APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1997, p. 775–778 0099-2240/97/$04.0010 Copyright q 1997, American Society for Microbiology
Vol. 63, No. 2
Comparison of Commercially Available Kits with Standard Methods for Detection of Salmonella Strains in Foods KATSUYUKI HANAI,1 MIKIO SATAKE,1 HISAO NAKANISHI,2
AND
KASTHURI VENKATESWARAN1*
Central Research Laboratory, Nippon Suisan Kaisha, Ltd., Hachioji City, Tokyo 192,1 and Public Health Research Institute of Kobe, Minatojima-Nakamachi, Kobe 650,2 Japan Received 3 June 1996/Accepted 25 November 1996
Six commercial kits were compared with the U.S. Food and Drug Administration (USFDA) method and the Japanese standard method for Salmonella isolation in foods. When only Salmonella serovars were tested, many of the methods performed well; however, when foods were artificially inoculated, only the USFDA method and immunomagnetic separation coupled with the xylose-lysine-brilliant green agar method (MS-XLBG) could positively detect Salmonella serovars. All seven wild-type Salmonella serovars were detected by the USFDA method, and the MS-XLBG method detected salmonellae from six samples. final concentrations of 102 to 103 CFU/ml. (ii) Pre-enriched Salmonella cultures were artificially inoculated into 250 ml of food homogenate to final concentrations of 102 to 103 CFU/ml. (iii) All food samples were subjected to Salmonella detection. Two internationally used standard procedures and six commercially available kits were compared for Salmonella recovery. The six kits used in this study were based on various principles, such as biochemical reactions (Oxoid Salmonella Rapid Test [OSRT] and Suncoli Test Paper), ELISA (Unique and Locate), immunoimmobilization (1-2 Test), and the MS-XLBG technique. Commercial sources and references for details of the methodology used are given in Table 1. Microcosms were prepared by homogenizing 25 g of analytical grade food in 250 ml of appropriate pre-enrichment medium. Lactose broth (LB; Difco Laboratories, Detroit, Mich.) for pre-enrichment and selenite cystine and tetrathionate broths (Nissui, Tokyo, Japan) for selective enrichment were used as described elsewhere for the USFDA method (1). The selectively enriched samples were streaked onto XLBG agar (6, 13) and BS agar (Difco). In the Japanese standard method, a pre-enriched culture grown in enterobacterial enrichment mannitol broth (EEM; Nissui) at 358C was selectively enriched in both selenite cystine broth (438C) and tetrathionate broth (358C) for 18 to 24 h (7) and then streaked onto DHL agar (Nissui) and MLCB-L agar (Nissui). MS-XLBG method. Samples were pre-enriched in sterile Trypticase soy broth and incubated at 358C for 6 to 16 h. Superparamagnetic, monosized, polystyrene, tosylactivated beads (diameter, 2.8 mm) coated with antisalmonella monoclonal antibodies were purchased (Dynabeads M-280; Dynal). A 20-ml volume of Dynabeads was added to 1 ml of a Trypticase soy broth pre-enriched sample and kept at room temperature for 15 min. The magnetic particles were recovered by magnetic force (MPC; Dynal) and washed twice with sterile buffer as recommended by the manufacturer (11). The resuspended magnetic beads in sterile phosphate-buffered saline were streaked onto XLBG agar and incubated at 358C for 24 h. This method is termed MS-XLBG in this communication. Although H2S-negative salmonellae were reported, only H2Sproducing salmonellae were picked and identified during this study. All black colonies from selective agar plates were picked and purified in Trypticase soy agar before being subjected to a battery of biochemical tests (13). Biolog identification system (Biolog, Hayward, Calif.) and EB-20 (Nissui) identification kits were used to identify the isolates (15). Serological typing of all
Many rapid methods have been developed for the identification of salmonella contamination (2, 8), but few have been shown to provide a high level of sensitivity by using simple, inexpensive techniques (for a review, see reference 5). Immunomagnetic separation (IMS) affords the advantage of relatively rapid antigenic capture (compared with enzyme-linked immunosorbent assay [ELISA]) via mobile suspended magnetic microbeads and has been used to easily and effectively capture and concentrate bacteria from a variety of complex biological media, including foods and feces, and environmental water samples (9, 11, 12, 18). This study was undertaken to compare six commercial methods with the U.S. Food and Drug Administration (USFDA) and Japanese standard methods for Salmonella detection in foods. In addition, we evaluated three commercial selective agar media that have been widely used for Salmonella isolation, i.e., bismuth sulfite (BS), desoxycholate (DHL), mannitol-lysine-crystal violet-brilliant green (MLCB-L) agar, and one noncommercial xylose-lysine-brilliant green (XLBG) agar, to optimize a protocol and obtain the largest number of isolates with the minimum amount of work and the lowest cost. Combination of IMS (Salmonella separation) with XLBG agar (differential isolation of organism) (MSXLBG) was used as a strategy for rapid detection and enumeration of salmonellae. Eighteen Salmonella strains representing 17 serovars, Citrobacter freundii IFO 12681, and Proteus vulgaris IFO 3045 were used in this study. All strains were either isolated by us (13) or purchased from the Institute of Fermentation, Osaka, Japan. Cultures grown in Trypticase soy broth (Eiken, Tokyo, Japan) at 358C for 24 h were serially diluted in sterile phosphate-buffered saline. Twenty food samples were obtained from various retail establishments, and bacteriological analysis was initiated within 2 to 3 h of purchase. The samples included chicken meat and meat products (14 samples), pork meat and meat products (3 samples), chicken eggs (2 samples), and spaghetti (1 sample). The performance capabilities of various methods for Salmonella detection were evaluated in three categories. (i) Salmonella cultures were grown in Trypticase soy broth overnight at 358C, and pre-enriched cultures were used as the inoculum at
