Growth of Inoculated Psychrotrophic Pathogens on Refrigerated Fillets of Aquacultured Rainbow Trout and Channel Catfish t

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1 313 Journal of Food Protection, Vol. 1, No.3, 199, Pages Growth of Inoculated Psychrotrophic Pathogens on Refrigerated Fillets of Aquacultured Rainbow Trout and Channel Catfish t CSTY F. FERNANDES,I* GEORGE J. FLlCK, AND TASHA B. THOMAS3 lexperimental Seafood Processing Laborato/); Coastal Research and Extension Center, Mississippi State niversity, Pascagoula, Mississippi ; Department of Food Science and Technology, Virginia Polytechnic 1nstitute and State niversity, Blacksburg, Virginia 1-1; and 3Department of Human Environment and Family Sciences, North Carolina Agricultural & Technical State niversity, Greensboro, North Carolina 755, SA MS 9-19: Received 19 July 199/Accepted 3 September 1997 ABSTRACT Aquacultured rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus) fillets were inoculated with the psychrotrophic pathogens Listeria monocytogenes and Aeromonas hydrophila: cell populations were monitored during refrigerated storage at to DC. Fillets of both species were placed individually in sterile plastic bags and inoculated with cell suspensions (1. 7 CF/lOO g of fish) of either A. hydrophila or L. monocytogenes or of both A. hydrophila and L. monocytogenes, for a total of three treatments for each species of fish. Each inoculum and fillet were mixed to ensure uniform distribution and then stored at to D C.A. hydrophila, L. monocytogenes, and aerobic cell populations were determined on days 1,3,,,1,13, and 15. Individually inoculated A. hydrophila and L. monocytogenes grew on catfish and trout fillets during the 15-day study. There was no inhibition of either pathogen by the natural flora on the fillets. Both psychrotrophic pathogens grew equally well in catfish and trout fillets inoculated with a combination of A. hydrophila and L. monocytogenes. In all three treatments, the counts of the psychrotrophic pathogens were lower than the aerobic plate counts. The growth of the psychrotrophic pathogens L. monocytogenes and/or A. hydrophila during refrigerated storage on aquacultured fish fillets could increase the food hazard risk, particularly where there is a possiity of cross-contamination with ready-to-eat food products. Listeria monocytogenes and Aeromonas hydrophila are omnipresent psychrotrophic pathogens which have been isolated from a variety of fish and in growing waters (,, 9, 1). Among the psychrotrophic pathogens, L. monocytogenes (3) is a recognized foodborne pathogen while A. hydrophila is an emerging pathogen. A. hydrophila has been identified as a cause of human gastroenteritis (1, 15) and an opportunistic pathogen (15, 1). Psychrotrophic pathogens are part of the natural microflora in aquacultured channel catfish (Ictalurus punctatus) and rainbow trout (Oncorhynchus mykiss) and hence have been isolated from fresh aquacultured products. Leung et ai. () isolated L. monocytogenes and A. hydrophila from the viscera of channel catfish. DePaola et al. () also isolated A. hydrophila from market channel catfish. McAdams (9) isolated L. monocytogenes in both whole fish and fillets of aquacultured rainbow trout. Paniagua et al. (13) reported the isolation of motile Aeromonas spp. which were virulent to rainbow trout following intraperitoneal and intramuscular injections. The sale of fish fillets and shellfish products containing psychrotrophic pathogens as their natural flora in supermarket display and other outlets presents several problems. The * Author for correspondence. Tel: ; Fax: -7-77; custyfer@ces.msstate.edu t Presented at the 3 rd Annual Meeting of the International Association of Milk, Food and Environmental Sanitarians in Seattle, WA, 3 June to 3 July 199. The.S. Government is authorized to produce and distribute reprints for government purposes notwithstanding any copyright notation that may appear hereon. chief concerns are the growth of these psychrotrophic pathogens at refrigeration temperatures and the possible cross-contamination of the fresh fillets with cooked ready-toeat products with the psychrotrophic pathogens during market handling or in the home refrigerator. Hence, the psychrotrophic pathogens are of concern as they may multiply to an undesirable level on refrigerated fresh fish during a normal refrigerated storage period. Foods appear to be a major vector of human infection with L. monocytogenes (15). Further, L. monocytogenes could proliferate on fish products (, 7); aquacultured fish fillets are an excellent nutrient-rich substrate for growth of psychrotrophic pathogens. Researchers have inoculated psychrotrophic pathogens on fish products