Food Microbiology 26 (2009) 317319

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Food Microbiology
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Comparison of culture media for enrichment and isolation of Salmonella spp. from frozen Channel catsh and Vietnamese basa llets
Amit Pal a, Douglas L. Marshall b, *
a b

Campbell Soup Company, 1 Campbell Place, Camden, NJ 08103, USA College of Natural and Health Sciences, University of Northern Colorado, Gunter 1000, Box 134, Greeley, CO 80639, USA

a r t i c l e i n f o
Article history: Received 21 February 2008 Received in revised form 10 December 2008 Accepted 11 December 2008 Available online 31 December 2008 Keywords: Salmonella Catsh Basa Isolation methods

a b s t r a c t
Frozen llets of Channel catsh and Vietnamese basa sh were used to compare Salmonella spp. recovery effectiveness of selective enrichment in RappaportVassiliadis (RV) broth and tetrathionate broth (TT) and selective isolation on Hekteon enteric (HE) agar, xylose lysine deoxycholate (XLD) agar, and bismuth sulte (BS) agar. Isolate conrmation was through fatty acid methyl ester analysis. Of 60 samples analyzed, 25 were found contaminated with Salmonella (42% incidence). Salmonella spp. recovery after enrichment in RV medium was 35% on HE agar, 30% on XLD agar, and 42% on BS agar. Similarly, after enrichment in TT broth, HE and XLD agars recovered 22% each and BS agar recovered 37%. No performance difference (p > 0.05) was observed in the recovery of Salmonella using the combinations of BS, HE, and XLD agars with RV broth and BS agar with TT broth. The combination of selective enrichment in RV and selective isolation on BS gave numerically greatest isolation of Salmonella from Channel catsh and Vietnamese basa sh compared to other isolation combinations. 2008 Elsevier Ltd. All rights reserved.

1. Introduction Salmonella has been previously isolated from catsh (Andrews et al., 1977; Wyatt et al., 1979; Hannah and McCaskey, 1995), and Channel catsh (Ictalurus punctatus) has been responsible for one human salmonellosis outbreak (CDC, 1991). The primary habitat of Salmonella is in the intestinal tract of animals, such as birds, reptiles, farm animals, humans, and occasionally insects (FloresAbuxapqui et al., 2003). Berg and Anderson (1972) determined that avian fecal material is a primary source of Salmonella in sh products. The potential source of Salmonella contamination in farm-raised catsh is likely due to poor water quality, farm runoff, fecal contamination from wild animals or livestock, feed (Ward, 1989; Gonzalez-Rodrguez et al., 2002), processing under poor sanitary conditions (DAoust et al., 1992), or poor distribution, retail marketing, and handling/preparation practices (Zhao et al., 2003). Wyatt et al. (1979) found that high stocking densities and high water temperature may be responsible for increased Salmonella contamination of farm-raised catsh. Salmonella has been isolated not only from domestic catsh but also from imported sh and sh products, including catsh (DAoust et al., 1992; Heinitz et al., 2000; Zhao et al., 2003). Widespread distribution of antibiotic-resistant Salmonella strains
* Corresponding author. Tel.: 1 970 351 2877; fax: 1 970 351 2176. E-mail address: douglas.marshall@unco.edu (D.L. Marshall). 0740-0020/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2008.12.003

due to international trade of Salmonella-contaminated seafood products (DAoust et al., 1992; Zhao et al., 2003; Ponce et al., 2008) is a public health concern. Survey studies in the USA have shown a low incidence (1.54.5%) of Salmonella contamination in retail catsh llets (Andrews et al., 1977; Hannah and McCaskey, 1995; Heinitz et al., 2000). Low Salmonella incidence was also seen with other U.S. seafood products (1.3% of 768 samples) (Heinitz et al., 2000). In contrast, Salmonella incidence in imported seafood products was greater (7.2% of 11,312 samples). Among all seafood products examined raw sh had the greatest Salmonella incidence, with Vietnamese seafood products having the greatest Salmonella incidence of all the import products tested (Heinitz et al., 2000). The U.S. Food and Drug Administration method for Salmonella isolation from sh and sh products recommends pre-enrichment with lactose broth (LB), followed by selective enrichment in RappaportVassiliadis (RV) broth and tetrathionate broth (TT), and then selective isolation of typical and atypical Salmonella colonies on Hekteon enteric (HE) agar, xylose lysine deoxycholate (XLD) agar, and bismuth sulte (BS) agar (Andrews and Hammack, 2006). Since the above protocol requires multiple enrichment and isolation media, the effort of Salmonella isolation from sh could be minimized if media selection is reduced. Therefore, the objective of the present study was to use this protocol to assess whether there were performance differences among the media to isolate Salmonella spp. from naturally contaminated U.S. farm-raised Channel catsh and Vietnamese basa sh.