* Corresponding author. Mailing address: Center for Great Lakes Studies, 600 E. Greenfield Avenue, Milwaukee, WI 53204. Phone: (414) 382-1712. Fax: (414) 382-1705. E-mail:
[email protected]. 775
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APPL. ENVIRON. MICROBIOL. TABLE 1. Methods and commercial kits employed for Salmonella isolation in this studya
Method
Principle
Preenrichment culture
Selectiveenrichment culture
Selective agar
Biochemical tests Biochemical tests Biochemical tests Biochemical tests ELISA ELISA Immunoimmobilization Imunomagnetic separation
LB EEM BPW EEC BPW BPW TT TSB
SC, TT SC, TT
XLBG, BS DHL, MLCB-L
Manufacturer
USFDA Japanese standard OSRT Suncoli Test Paper ELISA-Unique ELISA-Locate 1-2 Test MS-XLBG agar
Oxoidb Sun Chemicalsc Tecra Diagnosticsd Rhone-Poulence BioControl Systems, Inc.f
XLBG
Time taken (h)
.72 .72 48 24 72 48 48 30–40
Reference
1 13 11 10 26 18 This study
a
Abbreviations: TSB, Tryptosoy broth; BPW, buffered peptone water; SC, selenite cystine broth; TT, Hajna tetrathionate broth. Hampshire, United Kingdom. Tokyo, Japan. d Roseville, New South Wales, Australia. e Glasgow, Scotland. f Bothell, Wash. b c
identified Salmonella strains was carried out at the Tokyo Institute of Public Health, Tokyo, Japan. Various methodologies for Salmonella detection are compared in Table 2. When Salmonella cultures only were tested, the USFDA, ELISA, and MS-XLBG methods detected all of the serovars with no discrepancies. The Japanese standard method failed to differentiate the Schwarzengrund serovar. The DHL agar used for selective morphological differentiation of salmonellae in the Japanese standard method suppressed the H2S production of the Schwarzengrund serovar, thus producing grey coloration. Serovars Champaign and Krefeld were not identified by the OSRT method; likewise, the Champaign and Weltevreden serovars did not develop any detectable coloration in Suncoli Test Paper. No immunoband was noticed when serovars Champaign and Paratyphi B were inoculated in the 1-2 Test. Revival of these organisms was possible when a portion of the inoculated 1-2 Test motility chamber was streaked onto selective agar plates. All of the methods employed, except Suncoli Test Paper, differentiated nonsalmonella (Proteus spp., Citrobacter spp., etc.) organisms from salmonella serovars. When Salmonella serovars were artificially inoculated into foods, only the USFDA and MS-XLBG methods detected all of the salmonellae. As stated above, the Schwarzengrund serovar was not differentiated by the Japanese standard method; the Champaign serovar was not detected by the OSRT, Suncoli Test Paper, and 1-2 Test methods. It should also be noted that Paratyphi B isolates that did not show an immunoband produced a positive 1-2 Test result when the organism was spiked into foods. The Salmonella detection rate of the OSRT method was lower when organisms were artificially inoculated into foods (60%) than when salmonellae were tested without foods (90%). Of 20 samples tested,
9 were from an open market, 8 were from a supermarket, 2 were from a food development center, and 1 was purchased from a convenience store. Six samples of chicken meat and its products and one spaghetti sample were naturally contaminated with salmonellae. All of these seven samples were Salmonella positive by the USFDA method. The MS-XLBG method showed good performance among the rapid methods employed and isolated salmonellae from six samples; the ELISA method detected salmonellae in two samples, and the OSRT showed a positive reaction for one sample. Surprisingly, the 1-2 Test did not recover Salmonella cells from any of these naturally Salmonella-contaminated samples. Salmonella identification was done by PCR (14) for a total of 50 strains by amplifying a 163-bp fragment of the DNA replication oriC gene (16). Bacterial cells grown in Trypticase soy agar were resuspended in sterile phosphate-buffered saline to a concentration of 105 CFU/ml and used for template DNA (14). Serological characterization of salmonellae from naturally contaminated samples showed the existence of five different Salmonella serovars, namely, Champaign, Infantis, Montevideo, Schwarzengrund, and Typhimurium. Two chicken samples harbored more than one serovar. Interestingly, serovar Infantis was isolated from samples from the open market only and was isolated three times. However, samples procured from a supermarket did not exhibit such a pattern. Molecular-level studies intended to clarify the epidemiology are in progress in our laboratory. Commercial detection kits had no problem in detecting Salmonella organisms, although the detection level was lowered when Salmonella serovars were artificially inoculated into foods. Previous studies reported the influence of competitive microflora and their inhibition of Salmonella growth (13).
