ORIGINAL ARTICLE. P.-Y. Cheung, K.K. Kwok and K.M. Kam. Abstract

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1 Journal of Applied Microbiology ISSN ORIGINAL ARTICLE Application of BAX system, Tecra Unique TM test, and a conventional culture method for the detection of in ready-to-eat and raw foods P.-Y. Cheung, K.K. Kwok and K.M. Kam Food and Water Laboratory, Public Health Laboratory Services Branch, Department of Health, Hong Kong Keywords BAX-PCR, food pathogen,, Tecra Unique TM. Correspondence P.-Y. Cheung, Room 704, Public Health Laboratory Centre, 382 Nam Cheong Street, Shek Kip Mei, Kowloon, Hong Kong. pycheung@dh.gov.hk 2006/1027: received 16 July 2006, revised 4 September 2006 and accepted 21 September 2006 doi: /j x Abstract Aims: To compare the BAX system, the Tecra Unique TM test, and a conventional culture method for the detection of in various foods. Methods and Results: Ready-to-eat and raw foods were inoculated with serotype Typhimurium, serotype Enteritidis, serotype Typhi, or serotype Derby. Incubated pre-enrichment cultures were examined using the BAX system, the Tecra Unique TM test, and a conventional culture method. could be detected in all ready-to-eat food samples inoculated with S. Typhimurium, S. Enteritidis, or S. Derby, with any of the three test methods. However, false negatives were obtained with the Tecra test and the culture method when samples with higher background flora were inoculated with S. Typhi. Sensitivity test results suggested the two rapid tests performed as well as the culture method in the detection of 10 1 CFU of S. Typhimurium in 25-g cooked or raw food. Conclusions: The BAX system and the Tecra Unique TM test demonstrated results comparable with those of the culture method in the detection of serotypes used except S. Typhi. Significance and Impact of the Study: This is the first evaluation of the BAX system, the Tecra Unique TM test, and a culture method in the detection of in a variety of western and oriental foods. Introduction Foodborne illnesses caused by spp. have always been a significant health problem worldwide (Baumler et al. 2000; Allos et al. 2004). Among the more than 2500 serotypes, S. Typhimurium and S. Enteritidis are the two most frequently reported serotypes (33% of isolates) from human sources in the United States [Vugia et al. 2004; Centers for Disease Control and Prevention (CDC) 2005]. In Hong Kong, 22% of reported foodpoisoning outbreaks were caused by during , and among all the isolates, S. Enteritidis, S. Typhimurium, and S. Derby had been the top three serotypes during (Lam 2005). Ready-to-eat foods available in local retail outlets are under routine surveillance for organisms and other foodborne pathogens [Food and Environmental Hygiene Department (FEHD) 2006a]. The presence of in 25 g of a sample examined is regarded as potentially hazardous to consumers, and is unacceptable for consumption (FEHD 2006b). In 2004, the food surveillance program in Hong Kong identified in eight (32%) of 25 raw meat or meat product samples, and one (7%) of 14 raw fish samples examined. Out of over 6000 cooked food samples analysed, seven (0Æ1%) were positive (Lam 2005). Both food poisoning and typhoid fever are notifiable diseases in Hong Kong. Typhoid fever is an invasive disease caused by S. Typhi. In 2000, there were 106 cases of typhoid fever reported and 45 isolations of S. Typhi (Yeung and Kam 2001). Typhoid fever can occur as a result of the consumption of contaminated food. Cote Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007)

2 Rapid methods for detection P.-Y. Cheung et al. et al. (1995) reported an outbreak owing to the consumption of potato salad prepared by an infected food handler. A local typhoid fever outbreak of 14 cases was reported to occur between November 2005 and January 2006 in Yuen Long district, Hong Kong (Ma 2006). Although a number of samples, including water, food, food handlers stool/rectal swabs, and environmental swabs taken from food premises patronized by the cases were examined, no S. Typhi could be isolated. Unlike the more ubiquitous serotypes, such as S. Enteritidis and S. Typhimurium, very few reports were available on the detection of S. Typhi in environmental or food samples with rapid methods (Kumar et al. 2006). In contrast to the conventional standard culture method [e.g. Health Protection Agency (HPA) F13], which requires three working days to generate a negative result and five working days for a confirmed positive result, more rapid and specific methods, such as the commercial automated BAX-PCR system can reduce the turnaround time (including the overnight pre-enrichment step) to about 24 h (Bailey and Cosby 2003; HPA 2003). The BAX system