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2. Materials and methods 2.1. Fish source and microbial analysis Frozen farm-raised Channel catsh (Ictalurus punctatus) llets were purchased from four local retail stores. Frozen basa sh (Pangasius bocourti) llets were initially purchased from retail outlets and later, due to unavailability in the market because of import restrictions, were purchased from a local seafood distributor. Fillets were transported frozen within retail seafood bags to the laboratory and stored at 20  C until day of use. During the course of the study, 30 llets of each sh species from different retail bags were analyzed. The study was conducted from May 2003 through December 2004. Frozen sh llets were thawed overnight at 6  C before microbial analysis. Fillets were removed aseptically from bags and 25-g pieces from each llet were aseptically excised and transferred to sterile stomacher lter bags (ThermoFisher Scientic, Fairlawn, NJ, USA). Fifty milliliters of 0.1% sterile peptone water (BD Diagnostic Systems, Sparks, MD, USA) was added to each bag and blended for 2 min in a stomacher (Tekmar Company, Cincinnati, OH, USA). Decimal dilutions from the homogenates were prepared with 0.1% peptone water. One-milliliter aliquots from each dilution were then plated on duplicate 3M Petrilm Aerobic Count plates (3M, St. Paul, MN, USA) that were incubated at 35  C for 48 h and counted. Similarly, fecal coliform counts were measured using duplicate 3M Petrilm coliform count plates incubated at 44.5  C for 24 h. Mean count values were reported as log10 CFU/g. 2.2. Isolation of Salmonella spp. Twenty-ve grams of thawed sh llet was aseptically placed in 225 ml sterile lactose broth (LB; BD Diagnostic Systems) and blended in a stomacher for 2 min. Inoculated LB was aseptically transferred to capped 250 ml screw-cap jars and kept for 60 min at 25  C, then the jar cap was opened 1/4 turn before incubation at 35  C for 24 h. Following enrichment, jars were vigorously shaken for 5 s and 0.1 ml of LB was transferred into 10 ml Rappaport Vassiliadis broth (RV; Sigma Chemical Co., St. Louis, MO, USA) and 1 ml of LB was transferred into 10 ml tetrathionate broth (TT; Sigma Chemical Co.). RV tubes were incubated at 42  C and TT tubes at 43  C for 24 h. After mixing each tube, 3 mm loopfuls (10 ml) were streaked from RV and TT broths onto Hekteon enteric (HE) agar, xylose lysine deoxycholate (XLD) agar, and bismuth sulte (BS) agar plates (all from BD Diagnostic Systems). All plates were incubated at 35  C for 24 h. The presence of both typical and atypical Salmonella colonies was examined on each selective agar. Two or more typical Salmonella colonies per plate were picked and transferred via stabs to triple sugar iron agar (TSI; BD Diagnostic Systems) and lysine iron agar (LIA; BD Diagnostic Systems) slants, which were incubated for 24 h at 35  C. Color changes typical of Salmonella were noted for each agar slant. 2.3. Conrmation of Salmonella spp. Isolates that were presumptively identied as Salmonella were conrmed by fatty acid methyl ester analysis using gas chromatography (Pendergrass, 1998; Yuk and Marshall, 2003; OHara, 2005). Typical colonies from selective media were streaked onto triplicate trypticase soy agar plates (TSA; BD Diagnostic Systems). Upon complete determination of purity, approximately 40 mg (1520 loopfuls) of each isolate was harvested using a sterile disposable loop and placed into a 13 100 mm Kimax test tube and suspended in 1 ml of saponication solution consisting of 45 g of ACS grade NaOH pellets (#S318-500; ThermoFisher Scientic),

