supplemented with iron-bisulfite-pyruvate (11). All samples were incubated microaerophilically (85% N2, 10% CO2, 5% O2) for 24 h. One sample was enriched at 37°C, and one was enriched at 42°C. A 10-l loopful of each enrichment was then streaked onto a plate of Preston selective agar and reincubated at the original temperature for up to 72 h. The plates were examined at 24-h intervals, and two typical colonies per plate were selected for purification and confirmation using standard biotyping procedures (4). Confirmed campylobacters from chicken were then characterized using three molecular-typing techniques. Lamb liver isolates were typed using RAPD only. RAPD typing was based on the method of Madden et al. (15), using a 10-mer primer (5⬘-CCTGTTAGCC-3⬘). However, Gibco BRL Taq polythermase (Life Technologies, Paisley, United Kingdom) was used with 4 mM MgCl2 and an annealing time of 1 min. For PFGE, SmaI was used as the restriction enzyme (32). PCR-RFLP typing of the flaA gene (16) was conducted using DdeI. All profiles were analyzed using Gelcompar (version 4.1) software (Applied Maths, St. Martens-Latem, Belgium).
RESULTS To simplify subsequent references to the results obtained following incubation at 37 and 42°C, they will be presented only as results obtained at the specific temperature, with the understanding that the temperature refers to the incubation temperature of the enrichment and selection procedures. Twenty-four packs of chicken pieces were analyzed, representing 15 different processing plants in the United Kingdom, France, Ireland, and Northern Ireland; therefore, the results should not be biased by sampling being limited to a small number of flocks. Overall, 93% of the packs yielded campylobacters. The distributions of positive packs at both incubation temperatures were similar (Table 1). Of the 84 poultry isolates obtained, 81 were C. jejuni (96%) and the remainder were Campylobacter lari (4%). The latter species was found in only one pack, with two isolates obtained at 37°C and one obtained at 42°C. Eighty per cent of lamb liver samples were positive (Table 1), and all 74 isolates obtained were C. jejuni. Molecular typing of the C. jejuni isolates from the rawchicken packs revealed a total of 49 distinguishable RAPD genotypes and 47 PFGE genotypes, indicating similar levels of discrimination. fla typing produced 27 genotypes. Thirty RAPD genotypes were obtained from the lamb liver isolates. The effects of temperature on the distribution of genotypes are summarized in Table 2. Overall (from poultry and lamb), the use of a single incubation temperature, 37°C, gave 56% of the total number of RAPD C. jejuni genotypes; hence, 44% remained undetected. The effect was especially marked in the poultry samples, where incubation at 37°C gave 47% of the
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TABLE 2. Effect of incubation temperature (37 or 42°C) of Preston media on the number of genotypes of C. jejuni isolated from raw foodstuffs
Sample type
Genotyping method
RAPD Chicken pieces PFGE PCR-RFLP (flaA) Lamb liver RAPD
% of genotypes found 37°C only
37 and 42°C
42°C only
43 43 19 40
2 4 33 33
55 53 48 27
Total no. of genotypes
49 47 27 30
PFGE genotypes but 53% were exclusively recovered after incubation at 42°C. Two colonies per sample were characterized, and a greater diversity of genotypes, i.e., significantly fewer identical pairs, was found at 42°C (Table 3). DISCUSSION In the study reported here, the use of Preston media incubated at 42°C resulted in 11% of the foodstuffs being reported as false negatives, while incubation at only 37°C reduced this to 7%. Thus, incubation at both temperatures would be required to maximize recovery of campylobacters using these media. This would be in accord with the observation that more than one incubation temperature should be used for the isolation of campylobacters (28), but it contradicts the suggestion (7) that incubation could take place at 37°C instead of 42 or 43°C. Considering the results from the two incubation temperatures separately, there was no significant difference in terms of the number of positive samples detected (Table 1) or in the species detected. Only one sample yielded a Campylobacter sp. other than C. jejuni, i.e., C. lari, and isolates were obtained at both incubation temperatures. However, when the C. jejuni isolates were genotyped, the incubation temperature was shown to have a very marked effect on the isolates obtained (Table 2). With the poultry samples, RAPD and PFGE showed very similar levels of discrimination (Table 2), supporting the use of the former method in these studies. With both methods, ⬍5% of the genotypes from poultry were isolated at both incubation temperatures, emphasizing how the incubation temperature selects for a very different range of genotypes from the same samples. The diversity was higher at 42°C, where more than half of all genotypes were exclusively isolated. Hence, 42°C yielded a lower number of positive samples but the majority of genotypes. TABLE 3. Distribution of identical genotypes within replicate pairs isolated from retail packs of foodstuffs incubated in Preston media at two temperatures Sample type
Chicken Lamb
Method
RAPD PFGE PCR-RFLP (flaA) RAPD
% of identical replicate isolates 37°C
42°C
68 79 88 72
42 53 68 55
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To calculate the statistical significance of the effect of the incubation temperature on the distribution of the genotypes requires knowledge of the total number of genotypes present in each sample, and that cannot be determined. However, if one considers the hypothesis that three RAPD genotypes are present in the poultry samples, then the occasions where a sample yields identical genotypes at one incubation temperature but different genotypes at the other can be studied. Twelve samples showed this pattern. Calculations showed the probability of such an event occurring by chance: P ⫽ 0.000002. Thus, the incubation temperature is seen to have a statistically highly significant effect on the distribution of the genotypes. Since genotyping of campylobacters is often used to compare isolates from animals and humans in order to elucidate the route of infection (12, 17, 18, 24, 30), it is clear that the use of an enrichment process could mean that key genotypes are not isolated due to the choice of a single incubation temperature. This observation is supported by the flaA typing results (Table 2), which exhibit a lower level of discrimination, since they are based on a single gene (26). Using only 37°C would mean almost half of the genotypes would not be detected. Contamination of poultry meat by several types of campylobacters has been reported (30); therefore, another foodstuff known to be contaminated by campylobacters, lamb liver (4), was also studied (Table 2) to see if the selective effect was unique to poultry. An English survey (14) using both enrichment and direct plating found that 73% of lamb liver samples contained campylobacters, a level similar to the 80% found in this study. Locally, lamb livers are shipped in vacuum packs holding several kilograms; hence, there is ample opportunity for cross-contamination within a batch, reducing the potential for differences between individual batches of samples. This may account for the fact that a third of RAPD genotypes were found at both temperatures. However, almost a quarter of the genotypes found were isolated exclusively at 42°C, and therefore, the incubation temperature will again be a significant factor influencing the specific genotypes found. The presence of multiple types of Campylobacter spp. in human infection has been recognized and is seen as a significant epidemiological problem (29), since diagnostic laboratories may only isolate and characterize a single colony. Thus, a potential source of an outbreak may not be identified, as the data on some of the causative organisms will be unavailable. Similarly, when C. jejuni isolates are obtained from foodstuffs using enrichment at a single temperature, certain genotypes may escape detection and frustrate epidemiological investigations. Therefore, to obtain a wide range of types it will be necessary to enrich samples at both 37 and 42°C. It is also clear that genotyping results arising from the use of a single incubation temperature should be interpreted with caution, as the studies may have been performed on a limited group of isolates. REFERENCES 1. Altekruse, S. F. 1998. Campylobacter jejuni in foods. J. Am. Vet. Med. Assoc. 213:1734–1735. 2. Baggerman, W. I. and T. Koster. 1992. A comparison of enrichment and membrane filtration methods for the isolation of campylobacter from fresh and frozen foods. Food Microbiol. 9:87–94. 3. Bolton, F. J., and L. Robertson. 1982. A selective medium for isolation of Campylobacter jejuni/coli. J. Clin. Pathol. 35:462–467.
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