simplifies and streamlines the laboratory detection of in food samples by combining all the necessary reagents, such as primers, DNA polymerase, nucleotides, internal positive control, and fluorescent dye (SybrGreen), into a single lyophilized tablet in each PCR tube. This effectively reduces the time required for reagent transfers, and eliminates the potential for technical mistakes and cross-contamination. Moreover, the BAX system combines PCR with automated fluorescence detection, thus eliminating the time and labour for gel electrophoresis. Another commercialized rapid method for detection in foods is the immunoassay-based Tecra Unique TM test. This test provides the users with all the required reagents in a test module. Following h of sample pre-enrichment, samples can be applied to the test module, and then the immunoenrichment and detection steps can be carried out automatically with antibody-coated dipsticks inside the Unique Plus TM instrument. Presumptive results can be generated in <22 h, followed by confirmation of presumptive positives with culture methods (Hughes et al. 2001). Our laboratory tests a variety of ready-to-eat cooked and raw foods for on a daily basis, and the application of rapid test methods, such as BAX-PCR and Tecra Unique TM test to our routine work would enhance both productivity and analytical flexibility. Nevertheless, commercial rapid test system or test kits may not be suitable for analysing the food matrices that we encounter in routine surveillance tests. The culinary habits in Hong Kong contain diverse types of both western- and oriental-style foods, which are available conveniently in retail outlets. Evaluation studies on test methods for food pathogens seldom include oriental foods, such as, oyster sauce, sushi, or sashimi. In the present study, we evaluated the performance of the PCR-based BAX system, the immunoassay-based Tecra Unique TM test, and a conventional standard culture method for detection in naturally and artificially contaminated food matrices. Materials and methods Food samples Food samples, such as raw pork, raw poultry, and those in the nine major ready-to-eat food groups covered under the local food surveillance programme were selected (FEHD 2006b). The ready-to-eat raw or cooked food samples, and raw pork and poultry samples were purchased from local retail outlets, and delivered to the Food and Water Laboratory immediately (Table 1). Each sample was aseptically cut into small pieces and mixed well. Twenty-five-gram portions of each sample were placed in sterile stomacher bags (Seward, London, UK) and kept at )20 C until analysis. Bacterial strains and preparation of inoculum Four S. enterica serotypes were selected for this study: S. Typhimurium (ATCC 14028), S. Enteritidis (ATCC 13076), and two wild types of, S. Typhi Table 1 Aerobic mesophilic count of food samples selected for the detection of spp.* Food group Sample CFU g )1 Cooked meat Roast pork 3Æ Seafood Raw oyster 600 Grilled eel <200 Dessert Egg tart <200 Savoury Oyster sauce <200 Vegetable Cherry tomatoes Dairy Processed cheese slices <200 Ready-to-eat meals Pasta with meat <200 Fried rice with meatà <200 Sandwiches and filled rolls Egg sandwich Sushi and sashimi Salmon sashimi 600 Salmon sushià Raw meat/poultry Raw ground pork Raw ground chicken *Each sample homogenate was inoculated with serotype Typhimurium, serotype Enteritidis, serotype Typhi, or serotype Derby before pre-enrichment incubation. A replicate sample was inoculated with Enterobacter aerogenes as specificity negative control. àinoculated with S. Typhimurium only for sensitivity tests. 220 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007)

3 P.-Y. Cheung et al. Rapid methods for detection (M ) and S. Derby (M ), previously isolated by our laboratory. A strain of Enterobacter aerogenes (ATCC 13048) was used as the negative control in specificity tests. Frozen ()70 C) stock cultures of individual spp. and E. aerogenes strains preserved on Protect TM beads (Technical Service Consultants Ltd., Heywood, Lancashire, UK) were each inoculated onto Columbia blood agar (BA; Oxoid, Hampshire, UK) plates and incubated overnight at 35 C. Colonies were then transferred to tryptone water (Oxoid) and incubated at 35 C for 2 h. Each incubated culture was then used to prepare a or E. aerogenes inoculum at one McFarland density equivalent (c CFU ml )1 ) in maximum recovery diluent (MRD; Oxoid) or Butterfield s buffered phosphate diluent (BBPD) (AOAC 2002). The inocula were then decimally diluted with MRD or BBPD for the inoculation of sample homogenates. Inoculation and pre-enrichment For the analyses using the BAX system and conventional culture method, food sample portions in