150 ml HPLC grade methanol (#A452-4; ThermoFisher Scientic), and 150 ml of deionized distilled water by vortexing for 5 s. Teon capped tubes were then heated in a boiling water bath (100  C) for 5 min followed by vortexing for 5 s. The tubes were returned to the 100  C water bath for additional heating for 25 min followed by cooling in cold tap water. This combination of methanolic base and heat lysed cells, which released fatty acids from cell membranes that were then converted to their sodium salts. The second step, methylation, used 2 ml of a methylation reagent composed of 325 ml 6.0 N HCl (#LC15370-3; LabChem Inc., Pittsburgh, PA, USA) and 275 ml HPLC grade methanol (#A452-4; ThermoFisher Scientic). Tubes were recapped and heated for 10 min at 80  C followed by rapid cooling in tap water. The methylation step converted the fatty acids (salt form) to fatty acid methyl esters, which increased volatility for gas chromatography analysis. The third step was extraction, during which the fatty acid methyl esters were removed from the acidic aqueous phase and transferred to an organic phase. Extraction was achieved by the addition of 1.25 ml of the extraction reagent to each tube and then tumbling for 10 min. This reagent consisted of 200 ml HPLC grade hexane (#H302-4; ThermoFisher Scientic) and 200 ml HPLC grade methyl tert-butyl ether (#E127-4; ThermoFisher Scientic). After cessation of tumbling, the bottom phase of each sample was discarded. The top phase samples were washed with 3.0 ml of a mild base solution made of 10.8 g ACS grade NaOH pellets (ThermoFisher Scientic) and 900 ml deionized distilled water to remove free fatty acids and residual reagents from the organic extract. After recapping, tubes were tumbled for 5 min, then two-thirds of the organic phase was pipetted into a vial that was capped and ready for analysis. A HewlettPackard Model 6890 gas chromatograph (Wilmington, DE, USA) equipped with a split capillary injector and a ame ionization detector was used to analyze fatty acid methyl esters. Separations were obtained using a HewlettPackard Ultra 2 crosslinked 5% PHME siloxane column (25 m0.2 mm0.33 mm lm thickness) (HP part #19091B-102). Ramping of temperature program was from 170 to 270  C at 5  C per minute. Hydrogen was used as carrier gas with a ow rate of 30 ml/min. MIDI Sherlock Microbial Identication System (MIDI Inc., Newark, DE, USA) was used for analyzing fatty acid proles. Each similarity table provided a list of organism names along with a similarity value. Presence of Salmonella spp. was conrmed if the similarity value was at least 0.5 and if there was a minimum of one-tenth separation between the similarity values of other suggested genera. 2.4. Statistical analysis The Student t-test using the general linear models procedure (SAS 9.1.2, SAS Institute Inc., Cary, NC, USA) was used to assess differences (p 0.05) between aerobic and fecal coliform counts of the two sh species. Paired-wise Fisher exact test (from SAS PROC FREQ) was used to nd signicant differences (p 0.05) between Salmonella incidence (%) using different enrichment (RV or TT) and isolation media (HE, XLD, and BS). 3. Results and discussion Mean (standard deviation) aerobic plate count of the Channel catsh llets was 4.4 0.7 log10 CFU/g, which was signicantly greater (p < 0.05) than that of basa llets (3.8 0.4 log10 CFU/g) (Fig. 1). The majority of Channel catsh (24 out of 30) and basa sh (27 out of 30) samples had fecal coliform counts below 3 CFU/g. The remaining samples had mean fecal coliform counts of 1.1 0.2 log10 CFU/g for 6 Channel catsh samples and 1.0 0 log10 CFU/g for 3 basa sh samples, which were not signicantly different

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6 5 a b
Channel catfish Basa fish

