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Food Microbiology 26 (2009) 317–319

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Food Microbiology
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Comparison of culture media for enrichment and isolation of Salmonella spp. from frozen Channel catfish and Vietnamese basa fillets
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 Catfish Basa Isolation methods

a b s t r a c t
Frozen fillets of Channel catfish and Vietnamese basa fish were used to compare Salmonella spp. recovery effectiveness of selective enrichment in Rappaport–Vassiliadis (RV) broth and tetrathionate broth (TT) and selective isolation on Hekteon enteric (HE) agar, xylose lysine deoxycholate (XLD) agar, and bismuth sulfite (BS) agar. Isolate confirmation 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 catfish and Vietnamese basa fish compared to other isolation combinations. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Salmonella has been previously isolated from catfish (Andrews et al., 1977; Wyatt et al., 1979; Hannah and McCaskey, 1995), and Channel catfish (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 fish products. The potential source of Salmonella contamination in farm-raised catfish is likely due to poor water quality, farm runoff, fecal contamination from wild animals or livestock, feed (Ward, ´ ´ 1989; Gonzalez-Rodrıguez et al., 2002), processing under poor sanitary conditions (D’Aoust 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 catfish. Salmonella has been isolated not only from domestic catfish but also from imported fish and fish products, including catfish (D’Aoust et al., 1992; Heinitz et al., 2000; Zhao et al., 2003). Widespread distribution of antibiotic-resistant Salmonella strains
* Corresponding author. Tel.: þ1 970 351 2877; fax: þ1 970 351 2176. E-mail address: (D.L. Marshall). 0740-0020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/

due to international trade of Salmonella-contaminated seafood products (D’Aoust 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 (1.5–4.5%) of Salmonella contamination in retail catfish fillets (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 fish 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 fish and fish products recommends pre-enrichment with lactose broth (LB), followed by selective enrichment in Rappaport–Vassiliadis (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 sulfite (BS) agar (Andrews and Hammack, 2006). Since the above protocol requires multiple enrichment and isolation media, the effort of Salmonella isolation from fish 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 catfish and Vietnamese basa fish.