stomacher bags were homogenized with 225-ml buffered peptone water (BPW; Oxoid) for 1 min at 230 rev min )1 with StomacherÒ 400 (Seward, London, UK) (Fig. 1). In the specificity test, each sample homogenate was inoculated with 1 ml of a diluted inoculum, which contained 10 2 CFU of, and incubated for 20 h at 35 C (for BAX system analysis), or 37 C (for conventional culture method). A positive (BPW inoculated with 10 2 CFU of ) and a negative extraction control (uninoculated BPW) were incubated at the same conditions for BAX system analysis. The concentrations of the inocula were confirmed by plating on BA. Colonies were counted after incubation for 24 h at 35 C. For the analyses using Tecra Unique TM test kit, food samples were homogenized in modified buffered peptone water (MBPW; Tecra, Frenchs Forest, Australia) with Tecra supplement (for raw meats and vegetable samples only) (Tecra) and imbentin (for raw meats and fish samples only) (Tecra), according to the manufacturer s instruction (Fig. 1). After the inoculation with, sample homogenates were incubated for 20 h at 42 C (raw meats, fish and other seafoods, and vegetable samples) or 35 C (other samples). The homogenates of replicate egg tart samples were inoculated with 10 7 CFU of E. aerogenes as specificity negative controls, and incubated at the same conditions as BAX: Incubate at 35 C for 20 h Subculture to BHI and incubate at 37 C for 3 h Extract DNA and perform PCR with BAX system Homogenize 25 g sample in BPW (for BAX and culture method) or MBPW (for Tecra) Spike homogenates with Tecra: Incubate at 35 C or 42 C for 20 h Transfer culture to test kit module, perform test with Tecra UNIQUE Plus TM instrument Confirm presumptive positives by transferring broth from tube 3 to SC (incubate at 37 C for h) and RVS (incubate at 41 5 C for h) Culture: Incubate at 37 C for 20 h Subculture to SC (incubate at 37 C for h), and to RVS (incubate at 41 5 C for h) Figure 1 Procedure for the detection of using BAX system, Tecra Unique TM, and culture method. Abbreviations: MAC, MacConkey agar; NA, nutrient agar; XLD, xylose lysine desoxycholate agar; BGA, brilliant green agar; RVS, Rappaport-Vassiliadis soya peptone broth; DCA, desoxycholate citrate agar; SC, selenite cystine broth; BPW, buffered peptone water; MBPW, modified buffered peptone water. Subculture to XLD, BGA, or DCA (incubate at 37 C for 20 24h) Subculture typical colonies to NA and MAC Serological and biochemical confirmation Subculture to XLD, BGA, or DCA (incubate at 37 C for 20 24h) Subculture typical colonies to NA and MAC Serological and biochemical confirmation Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007)

4 Rapid methods for detection P.-Y. Cheung et al. described earlier for analyses by the BAX system, the Tecra test kit, and the conventional culture method, respectively. A set of uninoculated food sample homogenates was incubated at the same conditions, and tested for the presence of in the original selected foods. Aerobic mesophilic count of each original sample was determined using the Autoplate 4000 spiral plater (Spiral Biotech, Inc., MA, USA) according to standard method (HPA 2004). In the sensitivity tests, homogenates of the ready-to-eat meal sample (fried rice with meat) and the salmon sushi sample were inoculated with different concentrations of S. Typhimurium ( or CFU) before incubation at the same conditions as described earlier for the analyses using the BAX system, the Tecra test kit, and the conventional culture method. An uninoculated homogenate of each sample was incubated as the negative extraction control. Concentrations of the inocula were confirmed as described earlier. Detection of spp. in incubated pre-enrichment cultures using the BAX system After incubation, 10-ll pre-enrichment culture of all food samples, except meat and poultry, was transferred to 500-ll brain heart infusion broth (BHI; Oxoid) and incubated for 3 h at 37 C. The pre-enriched meat and poultry cultures, the regrown culture of other food samples, and the positive and negative extraction controls (5 ll each) were then extracted for bacterial DNA using the lysis buffer and the protease provided with the BAX system reagent package (DuPont Qualicon, Wilmington, DE, USA). Lysates (50 ll) were transferred to PCR tubes, each containing a tablet of primers, DNA polymerase, nucleotides, internal positive control, and fluorescent dye. PCR amplification, detection of products, and melting curve analysis were performed using the BAX system application software (v. 1.79) on the BAX cycler/detector (DuPont Qualicon, USA) according to the manufacturer s protocol for detecting. At the end of the test run, results of both samples and respective internal positive