TABLE 2. Sensitivities of methods and commercial kits used in this study for food-borne Salmonella isolation No. (%) of samples positive for salmonellae by: Condition
Testing of Salmonella serovars only Artificial Salmonella inoculation into foods Testing of naturally Salmonellacontaminated samples a b
No. of samples tested
USFDA method
Japanese standard method
OSRT
ELISAUnique
ELISALocate
Suncoli Test Paper
Salmonella 1-2 Test
MSXLBG agar
20 20 7/20a
20 (100) 20 (100) 7 (100)
19 (95) 19 (95) 1 (14)
18 (90) 12 (60) 1 (14)
20 (100) 19 (95) 2 (29)
20 (100) 19 (95) 2 (29)
18 (90) 18 (90) —b
18 (90) 19 (95) 0
20 (100) 20 (100) 6 (86)
Of the 20 foods were tested for salmonellae, 7 were found to be naturally contaminated with salmonellae. —, Suncoli Test Paper exhibited a positive reaction for all samples.
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TABLE 3. Sensitivity of immunomagnetic beads for Salmonella isolation Isolation agar or method
XLBG
Dynabead treatment
10
104
103
102
0
No Yes No Yes No Yes
1 1 1 1 1 1
1 1 1 1 1 1
1 1 1 1 1
1 1 1 1
No Yes No Yes No Yes
1 1 1 1 1 1
1 1 1 1 1 1
1 1 1 1 1
1 1 1 1
No Yes No Yes No Yes
1 1 1 1 1 1
1 1 1 1 1 1
1 1 1 1 1 1
1 1 1 1 1 1
No
1
1
Yes No Yes No Yes
1 1 1 1 1
1 1 1 1 1
No No No No No No
105/10 104:1 107/10 106:1 108/10 107:1
104/10 103:1 105/10 104:1 108/107 10:1
103/10 102:1 104/102 102:1 108/108 1:1
102/10 10:1 103/102 10:1 107/108 8:10
6 24 DHL
0 6 24
1-2 Test
0 6 24
ELISALocate
0 6 24
Populationb Ratioc Population Ratio Population Ratio a b c
Salmonellaa isolation with initial inoculum (CFU/ml of food slurry) in range of:
Pre-enrichment incubation time (h)
0 6 24
5
101
1
1 1
1 1
10/10 1:1 102/103 1:10 107/108 1:10
1/10 1:10 10/103 1:102 106/108 1:102
1, salmonellae isolated. The numerator denotes salmonellae, and the denominator denotes food microflora population (CFU per milliliter). [Ratio of salmonellae to other microflora.