4 3 2 1 0

APC

FCC

Microbial counts
Fig. 1. Aerobic (APC) and fecal coliform (FCC) counts of frozen Channel catsh and basa sh llets. Numbers within count type that have the same letter are not signicantly different (p > 0.05). N 30 for each sh type. FCC: 6 positive Channel catsh llets and 3 positive basa sh llets.

the presence of Salmonella on these sh. Additional work with larger sample sizes and further isolate conrmation using polyvalent antisera (O and H) will be needed to conrm this notion. If conrmed, valuable time and resources could be saved by using a single enrichment and isolation combination. It is notable that the Salmonella incidence levels reported here are greater than previous reports with sh (Andrews et al., 1977; Wyatt et al., 1979; Hannah and McCaskey, 1995; Heinitz et al., 2000). Although the reason for this is unknown, a possible explanation may be the use of different isolation and identication methods among the studies. Additionally, there may be differences in how sh were produced, processed, and handled over time. Nevertheless, the high incidence of Salmonella contamination of both sh species should merit reasonable caution when they are prepared for consumption. Acknowledgments

(p > 0.05). Fillets from both sh species were of acceptable microbiological quality with total counts below 6 log10 CFU/g (Nychas et al., 2007). The numbers of Salmonella-positive samples recovered by each selective isolation media after selective enrichment are summarized in Table 1. Twenty-ve sh samples out of 60 were Salmonella positive (42% incidence). The incidence of Salmonella in Channel catsh and Vietnamese basa was 33% and 50%, respectively. Regardless of the selective isolation media used, the percentages of positive samples after selective enrichment in RV were not different (p > 0.05) (Table 1). Salmonella recovery after selective enrichment in TT and selective isolation on BS was not different (p > 0.05) than the combination of selective enrichment in RV and selective isolation on BS, HE, or XLD; however, lower recovery (p < 0.05) was seen when samples were selectively enriched in TT and selectively isolated on HE or XLD. Numerically, selective enrichment in RV gave more Salmonella-positive samples than selective enrichment in TT. This result is consistent with other reports with high count foods, where RV consistently gave greater recovery of Salmonella (June et al., 1996; Andrews and Hammack, 2006), while foods with low microbial load are better suited to selective enrichment in TT (Hammack et al., 1999). The current nding that HE and BS were not signicantly different in selectively isolating Salmonella when selectively enriched in RV is consistent with previous work with shrimp (Kumar et al., 2003). The present study found no signicant (p > 0.05) isolation differences between media that were specic to the two sh species. Using BS for selective isolation resulted in numerically greater recovery of Salmonella than the other two selective isolation media when selectively enriched in either RV or TT. This observation coupled with numerically greater recovery after selective enrichment in RV suggests that use of RV for selective enrichment and BS for selective isolation may be the best combination to determine
Table 1 Number of conrmed Salmonella positive sh llets by each media (n 30 for each sh type). Selective enrichment media Selective isolation media Channel catsh Basa sh Total (60) RV HE 7 14 21 a XLD 6 12 18 a BS 10 15 25 a TT HE 4 9 13 b XLD 3 10 13 b BS 8 14 22 a