controls were displayed so that a true negative result could be differentiated from a failed reaction owing to PCR inhibition. PCR products with melting curve profiles showing temperature peaks specific for also indicated that the results were not false positive. Isolation and identification of spp. using conventional culture method spp. was isolated from samples using conventional standard culture method (HPA 2003). Each incubated pre-enrichment culture was subcultured to two selective enrichment broths: (i) 1-ml pre-enrichment culture was subcultured to 10-ml selenite cystine broth (SC; Difco) and incubated for h at 37 C; (ii) 100-ll pre-enrichment culture was subcultured to 10-ml Rappaport-Vassiliadis soya peptone broth (RVS; Oxoid), and incubated for h at 41Æ5 C. After incubation, both selective enrichment broths were streaked onto xylose lysine desoxycholate agar (XLD; Oxoid) and modified brilliant green agar (BGA; Oxoid) plates. As S. Typhi would not grow on solid media containing higher concentrations of brilliant green, SC broths were streaked onto XLD and desoxycholate citrate agar (DCA; Oxoid) plates in order to enhance the isolation of S. Typhi from samples. The plates were incubated for h at 37 C, and then examined for the presence of typical colonies. Typical colonies were subcultured to nutrient agar slopes (NA; Oxoid) and MacConkey (MAC; Oxoid) plates for serological tests (Remel Inc., Lenexa, KS, USA) and biochemical identification using Vitek System and GNI+ test cards (biomérieux, Marcy l Etoile, France). Detection of spp. in incubated pre-enrichment cultures using the Tecra Unique TM test kit One millilitre of the incubated pre-enrichment culture of each food sample was transferred together with an antibody-coated stick to tube 1 of the Tecra test kit module. Test for was performed using the A Protocard (v. 1.03) on the Unique Plus TM instrument (Tecra, Australia). Antibodies on the stick captured cells present in the pre-enrichment culture in tube 1. The stick was then automatically transferred to tube 3 after washing in tube 2. Tube 3 contained an enrichment broth, which allowed the replication of captured cells on the stick. The stick was then transferred to tubes 4, 5, and 6, where binding of enzyme-linked antibodies specific for, and reaction between the enzyme and substrate occurred. At the end of the test run, the presumptive results were read automatically and printed out, and could be crosschecked by manually reading the colour of the antibody-coated stick for each test sample. Following the manufacturer s instructions, presumptive positives were confirmed by transferring 100 ll of the enrichment broth from tube 3 of the kit modules to 10-ml RVS, and incubated for h at 41Æ5 C. As S. Typhi could be inhibited by the constituents of RVS (HPA 2003), for the isolation of S. Typhi, 500 ll of the tube 3 broth was also transferred to 10-ml SC, and incubated for h at 37 C. RVS broths were then streaked onto XLD and BGA, and incubated for h at 37 C; 222 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007)

5 P.-Y. Cheung et al. Rapid methods for detection Table 2 Detection of spp. using the BAX system, Tecra Unique TM test, or culture method in uninoculated food samples and samples artificially contaminated with different serotypes used for sample inoculation Uninoculated Typhimurium Enteritidis Typhi Derby Sample B T C B T C B T C B T C B T C Roast pork ) ) ) ) ) Raw oyster ) ) ) )* Grilled eel ) ) ) ) Egg tart ) ) ) Oyster sauce ) ) ) Cherry tomatoes ) ) ) ) Processed cheese slices ) ) ) Pasta with meat ) ) ) Egg sandwich ) ) ) Salmon sashimi ) ) ) )* Raw ground pork )à Raw ground chicken )à Egg tart ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) BAX-positive extraction control + na na + na na + na na + na na + na na BAX-negative extraction control (uninoculated BPW) ) na na ) na na ) na na ) na na ) na na B, BAX system results; T, Tecra Unique TM test presumptive results (before confirmation); C, culture method results; na, not applicable. +, organisms detected; ), no detectable organisms. * Typhi was isolated from the incubated broth in tube 3 of the test kit module. Typhi was not isolated from the incubated broth in tube 3 of the test kit module. à Typhi was not isolated from the culture although other serotypes might be present on selective plates. Inoculated with Enterobacter aerogenes only (specificity negative control). while SC broths were streaked onto XLD and DCA and incubated at the same conditions. Suspect colonies were subcultured to NA and MAC, and confirmed by serological and biochemical tests, respectively, as mentioned earlier. Results Detection of in ready-to-eat food samples Results of the detection of