and subsequently studied their proliferation (, 7, ). However, in reality more than one psychrotrophic pathogen may be present in a given food product. There is a paucity of data on the combined growth of important psychrotrophic pathogens such as L. monocytogenes and an emerging psychrotrophic pathogen such as A. hydrophila on fish fillets. Therefore, the objective of this study was to evaluate the individual and combined inoculation of A. hydrophila and L. monocytogenes on aquacultured channel catfish and rainbow trout fillets and to monitor their growth during refrigerated storage. MATERIALS AND METHODS Inoculum for spiking. The psychrotrophic pathogens Aeromonas hydrophila ATCC 795 and Listeria monocytogenes ATCC 7 were propagated in nutrient broth. About 1 fil of

2 3 FERNANDES ET AL. J. Food Prot., Vol. 1, No.3 each culture (stored at - C) was inoculated into 5. ml of nutrient broth # (Difco Laboratories, Detroit, MI) in 15-ml flasks and incubated at 35 C for 1 h. The flasks were refrigerated ( to C) for up to h until analyzed. L. monocytogenes cell numbers were determined by plating on Oxford medium base (Difco); A. hydrophila CF were enumerated by plating on e salts brilliant green starch agar (ph 7.) (1). Aquacultured fish fillets. Two aquacultured fish species were used in these studies. Fresh rainbow trout (Oncorhynchus mykiss) fillets were procured from an aquaculture farm in Virginia 1 day before the study began. Fillets were packaged in a polyethylene bag and transported on ice to the Virginia Tech campus. pon arrival in the laboratory, 3 fillets were mixed by hand with a sterile spatula to achieve a uniform distribution of the indigenous microflora. Each fillet was trimmed to a weight of about 1 g (::+: g) and placed individually in a sterile 7 by 1 in stomacher bag (Seward, London, K). Individual quick frozen channel catfish (Ictalurus punctatus) fillets, about 15 g, were obtained from a local wholesale distributor and held frozen for a maximum of 15 days at - C. Two days prior to the initiation of the study, the fillets were allowed to thaw in a refrigerator ( to C). Thawed fillets were mixed with a sterile spatula to obtain a uniform distribution of microflora. Each fillet was trimmed to a weight of about 1 g (::+:g) and packaged in a sterile stomacher bag. Quantitative analyses of psychrotrophic pathogens. Preliminary studies were performed to determine the base level of indigenous species of Listeria and Aeromonas. Black Listeria-like colonies were determined by plating samples on Oxford medium base supplemented with Oxford antimicrobic supplement (Difco). Aeromonas-like colonies, identified by the appearance of a clear surrounding zone upon addition of Lugol's iodine (Sigma Chemical Co., St. Louis, MO), were enumerated by plating samples on e salts brilliant green starch agar (BSGA), ph 7. (1). Both sets of plates were incubated at 35 C for h. Samples for plating were prepared as described below. Spiking fish fillets. Thirty-six fish fillets each of rainbow trout and channel catfish were randomly distributed for three treatments for each aquacultured species: inoculation of L. monocytogenes or of A. hydrophila, or of a combination of L. monocytogenes and A. hydrophila). Individual fillets were each placed in a sterile stomacher bag and each fillet in the lot was inoculated with a standard volume (1. ml, about 1. 7 CF/lOO g of the fillet) of the psychrotrophic pathogen culture and 1. ml of sterile distilled water. Fillets spiked with a combination of both psychrotrophic pathogens were inoculated with 1. ml of the A. hydrophila and 1. ml of L. monocytogenes cultures. All spiked samples were massaged manually for. min and stored in a refrigerator ( to C). Microbial analyses. One fillet from each treatment was analyzed initially and at each time point of the refrigerated storage. To each fillet, an equal volume of sterile cold ( to C) 15 mm phosphate buffer was added and the bag contents were stomached for. min (Laboratory Blender Stomacher, Seward Medical, London, K). The homogenate was used for microbial analyses, which included aerobic plate, A. hydrophila and L. monocytogenes counts. Aerobic plate counts were determined in duplicate on standard methods agar (Difco) following a -h incubation at 35 C (19). The number of CF of L. monocytogenes and A. hydrophila were determined as described above. The counts were averaged for statistical analyses. The fillets were analyzed for their microbial quality during 15 days of refrigerated ( to C) storage; counts were determined on day 1,3,,, 1, 13, and 15 after inoculation. On each sampling day the fillets were also checked for odor; by these subjective analyses, the fillets were classified as either