One-milliliter aliquots from each dilution were then plated on duplicate 3M PetrifilmÔ Aerobic Count plates (3M. OH.25 ml of the extraction reagent to each tube and then tumbling for 10 min.3. Decimal dilutions from the homogenates were prepared with 0. Sigma Chemical Co. Cincinnati. 30 fillets of each fish species from different retail bags were analyzed. Hydrogen was used as carrier gas with a flow rate of 30 ml/min. St.4. Similarly.2 mmÂ0.L.. and 150 ml of deionized distilled water by vortexing for 5 s. The second step.0 Æ 0 log10 CFU/g for 3 basa fish samples. 2. MN. Presence of Salmonella spp. BD Diagnostic Systems). DE. The majority of Channel catfish (24 out of 30) and basa fish (27 out of 30) samples had fecal coliform counts below 3 CFU/g.. Fillets were removed aseptically from bags and 25-g pieces from each fillet were aseptically excised and transferred to sterile stomacher filter bags (ThermoFisher Scientific. which released fatty acids from cell membranes that were then converted to their sodium salts.2. approximately 40 mg (15–20 loopfuls) of each isolate was harvested using a sterile disposable loop and placed into a 13 Â 100 mm Kimax test tube and suspended in 1 ml of saponification solution consisting of 45 g of ACS grade NaOH pellets (#S318-500. Two or more typical Salmonella colonies per plate were picked and transferred via stabs to triple sugar iron agar (TSI. The top phase samples were washed with 3.1.318 A. St. ThermoFisher Scientific).1 Æ 0. XLD. Fifty milliliters of 0. NC. MD. 2.05) between Salmonella incidence (%) using different enrichment (RV or TT) and isolation media (HE. ThermoFisher Scientific). The remaining samples had mean fecal coliform counts of 1. After cessation of tumbling. Color changes typical of Salmonella were noted for each agar slant. USA) was added to each bag and blended for 2 min in a stomacher (Tekmar Company. D. Typical colonies from selective media were streaked onto triplicate trypticase soy agar plates (TSA. Teflon capped tubes were then heated in a boiling water bath (100  C) for 5 min followed by vortexing for 5 s. Pal. Louis. All plates were incubated at 35  C for 24 h. Tubes were recapped and heated for 10 min at 80  C followed by rapid cooling in tap water. USA) was used for analyzing fatty acid profiles. USA) was used to assess differences (p 0. Fairlawn. 150 ml HPLC grade methanol (#A452-4.05) than that of basa fillets (3. SAS Institute Inc. xylose lysine deoxycholate (XLD) agar. Marshall / Food Microbiology 26 (2009) 317–319 2. Following enrichment. LabChem Inc. Sigma Chemical Co.5 and if there was a minimum of one-tenth separation between the similarity values of other suggested genera.33 mm film thickness) (HP part #19091B-102). The presence of both typical and atypical Salmonella colonies was examined on each selective agar. tubes were tumbled for 5 min. USA) equipped with a split capillary injector and a flame ionization detector was used to analyze fatty acid methyl esters. 1).1 ml of LB was transferred into 10 ml Rappaport– Vassiliadis broth (RV. The methylation step converted the fatty acids (salt form) to fatty acid methyl esters.. This reagent consisted of 200 ml HPLC grade hexane (#H302-4. 2. Confirmation of Salmonella spp. the bottom phase of each sample was discarded. 2005). USA) and 1 ml of LB was transferred into 10 ml tetrathionate broth (TT. Each similarity table provided a list of organism names along with a similarity value. NJ. during which the fatty acid methyl esters were removed from the acidic aqueous phase and transferred to an organic phase. MO. 3.2.5  C for 24 h. Separations were obtained using a Hewlett–Packard Ultra 2 crosslinked 5% PHME siloxane column (25 mÂ0. was confirmed if the similarity value was at least 0. which was significantly greater (p < 0. After mixing each tube.05) between aerobic and fecal coliform counts of the two fish species. This combination of methanolic base and heat lysed cells. Newark. Results and discussion Mean (Æstandard deviation) aerobic plate count of the Channel catfish fillets was 4. A Hewlett–Packard Model 6890 gas chromatograph (Wilmington. USA) and 275 ml HPLC grade methanol (#A452-4. DE.4 Æ 0. The tubes were returned to the 100  C water bath for additional heating for 25 min followed by cooling in cold tap water. Isolates that were presumptively identified as Salmonella were confirmed by fatty acid methyl ester analysis using gas chromatography (Pendergrass. Extraction was achieved by the addition of 1. USA). and bismuth sulfite (BS) agar plates (all from BD Diagnostic Systems). used 2 ml of a methylation reagent composed of 325 ml 6. and BS). then the jar cap was opened 1/4 turn before incubation at 35  C for 24 h. BD Diagnostic Systems) and lysine iron agar (LIA.2 log10 CFU/g for 6 Channel catfish samples and 1.). BD Diagnostic Systems) and blended in a stomacher for 2 min. ThermoFisher Scientific). Twenty-five grams of thawed fish fillet was aseptically placed in 225 ml sterile lactose broth (LB. During the course of the study. which increased volatility for gas chromatography analysis. Sparks. ThermoFisher Scientific) and 200 ml HPLC grade methyl tert-butyl ether (#E127-4. Yuk and Marshall.1. Ramping of temperature program was from 170 to 270  C at 5  C per minute. due to unavailability in the market because of import restrictions. USA) that were incubated at 35  C for 48 h and counted.8 g ACS grade NaOH pellets (ThermoFisher Scientific) and 900 ml deionized distilled water to remove free fatty acids and residual reagents from the organic extract. Pittsburgh.0 ml of a mild base solution made of 10. 3 mm loopfuls (10 ml) were streaked from RV and TT broths onto Hekteon enteric (HE) agar.1% sterile peptone water (BD Diagnostic Systems. The study was conducted from May 2003 through December 2004. Fillets were transported frozen within retail seafood bags to the laboratory and stored at À20  C until day of use. RV tubes were incubated at 42  C and TT tubes at 43  C for 24 h. Cary. 1998. were purchased from a local seafood distributor.1% peptone water. The third step was extraction. Statistical analysis The Student t-test using the general linear models procedure (SASÒ 9. After recapping. fecal coliform counts were measured using duplicate 3M Petrifilm coliform count plates incubated at 44. Fish source and microbial analysis Frozen farm-raised Channel catfish (Ictalurus punctatus) fillets were purchased from four local retail stores. which were incubated for 24 h at 35  C.0 N HCl (#LC15370-3. Frozen fish fillets were thawed overnight at 6  C before microbial analysis. Frozen basa fish (Pangasius bocourti) fillets were initially purchased from retail outlets and later. Paired-wise Fisher exact test (from SAS PROC FREQ) was used to find significant differences (p 0. PA.. jars were vigorously shaken for 5 s and 0. Paul. Materials and methods 2. which were not significantly different .7 log10 CFU/g.8 Æ 0. ThermoFisher Scientific). MIDI Sherlock Microbial Identification System (MIDI Inc. Upon complete determination of purity. then two-thirds of the organic phase was pipetted into a vial that was capped and ready for analysis. USA).4 log10 CFU/g) (Fig. 2003. methylation. BD Diagnostic Systems) slants. O’Hara. Isolation of Salmonella spp. Mean count values were reported as log10 CFU/g. Inoculated LB was aseptically transferred to capped 250 ml screw-cap jars and kept for 60 min at 25  C.