spp. in food samples using the BAX system, the Tecra Unique TM test kit, and the conventional culture method are shown in Table 2. No could be detected with the methods in any uninoculated ready-to-eat food samples, in the egg tart samples (specificity negative controls) inoculated with E. aerogenes only, or in the negative extraction controls (for BAX only). The internal positive control used in every BAX-PCR ensured that all negative reaction results were not false negatives owing to PCR inhibition. In the present study, no invalid results were produced by the BAX system. When the sample pre-enrichment cultures were artificially contaminated with 10 2 CFU of S. Typhimurium, S. Enteritidis, or S. Derby, positive results could be obtained for all samples with the three methods used. All presumptive positive samples obtained using the Tecra test kit were also confirmed positive when the enrichment broth from tube 3 of the test kit modules were subcultured. However, when the samples were inoculated with S. Typhi, four and two negative results were obtained with the Tecra test kit and the conventional culture method, respectively, although DNA could be detected in all samples with the BAX system. The Tecra test kit could not detect S. Typhi in inoculated roast pork, raw oyster, cherry tomatoes, and salmon sashimi samples. Nevertheless, when the tube 3 broth from the test kit modules were subcultured to selective enrichment medium RVS, S. Typhi could be isolated from the raw oyster and salmon sashimi samples. For the conventional culture method, S. Typhi could not be detected from inoculated roast pork and grilled eel samples. Colonies with typical S. Typhi characteristics were not observed on the XLD, DCA, and BGA plates of the roast pork samples, regardless of which selective enrichment broth was used. On the other hand, overgrowth of other bacteria was found on all selective agar plates of the grilled eel sample. Hence, no suspect S. Typhi colonies could be isolated. Visual Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007)

6 Rapid methods for detection P.-Y. Cheung et al. Sample serotypes used for sample inoculation Uninoculated* Typhimurium Enteritidis Typhi Derby Raw ground pork T Saintpaul (2) Typhimurium (2) na na Derby (2) C Weltevreden (4) Saintpaul (3) Saintpaul (2) Saintpaul (1) Derby (2) Raw ground chicken T Agona (2) na na Agona (2) Derby (2) C Agona (4) na Agona (2) na Agona (2), Derby (2) Table 3 spp. serotypes isolated from the tube 3 broth of Tecra Unique TM test kit modules and conventional enrichment cultures of naturally or artificially contaminated raw ground pork or chicken samples T, Tecra Unique TM test; C, culture method; na, data not available. *Naturally contaminated samples. Number of isolates in parentheses. analysis of the BAX system melting curve profile of samples that were BAX positive but Tecra test- or culture negative verified that the BAX results were not false positive. An oriental food sample, oyster sauce, was selected for the present study. PCR results showed that the dark brown colour of this sample did not interfere with the fluorescence signal detection of the BAX system (Table 2). The present study also indicated that samples with high fat content, such as roast pork, cheese, and raw ground pork, were not inhibitory to DNA extraction and the subsequent PCR amplification with the BAX system, as positive results could be obtained for all inoculated samples (Table 2). Detection of in raw pork and poultry Our results showed that the raw ground pork and raw ground chicken samples were naturally contaminated with spp. (Table 2). Isolates obtained from the tube 3 broths of the Tecra kit modules and from the conventional cultures of the uninoculated raw pork samples were identified as serotype Saintpaul and serotype Weltevreden, respectively (Table 3). Furthermore, both test methods also isolated serotype Agona from the uninoculated raw chicken samples (Table 3). Positive results could be obtained with the BAX system when the raw pork and chicken samples were inoculated with different serotypes (Table 2). As the system only detected DNA, the presence of which serotypes in the samples was not indicated. The Tecra test kit and the conventional culture method could also detect the presence of in the raw pork and chicken samples inoculated with S. Typhimurium, S. Enteritidis, and S. Derby (Table 2). However, S. Typhi could not be isolated from the tube 3 broth of the Tecra kit module for the S. Typhi-inoculated raw samples, although presumptive positive immunodetection results were obtained probably owing to the presence of other serotypes in the original samples. Similarly, S. Typhi could not be isolated from the