log10 CFU/g

This work was supported in part by a USDA-CSREES Special Grant (2003-34231-13064). References
Andrews, W.H., Hammack, T.S., 2006. Salmonella. In Bacteriological Analytical Manual Online, Chapter 5. Available at http://www.cfsan.fda.gov/webam/bam5.html. Accessed on 12/25/07. Andrews, W.H., Wilson, C.R., Poelma, P.L., Romero, A., 1977. Bacteriological survery of the channel catsh (Ictalurus punctatus) at the retail level. J. Food Sci. 42, 359363. Berg, R.W., Anderson, A.W., 1972. Salmonellae and Edwardsiella tarda in gull feces: a source of contamination in sh processing plants. Appl. Microbiol. 24, 501503. CDC. 1991. Annual listing of foodborne disease outbreaks, United States. Available at http://www.cdc.gov/foodborneoutbreaks/us_outb/fbo1991/fbonal1991.pdf. Accessed on 01/28/08. DAoust, J., Sewell, A.M., Daley, E., Greco, P., 1992. Antibiotic resistance of agricultural and foodborne Salmonella isolates in Canada: 19861989. J. Food Prot. 55, 428434. Flores-Abuxapqui, J.J., Puc-Franco, M.A., Heredia-Navarrete, M.R., de la VivasRosel, M.L., Franco-Monsreal, J., 2003. Comparison between sodium selenite and sodium tetrathionate broths, incubated at 37 C and 42 C for the isolation of Salmonella spp. from faeces of carries. Rev. Biomed. 14, 215220. Gonzalez-Rodrguez, M.N., Sanz, J.J., Santos, J.A., Otero, A., Garca-Lopez, M.L., 2002. Foodborne pathogenic bacteria in prepackaged fresh retail portions of farmed rainbow trout and salmon stored at 3 C. Int. J. Food Microbiol. 76, 135141. Hammack, T.S., Amaguana, R.M., June, G.A., Sherrod, P.S., Andrews, W.H., 1999. Effectiveness of selenite cystine broth, tetrathionate broth, and RappportVassiliadis medium for the recovery of Salmonella spp. from foods with a low microbial load. J. Food Prot. 62, 1621. Hannah, T.C., McCaskey, T.A., 1995. Evaluation of the microbial quality and safety of retail channel catsh llets. Southern Assoc. Agric. Sci. Abstr 32, 20. Heinitz, M.L., Ruble, R.D., Wagner, D.E., Tatini, S.R., 2000. Incidence of Salmonella in sh and seafood. Int. J. Food Microbiol. 63, 579592. June, G.A., Sherrod, P.S., Hammack, T.S., Amaguana, R.M., Andrews, W.H.,1996. Relative effectiveness of selenite cystine broth, tetrathionate broth, and Rappport-Vassiliadis medium for recovery of Salmonella spp. from raw esh, highly contaminated foods, and poultry feed: Collaborative study. J. AOAC Int. 79, 13071323. Kumar, H.S., Sunil, R., Venugopal, M.N., Karunasagar, I., Karunasagar, I., 2003. Detection of Salmonella spp. in tropical seafood by polymerase chain reaction. Int. J. Food Microbiol. 88, 9195. Nychas, G.J.E., Marshall, D.L., Sofos, J.N., 2007. Meat, poultry, and seafood. In: Doyle, M.P., Beuchat, L.R. (Eds.), Food Microbiology: Fundamentals and Frontiers, third ed. ASM Press, Washington, DC, pp. 105140. OHara, C.M., 2005. Manual and automated instrumentation for identication of Enterobacteriaceae and other aerobic Gram-negative bacilli. J. Clin. Microbiol. 18, 147162. Pendergrass, S.M. 1998. Aerobic bacteria by GC-FAME. Method 0801. NIOSH Manual of Analytical Methods, fourth ed. Available at http://www.cdc.gov/niosh/nmam/ pdfs/0801.pdf. Accessed on 12/5/08. Ponce, E., Khan, A.A., Cheng, C.M., Summage-West, C., Cerniglia, C.E., 2008. Prevalence and characterization of Salmonella enterica serovar Weltevreden from imported seafood. Food Microbiol. 1, 2935. Ward, D.R.,1989. Microbiology of aquacultured products. Food Technol. 43 (11), 8286. Wyatt, L.E., Nickelson, R., Vanderzant, C., 1979. Occurrence and control of Salmonella in fresh water catsh. J. Food Sci. 44, 10671069. 1073. Yuk, H.G., Marshall, D.L., 2003. Heat adaptation alters Escherichia coli O157:H7 membrane lipid composition and verotoxin production. Appl. Environ. Microbiol. 69, 51155119. Zhao, S., Datta, A.R., Ayers, S., Friedman, S., Walker, R.D., White, D.G., 2003. Antimicrobial-resistant Salmonella serovars isolated from imported foods. Int. J. Food Microbiol. 84, 8792.

Media: RappaportVassiliadis (RV) broth, tetrathionate broth (TT), Hekteon enteric (HE) agar, xylose lysine deoxycholate (XLD) agar, and bismuth sulte (BS) agar. Numbers with the same letter are not signicantly different (p > 0.05).