. R. Vanderzant.. Datta.E.A. Wagner. 84. Abstr 32. P. Clin.R. ˜ Hammack. Sewell. Acknowledgments (p > 0.D... and seafood. where RV consistently gave greater recovery of Salmonella (June et al. 82–86.. Media: Rappaport–Vassiliadis (RV) broth..C.05). 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. Walker.. W. from foods with a low microbial load. from faeces of carries. Antimicrobial-resistant Salmonella serovars isolated from imported foods.M. A. Ayers. Annual listing of foodborne disease outbreaks. T. Sherrod. It is notable that the Salmonella incidence levels reported here are greater than previous reports with fish (Andrews et al. McCaskey. Anderson. 2007). Microbiology of aquacultured products. Otero. Microbiol..S. Evaluation of the microbial quality and safety of retail channel catfish fillets. Detection of Salmonella spp. P.05) (Table 1). Franco-Monsreal. Foodborne pathogenic bacteria in prepackaged fresh retail portions of farmed rainbow trout and salmon stored at 3 C.N.05) isolation differences between media that were specific to the two fish species. however. 79. R.E.. S. HE. Marshall.. This result is consistent with other reports with high count foods. Sanz. Poelma.. Southern Assoc. M. Rev. Numbers within count type that have the same letter are not significantly different (p > 0. DC. J.L. valuable time and resources could be saved by using a single enrichment and isolation combination.. Pal. Int.H. Microbiol. A.W.. Washington. T. Daley. 2002. R..D. J.. lower recovery (p < 0.L. a possible explanation may be the use of different isolation and identification methods among the studies. the presence of Salmonella on these fish. 501–503. A.cdc.N.H. Microbiol. Manual and automated instrumentation for identification of Enterobacteriaceae and other aerobic Gram-negative bacilli. Beuchat.A. .A. Yuk.S.S. Friedman.. 1979.. H.R. J. selective enrichment in RV gave more Salmonella-positive samples than selective enrichment in TT. 135–141. M. Twenty-five fish samples out of 60 were Salmonella positive (42% incidence). Sherrod. G. Ruble. Food Microbiol. 18. M.. and Rappport-Vassiliadis medium for recovery of Salmonella spp. M.. 147–162. 24.. Heinitz et al.J. Berg.. and bismuth sulfite (BS) agar.. the high incidence of Salmonella contamination of both fish species should merit reasonable caution when they are prepared for consumption. xylose lysine deoxycholate (XLD) agar..G.. R. Marshall. Cheng.L. J. T. Available at http://www. Food Sci. C.. pp. 87–92.. Additionally..N. Selective enrichment media Selective isolation media Channel catfish Basa fish 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).. Agric. Int.. ´ ´ ´ ´ Gonzalez-Rodrıguez.05).R. and poultry feed: Collaborative study. 2006.A. J. June... Food Technol. in tropical seafood by polymerase chain reaction. Hannah. 91–95. incubated at 37 C and 42 C for the isolation of Salmonella spp. Comparison between sodium selenite and sodium tetrathionate broths. 14..pdf.).. 2006). 1. Appl.A. Food Microbiol. 69.. 215–220. J.cdc. L. 428–434. Wyatt. ASM Press. Romero. M... or XLD. Amaguana. 2000. Andrews.G. 1998. Ponce. 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 confirmed Salmonella positive fish fillets by each media (n ¼ 30 for each fish type).. Regardless of the selective isolation media used. Marshall / Food Microbiology 26 (2009) 317–319 319 6 5 a b Channel catfish Basa fish 4 3 2 1 0 a a APC FCC Microbial counts Fig. NIOSH Manual of Analytical Methods. J. Salmonellae and Edwardsiella tarda in gull feces: a source of contamination in fish processing plants.. J.W. M. Venugopal.. E.L. Method 0801. P. 1999. Flores-Abuxapqui. Hannah and McCaskey. 16–21. from raw flesh. A. Int. 105–140. de la VivasRosel. processed. Karunasagar. 20. J.. Incidence of Salmonella in fish and seafood. M. Food Microbiol.R. 359–363.. Amaguana. J..pdf. Appl. Although the reason for this is unknown. Karunasagar.. poultry. D. (Eds. Antibiotic resistance of agricultural and foodborne Salmonella isolates in Canada: 1986–1989..05) was seen when samples were selectively enriched in TT and selectively isolated on HE or XLD. third ed.A. The present study found no significant (p > 0. Andrews..M. Food Sci.L. W. J. S. 1995. 1067–1069. Greco. Food Microbiology: Fundamentals and Frontiers. Sci.cfsan. Prevalence and characterization of Salmonella enterica serovar Weltevreden from imported seafood. 1972. S. The current finding that HE and BS were not significantly different in selectively isolating Salmonella when selectively enriched in RV is consistent with previous work with shrimp (Kumar et al. highly contaminated foods. tetrathionate broth. C. C. W. 1995. N ¼ 30 for each fish Food Prot. P.... 579–592. R. 2003).05). C. 2003.R. and handled over time. Nevertheless. If confirmed. 2000).M.1996. Hammack. Accessed on 01/28/08. G. The incidence of Salmonella in Channel catfish and Vietnamese basa was 33% and 50%. Pendergrass. 42.E. Heredia-Navarrete. 2003. Heat adaptation alters Escherichia coli O157:H7 membrane lipid composition and verotoxin production. The numbers of Salmonella-positive samples recovered by each selective isolation media after selective enrichment are summarized in Table 1.. D. 55. J. fourth ed. CDC. D. W. pdfs/0801... Aerobic bacteria by GC-FAME. 1977.. Ward. 1992. Numbers with the same letter are not significantly different (p > 0. 1979. 1996. 2003. 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