inoculated raw pork and chicken samples with conventional culture method (Table 2). Isolates from the Tecra kit tube 3 enrichment broths and the conventional cultures of the inoculated raw pork and chicken samples were frequently identified as the serotypes present in the original naturally contaminated samples (Table 3). For example, S. Saintpaul and S. Agona could be isolated from the inoculated pork and chicken samples, respectively, most of the time. On the other hand, both naturally present and inoculated serotypes in the samples were isolated from S. Typhimurium-inoculated pork, and from S. Derby-inoculated chicken samples. Sensitivity of detection methods on Typhimurium-inoculated samples Results of the sensitivity test on two ready-to-eat cooked (fried rice with meat) or raw (salmon sushi) food samples using the BAX system, the Tecra test kit, and the conventional culture method are shown in Table 4. Sample homogenates were inoculated with different concentrations of S. Typhimurium before incubation. Results indicated that could be detected in all inoculated samples with any of the three methods used in the study. Negative results were obtained from all uninoculated samples and the negative extraction controls (for BAX). was detected in samples inoculated with as low as 10 1 CFU of the bacteria before incubation (i.e., 10 1 CFU per 25-g sample or c. 10 )1 CFU ml )1 sample homogenate equivalent). Discussion To date, very few reports on the detection of S. Typhi in foods by rapid methods are available, although the detection of other serovars by PCR or immunoassaybased methods have been described frequently (Hughes 224 Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007)

7 P.-Y. Cheung et al. Rapid methods for detection Table 4 Detection of serotype Typhimurium in 25 g of food samples inoculated with different concentrations of the organism before pre-enrichment Initial inoculum (CFU per 25-g sample) 0* Sample B T C B T C B T C B T C B T C B T C B T C Fried rice with meat ) ) ) Salmon sushi ) ) ) na na na na na na BAX-positive extraction control + na na + na na + na na + na na + na na + na na + na na BAX-negative extraction control (uninoculated buffered peptone water) ) na na ) na na ) na na ) na na ) na na ) na na ) na na B, BAX system results; T, Tecra Unique TM test confirmed results; C, culture method results; na, not applicable. +, organisms detected; ), No detectable organisms. *Uninoculated pre-enrichment culture of the sample. et al. 2001; Bailey and Cosby 2003; Uyttendaele et al. 2003; Cheung et al. 2004; Bansal et al. 2006; Kumar et al. 2006; Seo et al. 2006). Typhi could be recovered from the tube 3 enrichment broth of the Tecra test kit module of presumptive negative samples. This indicated that the organism was not only present in the incubated pre-enrichment cultures, but also in the tube 3 broth of the kit modules. A collaborative study conducted to evaluate the performance of Tecra Unique TM test for the detection of S. Enteritidis in dried egg also revealed that some test samples reported as negative became positive when subcultured from tube 3 broth of the modules (Hughes et al. 2001). The sensitivity limit of the Tecra test was demonstrated to be one to five cells per 25 g of sample, or cells per ml after enrichment, depending on serotypes (Tecra Unique TM test manufacturer s instructions). It was possible that the enrichment steps of the test method could not produce enough S. Typhi cells to generate a positive result in the detection step. Aerobic mesophilic count results showed that the roast pork, raw oyster, cherry tomatoes, and salmon sashimi samples contained a relatively higher number of bacteria than other ready-to-eat food samples. Background flora present in these samples might have suppressed the growth of S. Typhi in the cultures owing to competition. A study by Kumar et al. (2006) first demonstrated that a two log higher detection limit was observed in the PCR detection of S. Typhi in artificially contaminated meat rinse samples when compared with that in potable water. The authors suggested that the presence of large number of competing bacteria in foods might have restricted the growth of S. Typhi in the preenrichment step. Hanai et al. (1997) also reported that cells in the less selective pre-enrichment medium, such as peptone water, might not attain the level required for the detection in ELISA-based system (>10 6 CFU g )1 ) because of competitive microflora. On the other hand, the presence of a high number of non- bacteria in the incubated cultures might have also interfered with the detection steps of immunoassaybased tests (Hanai et al. 1997; Keith 1997; Huang et al. 2005). As the Tecra Unique TM test is marketed