However, Salmonella detection levels varied with method employed when naturally Salmonella-contaminated samples were tested. It is possible that laboratory-grown cells differ from cells in a natural setting. This uncertainty depends on the conditions governing expression of cell surface antigens recognized by ELISA and the IMS method, as well as injury and stress factors. The EEM used as a pre-enrichment medium in the Japanese standard method had 13.5 ppm brilliant green, which might be lethal to injured organism (6). In turn, the less selective LB medium employed in the USFDA method for pre-enrichment would have resuscitated the stressed salmonellae. Although the ELISA used peptone water, a less selective pre-enrichment medium, Salmonella cells would not have attained the level required for detection (.106 CFU/g) in this system because of competitive microflora. The 10 ppm brilliant green concentration in the tetrathionate pre-enrichment medium employed in the immunoimmobilization assay would have been lethal to the salmonellae in naturally contaminated samples (6). The inability of the 1-2 Test to detect salmonellae in the presence of large numbers of competing microflora has been documented (3). A total of 109 Salmonella strains were isolated from 20 food samples for identification. The best results with regard to both sensitivity (100%) and positive predictive value (89.3%) were
obtained with XLBG agar. The performance of the other agar media used for isolation of Salmonella strains during this study was, in descending order, MLCB-L, BS, and DHL agar media. Suppression of H2S-positive nonsalmonellae which were grown along with Salmonella strains by these agar media was not possible; thus, differentiation of salmonellae was not easy. Studies have shown that commercial brilliant green agar suffers from autoclaving (4, 6, 10). However, when unheated brilliant green (7 ppm) was added to xylose lysine agar, only salmonellae grew with black-centered colonies. This was particularly impressive, since Citrobacter freundii often mimics salmonellae on commercial brilliant green agars (6, 13). The nearly complete inhibition of Proteus vulgaris and P. mirabilis on XLBG agar greatly helped colony picks for biochemical testing. Sensitivity of magnetic beads for Salmonella isolation. Salmonella serovar Typhimurium IFO 12529 was pre-enriched in Trypticase soy broth overnight at 378C and serially diluted in sterile phosphate-buffered saline. A 25-g sample of chicken meat that was free from natural Salmonella contamination was homogenized for 1 min with 225 ml of Trypticase soy broth. Bacterial suspensions of 105, 104, 103, 102, 101, 1, and 0 CFU/ml (final concentrations) of slurry were inoculated into chicken slurry microcosms (250-ml volume) and incubated at 378C for 24 h. A 10-ml portion of each sample was removed at
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0, 6, and 24 h of incubation, and serovar Typhimurium was separated from the sample by using antibody-coated magnetic beads as described above. The magnetic bead-treated and nontreated samples were streaked onto XLBG agar and DHL agar. Furthermore, the immunoimmobilization test and ELISA-Locate were also carried out to detect salmonellae. Total microflora were checked in Trypticase soy agar. All black-centered colonies in selective agars were recorded as serovar Typhimurium colonies, and other colonies were counted as nonsalmonellae (Table 3). The results showed that the Salmonella detection level was high when the food sample was pretreated with magnetic beads. However, only 10% of the salmonellae present in a sample were recovered by the IMS technique (unpublished data). This was evident with all four of the methods employed, at any pre-enrichment incubation level. Salmonella detection varied with the methods employed, even after salmonellae were separated by use of antibody-coated magnetic beads. When XLBG agar was used as a selective agar medium, a minimum of 103 CFU of Salmonella cells per g was required to isolate the organisms, or the ratio of salmonellae to other, competitive microflora in the sample was in the range of 1:10. Although DHL agar needed 103 CFU of Salmonella cells per g, isolation of the organism was made possible when the ratio of salmonellae to other, competitive microflora was in the range of 10:1. In addition, the BS and MLCB-L agar media showed Salmonella detection levels similar to that of DHL (data not shown); hence, coupling of XLBG agar with the IMS technique was found to be superior for Salmonella isolation. Of the four methods employed, the 1-2 Test showed higher recovery of Salmonella cells than ELISA, which needed a higher density of Salmonella cells in the sample for detection of the target organism. The 1-2 Test isolated salmonellae when the Salmonella population level was 102 CFU/g or when salmonellae and other food microflora were present at a ratio of 1:10. However, ELISA-Locate required a minimum of 104 CFU/g for effective Salmonella detection in a given sample. A 6-h pre-enrichment in nonselective medium was sufficient for Salmonella capture by the IMS technique, and further incubation for up to 24 h did not increase the sensitivity of the procedure. For effective Salmonella isolation, an increase in Salmonella cells was not the only criterion, since the number of competitive microfloral organisms present in the sample also played a major role. Furthermore, when Salmonella cells were separated by the use of immunomagnetic beads, competition of the targeted organism with the other microflora became less, thus increasing the efficiency of the method used for Salmonella isolation. Widjojoatmodjo et al. (16, 17) isolated salmonellae by using the IMS technique and identified the species by PCR. The sensitivity level of their assay system, combining the IMS technique and PCR, was 105 organisms per ml of spiked feces, but the MS-XLBG technique required 103 CFU/ml in spiked foods. Furthermore, our unpublished results have shown that food particles inhibit PCR amplification. We conclude that XLBG agar, in combination with the IMS technique, is an excellent procedure for Salmonella isolation and rapid differentiation of the organism from competitive microflora. This combination needs 6 h of pre-enrichment in
APPL. ENVIRON. MICROBIOL.