acceptable or spoiled. Statistical design and analyses. Thirty-six fillets of each species were randomly divided into three whole plots (three treatments). The treatment design was split-plot with time (day of analysis) (17). The entire experiment was replicated three times and the data was analyzed as randomized complete blocks as the experimental design (1). I>IJ Q Q... :;s 7. I>IJ..s. RESLTS AND DISCSSION Pathogenic quality of fillets. Aeromonas- and Listerialike colonies from samples before inoculation of rainbow trout and channel catfish fillets ranged from to 7 and to 51 CF/1 g of fillet, respectively. Farber () reported that the L. monocytogenes count in cooked shrimp and cooked lobster was <1 CF/g. McAdams (9) determined the frequency and number of several pathogens in whole fish and fillets of rainbow trout. L. monocytogenes was identified in to 9% of whole fish and 55 to 5% of fillets examined; however, the counts were very low in both whole fish (.3 to.3 MPN/g) and fillets (.73 to 3.9 MPN/g) (9). Leung et a1. () observed that the visceral counts of A. hydrophila and Listeria spp. of pond-cultured catfish was 1. and CF/g (wet weight), respectively. Nedoluha and Westhoff (1) observed that % of the flora in aquacultured hybrid striped bass was due to Aeromonas spp. Since the indigenous background of Listeria-like and Aeromonas-like species were low, the catfish and trout fillets were inoculated with 1. 7 CF of each psychrotrophic pathogen per g. This inoculum level probably reduced and/or eliminated the background effect of the pathogens under investigation. Growth of psychrotrophic pathogens on rainbow trout (Oncorhynchus mykiss). Figure 1 illustrates the geometric means of A. hydrophila and aerobic plate counts on rainbow trout fillets during the IS-day storage period following inoculation. The aerobic plate counts increased significantly (P <.5) from. log CF/1 g on day 1 to about log CF/1 g on day 15; thea. hydrophila counts - - A. hydrophila. f -. -! FIGRE 1. Population of A. hydrophila and aerobic plate counts on aquacultured rainbow trout fillets under refrigeration at C for 15 days. Vertical bars: SE = ::+:

3 J. Food Prot., Vol. 1, No.3 GROWTH OF PATHOGENS AT C ON FILLETS OF AQACLTRED FISH 315 increased significantly (P <.5) from.5 log CF/lOO g to 7. log CP/lOa g during the same period. Inoculated A. hydrophila grew during the IS-day refrigerated storage in the presence of the indigenous microflora. However, after 1 to 13 days of storage, the fillets were considered to be unacceptable due to spoilage, a result of microbial decomposition as observed by subjective sensory analyses. Hazen et al. () observed that A. hydrophila can survive under a wide variety of environmental conditions. Abeyta and Wekell (1) showed that A. hydrophila is one of the components in the intestines of healthy fish and that the organism is widely distributed in nature. The presence of psychrotrophic pathogens such as A. hydrophila in fish products and their growth under refrigeration indicates that they could become a source of cross-contamination. Figure illustrates the geometric means for L. monocytogenes and aerobic plate counts following L. monocytogenes inoculation of rainbow trout fillets subsequently stored under refrigeration over a IS-day period. L. monocytogenes counts increased significantly (P <.5) by log CF/lOO g; the aerobic plate counts increased significantly (P <.5) by approximately 3 log CP/lOa g during refrigerated storage. Around 13 days the fillets emitted a putrid odor and were classified as unacceptable. Thus, L. monocytogenes proliferated on trout fillets in the presence of the natural microflora. The presence and proliferation of psychrotrophic pathogens such as L. monocytogenes could pose a problem if pathogens are transferred from the fish to the further processed food products (5). Figure 3 shows geometric means of A. hydrophila, L. monocytogenes, and aerobic plate counts following simultaneous inoculation with A. hydrophila and L. monocytogenes on rainbow trout fillets. The aerobic plate counts increased significantly (P <.5) from 1.3 CP/lOO g on day 1 to 1. CF/lOO g on day 15. The A. hydrophila counts increased from 1. 7 CP/lOa g to 1. 