for testing commonly transmitted in food and S. Typhi has not been included in the validation study of the kit (personal communications, L. Gleeson, Tecra), it appears that using the BAX-PCR or the culture method is more suitable when S. Typhi is suspected to be present in samples. According to the manufacturer s instructions for the Tecra Unique TM test, the limit of sensitivity was demonstrated to be cells per ml after enrichment. A validation study for the Unique TM test on orange juice indicated that there was no significant difference between the Unique TM test and the conventional standard culture method on the detection of in 25-g samples inoculated with one to five cells (Dailianis and Hughes 2002). Hughes et al. (2001) also reported that the Tecra Unique TM test was comparable with the standard culture method in the detection of various serotypes of in five different food types (milk powder, pepper, soy flour, milk chocolate, and dried egg) inoculated with one to five cells per 25-g sample. Bailey and Cosby (2003) compared the detection of serotypes from inoculated chicken rinses and chicken hotdogs (1 250 cells per ml or g) with the BAX system and the standard cultural procedures, and concluded that the PCR system provided reliable screening. Lantz et al. (1998) reported that the rapid growth of background flora in nonselective enrichment medium interfered with the detection of target bacteria by PCR. In the present study, although the salmon sushi sample contained a relatively high aerobic mesophilic count (Table 1), it did not appear that the background flora had inhibited any of the three methods used for the Journal compilation ª 2006 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007)

8 Rapid methods for detection P.-Y. Cheung et al. detection of S. Typhimurium, even when the level of inoculum was as low as 10 1 CFU per 25-g sample. Our study included an oriental coloured food, oyster sauce, and foods that were of higher fat contents, such as roast pork, cheese, and raw ground pork. Coloured substances originally present in samples might interfere with the fluorescence signal detection of the PCR system. However, the procedure of re-growing the cultures in BHI and preparing cell lysates as DNA templates for BAX analysis can significantly dilute the effect of substances in sample matrices that might interfere with signal detection. Rossen et al. (1992) and Wilson (1997) reported that food constituents, such as organic and phenolic compounds, and fats might inhibit DNA amplification. In this study, could be detected by BAX-PCR from these particular food samples without difficulty. Our results were in congruent with the study by Bennett et al. (1998), which demonstrated that the BAX system was optimized for the detection of meat and poultry products without any inhibition. In their study, could also be isolated from the BHI cultures of samples tested negative by conventional culture method. isolated from the Tecra kit enrichment broths and the conventional cultures of the inoculated raw pork and poultry samples were mostly the serotypes present in the original naturally contaminated samples. Previous research suggested that pre-enrichment and enrichment broths, selective plates and the incubation conditions used in different detection methods might probably favour the growth and isolation of some serotypes or bacterial strains over others (Maijala et al. 1992; Vimont et al. 2006). Our results showed that most naturally contaminating cells in the raw pork and poultry samples had selective advantage over the inoculated serotypes when grown in the enrichment and culture/selective media used in the present study. Although the present study only included a limited number of food samples and matrices for the detection of a few serotypes, the results suggested that the performance of the BAX system and the Tecra test kit was comparable with that of the conventional culture method in the detection of serotypes, such as S. Typhimurium, S. Enteritidis, and S. Derby. However, when matrices containing a higher background flora were inoculated with S. Typhi, the immunoassay-based method and the conventional culture method produced false-negative results, while the BAX-PCR system was still able to detect the pathogen. To our knowledge, this is the first report on the evaluation of the BAX system, the Tecra Unique TM test, and a conventional culture method for the detection of serotypes, including S. Typhi, in a variety of western- and oriental-style ready-to-eat raw or cooked foods, and raw food commodities. Acknowledgements We are thankful to W.P. Chan for technical assistance in the laboratory. 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