nonselective medium, 30 min for Salmonella capture by the IMS technique, and overnight incubation in XLBG agar for differential isolation of the organism; thus, 30 h is required for presumptive Salmonella identification in foods. This method is not only more sensitive than commercially available kits but also more specific and should therefore reduce the diagnostic laboratory workload considerably. We are grateful to Takeshi Itoh, Tokyo Institute of Public Health, for the serological typing of Salmonella strains. We are thankful to Takashi Kurusu, Akiko Murakoshi, and Yuri Kamijoh for technical assistance. REFERENCES 1. Association of Official Analytical Chemists. 1992. Bacteriological analytical manual, 7th ed. Association of Official Analytical Chemists, Arlington, Va. 2. D’Aoust, J.-Y. 1992. Commercial diagnostic kits for the detection of foodborne Salmonella, p. 9–19. Proceedings of a Conference on Salmonella and salmonellosis. Ploufragen, Saint-Brieuc, France. 3. D’Aoust, J.-Y., and A. M. Sewell. 1988. Reliability of the immunodiffusion 1-2 test system for detection of Salmonella in foods. J. Food Prot. 51:853–856. 4. Edel, W., and E. H. Kampelmacher. 1973. Comparative studies on the isolation of “sublethaly injured” salmonellae in nine European laboratories. Bull. W. H. O. 48:167–174. 5. Feng, P. 1992. Commercial assay systems for detecting foodborne salmonella. J. Food Prot. 55:927–934. 6. Hussong, D., N. K. Enkiri, and W. D. Burge. 1984. Modified agar medium for detecting environmental salmonellae by the most-probable-number method. Appl. Environ. Microbiol. 48:1026–1030. 7. Japanese Food Hygiene Association. 1990. Standard methods of analysis in food safety regulation. Ministry of Health and Welfare, Shibuya, Tokyo, Japan. 8. Joosten, H. M. L. J., W. G. F. M. van Dick, and F. van der Velde. 1994. Evaluation of motility enrichment on modified semi-solid Rappaport-Vassiliadis medium (MSRV) and automated conductance in combination with Rambach agar for Salmonella detection in environmental samples of milk powder factory. Int. J. Food Microbiol. 22:201–206. 9. Luk, J. C., and A. A. Lindberg. 1991. Rapid and sensitive detection of Salmonella (O:6,7) by immunomagnetic monoclonal antibody-based assays. J. Immunol. Methods 137:1–8. 10. Moats, W. A., J. A. Kinner, and S. E. Maddox, Jr. 1974. Effect of heat on the antimicrobial activity of brilliant green dye. Appl. Microbiol. 27:844–847. 11. Olsvik, O., T. Popovic, E. Skjerve, K. S. Cudjoe, E. Hornes, J. Ugelstad, and M. Uhlen. 1994. Magnetic separation techniques in diagnostic microbiology. Clin. Microbiol. Rev. 7:43–54. 12. Skjerve, E. L., M. Rorvik, and O. Olsvik. 1990. Detection of Listeria monocytogenes in foods by immunomagnetic separation. Appl. Environ. Microbiol. 56:3478–3481. 13. Venkateswaran, K., and H. Hashimoto. 1988. Influence of indicator bacteria on the incidence of Salmonella in aquatic environment. Nippon Suisan Gakkaishi 54:253–258. 14. Venkateswaran, K., A. Murakoshi, and M. Satake. 1996. Comparison of commercially available kits with standard methods for the detection of coliforms and Escherichia coli in foods. Appl. Environ. Microbiol. 62:2236– 2243. 15. Venkateswaran, K., A. Shimada, A. Maruyama, T. Higashihara, H. Sakou, and T. Maruyama. 1993. Microbial characteristics of Palau Jellyfish Lake. Can. J. Microbiol. 39:506–512. 16. Widjojoatmodjo, M. N., A. C. Fluit, R. Torensma, B. H. I. Keller, and J. Verhoef. 1991. Evaluation of the magnetic immuno PCR assay for rapid detection of Salmonella. Eur. J. Clin. Microbiol. Infect. Dis. 10:935–938. 17. Widjojoatmodjo, M. N., A. C. Fluit, R. Torensma, G. P. H. T. Verdonk, and J. Verhoef. 1992. The magnetic immuno polymerase chain reaction assay for direct detection of salmonellae in fecal samples. J. Clin. Microbiol. 30:3195– 3199. 18. Wright, D. J., P. A. Chapman, and C. A. Siddons. 1994. Immunomagnetic separation as a sensitive method for isolating Escherichia coli O157 from food samples. Epidemiol. Infect. 113:31–39.
