Journal of Applied Microbiology 2000, 89, 884ÿ891
Comparison of three enrichment media for the isolation of Campylobacter spp. from foods C.L.Baylis1, S.MacPhee1, K.W.Martin2, T.J.Humphrey2 and R.P.Betts1 1
Campden & Chorleywood Food Research Association, Chipping Campden, and 2PHLS Food Microbiology Research Unit, Exeter, UK 383/5/00: received 30 May 2000, revised 4 August 2000 and accepted 21 August 2000 C . L . B A Y L I S , S . M A C P H E E , K . W . M A R T I N , T . J . H U M P H R E Y A N D R . P . B E T T S . 2000.
Aim: This study compared the performance of three Campylobacter enrichment broths: Bolton broth (BB), Campylobacter Enrichment broth (CEB) and Preston broth (PB). Methods and Results: Pure cultures of target and competitor organisms, and naturallycontaminated food samples, were used to establish the performance of these media. In pure culture the PB supported the growth of the greatest number of strains of Campylobacter spp. but failed to inhibit some competitor organisms. The CEB showed the opposite result, inhibiting all 15 competitor organisms used but failing to support the growth of ®ve Campylobacter strains. By comparison, BB showed the best compromise between inhibition of competitors and growth of Campylobacter. Conclusions: Plates inoculated with BB and CEB food enrichments resulted in more Campylobacter growth than those inoculated with PB, which supported signi®cantly less typical growth (P R 0001). The most common competitor organism isolated from PB was Escherichia coli, and Pseudomonas spp. were frequently isolated from BB and CEB. Both BB and CEB were better than PB for the isolation of Campylobacter from naturallycontaminated foods, although BB yielded more con®rmed Campylobacter growth than CEB. Signi®cance and Impact of the Study: This study highlighted differences in performance of media used to isolate Campylobacter spp. from foods. INTRODUCTION
Campylobacter species continue to be the most common cause of human bacterial gastroenteritis in the UK, accounting for almost 55 000 cases in England and Wales during 1999. The most important pathogenic strains belong to the group of thermotolerant campylobacters, notably Camp. jejuni, Camp. coli and to a lesser extent, Camp. lari (Grif®ths and Park 1990). These organisms are microaerophilic, requiring an oxygen-reduced atmosphere for growth. The thermophilic species grow optimally at 42 C but do not grow below 30 C, whereas the mesophilic Campylobacter spp. grow suboptimally at 42 C. Campylobacter spp. are widely distributed in the environment and are commensals in the intestinal tract of a variety of domestic and wild animals, including cattle, sheep, pigs and birds. Consequently, foods and other products derived from these animals may become contaminated with Correspondence to: Mr Christopher L. Baylis, Campden & Chorleywood Food Research Association, Chipping Campden, Gloucestershire, GL55 6LD, UK (e-mail:
[email protected]).
Campylobacter spp. and may give rise to infection. Common vehicles have included untreated or contaminated water, raw and improperly pasteurized milk, and undercooked meat, particularly poultry which is a primary source of Campylobacter spp. Cross contamination to other foods is also responsible for these foods being implicated as vehicles of infection (Grif®ths and Park 1990; Park et al. 1991). Current conventional methods for detection of Campylobacter in foods involve selective enrichment followed by plating onto selective media and biochemical con®rmation. Although enrichment media and selective agars have continued to develop during the last two decades, all still rely on antibiotics to suppress the growth of competing organisms. Typically, cefoperazone, cycloheximide, trimethoprim, rifampicin, vancomycin and polymyxin B are used in various combinations (Corry et al. 1995). In common with other food-borne pathogens, the isolation of Campylobacter from foods is often dif®cult because these bacteria may be present in low numbers, often in the presence of high numbers of competitor organisms. Furthermore, cells may also be sublethally injured by food = 2000 The Society for Applied Microbiology
CAMPYLOBACTER ENRICHMENT MEDIA
processing conditions or by intrinsic factors associated with a particular food. These sublethally-injured cells often exhibit greater sensitivity to hydrogen peroxide and photochemically-induced oxygen radicals, and to the selective agents and antibiotics, particularly rifampicin, used in traditional culture media (Ray and Johnson 1984; Humphrey 1986a,b, 1989, 1990). The effects of sublethal injury, together with the speci®c growth requirements of Campylobacter, have led to dif®culties in isolating these organisms from foods. To improve the recovery of Campylobacter spp. during enrichment, various modi®cations to existing procedures and enrichment media have been proposed. These include changes to the concentration and the combination of antibiotics and other selective agents used, the delayed introduction of selective agents or elevated incubation temperatures during incubation, and the addition of compounds that appear to reduce the toxic effects of oxygen derivatives during enrichment (Bolton et al. 1984; Humphrey 1986a,b, 1990; Humphrey and Muscat 1989; Corry et al. 1995). More recently, two media, Bolton Broth (BB, Oxoid) and Campylobacter Enrichment Broth (CEB, LabM), have been developed for the selective enrichment of Campylobacter spp. from food. These media are designed to aid both recovery of injured cells and to avoid the need for a microaerobic atmosphere. The purpose of this study was to assess the performance of BB against CEB and Preston broth (PB, Oxoid) using pure strains of different Campylobacter spp. and selected competitor organisms, as well as food samples naturally-contaminated with Campylobacter. In addition, Nutrient Broth No. 2 containing 5% lysed horse blood was used as the reference broth for the pure culture study. The PHLS Food Microbiology Research Unit (FMRU), Exeter, performed the assessment using pure cultures, and the Methods Research Group, Microbiology Department, Campden & Chorleywood Food Research Association carried out the work with naturallycontaminated samples. MATERIALS AND METHODS Enrichment broth
Bolton Broth (BB, Oxoid CM983 with supplement SR183), Preston Broth (PB) prepared with Nutrient broth No. 2 (NB, Oxoid, CM67) Preston Campylobacter Selective Supplement (Oxoid SR117) and growth supplement (Oxoid SR084), Campylobacter Enrichment Broth (CEB LabM; Lab135, with supplement X131) and NB, all containing 5% (v/v) lysed horse blood (Oxoid, SR048), were prepared according to the manufacturers instructions. The broths used for the pure culture studies were dispensed
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into universal containers such that there was a ®nal `headspace' of approximately 15 cm. Bacterial strains
The strains used for the pure culture study were all held in the culture collection of the FMRU. These included strains previously isolated from foods, chickens, human cases of disease and strains from the National Collection of Type Cultures (NCTC, Colindale, UK). Cultures were grown on Columbia blood agar base supplemented with 5% (v/v) defribrinated horse blood (CBA, Oxoid CM 331) at 37 C prior to use. The strains used in this study are shown in Table 1. Determining enrichment efficiency
All Campylobacter strains were cultured on CBA incubated for 48 h under microaerobic conditions obtained using the CampyGenTM gas generating system (Oxoid: CN25). Thermotolerant Campylobacter strains were incubated at 42 C and non-thermotolerant Campylobacter spp. at 37 C. The competitor organisms were all grown on CBA at 37 C for 24 h in aerobic conditions. A suspension of each organism was prepared in Maximum Recovery Diluent (MRD, Oxoid CM733) and then further diluted (serial decimal) to obtain ®nal suspensions containing