9 CF/g in 15 days; during the same time interval L. monocytogenes counts rose from 1 5. CP/lOa g to 1. CP/lOa g. Both psychrotrophic pathogens could proliferate simultaneously on trout fillets in the presence of natural indigenous microflora. It did not appear that either of the inoculated psychrotrophic. f.i <>" A. hydrophila, - L. monocytogenes!...y.. I I.... I""I._,.y f-"--"-. FIGRE 3. Populations of A. hydrophila and L. monocytogenes and aerobic plate counts on aquacultured rainbow trout fillets under refrigeration at C for 15 days. Vertical bars: SE = O.3. pathogens inhibited the growth of the other. At about 13 days the fillets were considered unacceptable due to decomposition (odor). The combined growth of the psychrotrophic bacterial pathogens during refrigerated storage compounds the problems associated with food safety. Channel catfish (Ictalurus punctatus). The growth of individual and combined inoculated A. hydrophila and L. monocytogenes on channel catfish fillets stored at refrigeration temperature is shown in Figures to. In catfish fillets inoculated with A. hydrophila (Fig. ), thea. hydrophila and aerobic plate counts were similar to that of trout. The aerobic plate counts increased by 1,OOO-foldover a IS-day period, while the A. hydrophila count increased by.5 log CF/lOO g during the same period. A. hydrophila also proliferated in the presence of the natural microflora. Ventura and Grizzle () observed that A. hydrophila gained access to the intestinal organs of catfish through the upper digestive tract and can result in motile Aeromonas septicemia. DePaola et al. () isolated oxytetracycline- and tetracycline-resistant A. hydrophila from cultured channel catfish. Leung et al. () isolated A. hydrophila from pond water and sediments as well as catfish viscera. Since these organisms are natural flora of catfish they would be naturally present and proliferate on aquacultured channel catfish. Leung et al. (7) r..'.f -! r,/.' :i. -- L. monocytogencs.l - t- -I f-.. f..-.1-,.r :i. " - A. hydrophila ft. '.l'''',.i''.. ' FIGRE. Population of L. monocytogenes and aerobic plate counts on aquacultured rainbow trout fillets under refrigeration at Cfor 15 days. Vertical bars: SE = O.9. FIGRE. Population of A. hydrophila and aerobic plate counts on aquacultured channel catfish fillets under refrigeration at C for 15 days. Vertical bars: SE = O..

4 31 FERNANDES ET AL. J. Food Prot.. Vol. 1. No.3 observed that when catfish samples were inoculated with A. hydrophila the population grew rapidly and increased from log CF/cm on day 1 to.5 to 7. log CF/cm on day 1. Additionally, A. hydrophila grew well under aerobic conditions (7, 11). The conditions in this study were aerobic, which may have facilitated A. hydrophila growth. The geometric means of L. monocytogenes and aerobic plate counts following inoculation of catfish fillets are shown in Fig. 5. L. monocytogenes counts increased significantly (P <.5) from 1 51 CF/lOO g on day 1 to 1. 9 CF/1 g on day 15, while the aerobic plate counts increased significantly (P <.5) from 1. 1 CF/1 g to 1. CP/1 g during the same period. Inoculated L. monocytogenes proliferated on catfish fillets in the presence of the inherent catfish microflora, just as did the growth of L. monocytogenes on rainbow trout fillets observed in this study. The catfish fillet samples emitted a bad odor by about the 1th day of storage. Leung et al. (7) observed that the L. monocytogenes population increased slowly by approximately 1 to 1.5 log cycles over the first 1 days when L. monocytogenes was inoculated on channel catfish. However, their count decreased by about 1.5 log cycles on the 1th d. The differences between this and the study by Leung et al. (7) were due to the microbial species, packaging conditions and microbial enumerations. When L. monocytogenes anda. hydrophila were concurrently inoculated on catfish fillets their numbers increased (Fig. ). The A. hydrophila count increased from. log CF/lOO g to.5 log CF/1 g over a l5-day interval. During the same period, the counts of L. monocytogenes and aerobic plate counts increased by about. and.5 log CF/1 g respectively. When L. monocytogenes and A. hydrophila were inoculated simultaneously on the catfish fillets, both microorganisms produced similar growth. These results are similar to those obtained for trout fillets when a combination of the psychrotrophic pathogens were inoculated. It is evident that both individually and simultaneously inoculated psychrotrophic pathogens L. monocytogenes and A. hydrophila can grow on aquacultured catfish and rainbow trout fillets at refrigeration temperatures ( to DC). However, the inherent natural flora associated with the fillets was =. = L. monocytogenes 7. "r-- I 5.,.. -!' r - -! ! FIGRE 5. Population of L. monocytogenes and aerobic plate counts on aquacultured channel catfish fillets under refrigeration at Cfor 15days. Vertical bars: SE = ±.3. =. = A. hydrophila - - L. monocytogenes FIGRE. Populations ola. hydrophila and L. monocytogenes and aerobic plate counts on aquacultured channel catfish fillets under refrigeration at C for 15 days. Vertical bars: SE = ±O.. REFERENCES 1 1 "t. 1 t, I'"f /:'' r '. 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