Bacterial Microflora of Wild Brown Trout (Salmo trutta), Wild Pike (Esox lucius), and Aquacultured Rainbow Trout (Oncorhynchus mykiss)

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1 1270 Journal of Food Protection, Vol. 62, No. 11, 1999, Pages Copyright, International Association of Milk, Food and Environmental Sanitarians Bacterial Microflora of Wild Brown Trout (Salmo trutta), Wild Pike (Esox lucius), and Aquacultured Rainbow Trout (Oncorhynchus mykiss) CÉSAR-JAVIER GONZÁLEZ, TERESA-MARÍA LÓPEZ-DÍAZ, MARÍA-LUISA GARCÍA-LÓPEZ, MIGUEL PRIETO, AND ANDRÉS OTERO* Department of Food Hygiene and Food Technology, Veterinary Faculty, University of León, E León, Spain MS 99-10: Received 19 January 1999/Accepted 16 May 1999 ABSTRACT Initial numbers of bacteria associated with wild (brown trout and pike) and cultured (rainbow trout) freshwater fish as well as with the water in which they were caught were determined. Subsequently, a total of 979 randomly selected isolates were characterized and identified to the genus level. For all counts performed (aerobes, psychrotrophs, anaerobes, Enterobacteriaceae, and enterococci), no significant differences were observed in water samples, the highest level corresponding to psychrotrophs in pike environments ( CFU/ml). Overall, the skin and intestinal content of brown trout were the most contaminated, while rainbow trout specimens (gills and gut) yielded the lowest numbers. For all bacterial groups, pike gills had the highest numbers. Counts for all of the sampling sites compare well with findings in other temperate geographical environments. Biological characteristics (feeding and skin properties) and the use of antimicrobials in aquaculture might have influenced these results. Motile and nonmotile aerobic gram-negative bacteria together with Enterobacteriaceae accounted for 50 to 70% of the psychrotrophs isolated from water. Micrococcaceae, lactic acid bacteria, Bacillus, and coryneforms were also found. The groups represented in psychrotrophic isolates from the outer surfaces do not reflected those detected in water, so it was common that those organisms recovered in significant numbers from fish were not detected in surrounding habitat of the fish. Motile aeromonads and Carnobacterium were the dominant psychrotrophs in the guts of pike and brown trout, respectively. The intestinal content of reared fish gave a high incidence of Bacillus and coryneforms, while Enterobacteriaceae was absent. Again, rearing practices could have influenced this finding. Listeria monocytogenes was not detected in any of the examined samples. Two strains of Salmonella, which belonged to the same serovar and lysotype, were recovered from pond-water samples taken from one facility on different sampling days. From the gut of a pike specimen and from the pike s environment, two Plesiomonas shigelloides strains of different serovars were recovered. These latter four strains were resistant to a considerable number of antimicrobial compounds (multiple antibiotic resistance indices 0.2). As in other Mediterranean countries, fish and fish products are common items in the Spanish diet, the average consumption figure being approximately 40 kg per person and year (35). The majority of the fish supply is marine in origin and is harvested from wild populations, but as a result of the rapid depletion of feral stocks, aquaculture is a rapidly expanding industry in Spain. Currently, the aquacultured fish species, primarily rainbow trout, account for more than 15% of the total Spanish fishery products (15). Although much information is available on the numbers and types of bacteria associated with marine fish, similar data for healthy freshwater fish, both reared and wild, are more scarce. Moreover, there have been relatively few studies comparing the microbiological profile of cultured and wild animals. Likewise, it is generally believed that the environment can influence numbers and types of microorganisms of fish (31), but similar microbial loads have been found in aquacultured and wild freshwater fishes of the same species (32). Although the presence of human bacterial pathogens in * Author for correspondence. Tel: ; Fax: ; dhtaoc@unileon.es. freshwater fish is dependent on a number of factors, including species, water conditions, rearing practices, etc., isolation of certain organisms, such as motile aeromonads, Plesiomonas shigelloides, Listeria monocytogenes, and Salmonella, is not uncommon (8, 19, 29, 30). The occurrence of antibiotic-resistant pathogenic bacteria in surface waters and aquaculture environments is also a well-known phenomenon that carries a negative impact for public health and for the safety of the fish supply (44). This paper reports on the initial bacterial flora associated with wild (brown trout and pike) and cultured (rainbow trout) freshwater fish derived from rivers and artificial impoundments located at a temperate region within the northwest area of Spain. The microbial quality of the surrounding water and the occurrence of certain foodborne pathogens (and their antibiotic sensitivity patterns) were also investigated. MATERIALS AND METHODS Collection of samples. Between April and July 1997, 30 specimens of wild brown trout (Salmo trutta) and 12 specimens of wild pike (Esox lucius) were fished in rivers of the province of León (northwest region of Spain). On each sampling day, two

2 J. Food Prot., Vol. 62, No. 11 BACTERIAL MICROFLORA OF TROUT AND PIKE 1271 to three specimens of brown trout were caught by hook and line; the water temperatures of the nine rivers (Selmo, Eria, Esla, Curueño, Torío, Sil, Burbia, Porma, and Órbigo) were between 5 and 15 C. One to two pikes per day were taken by net from the Esla and Porma Rivers (water temperature ranged between 8 and 12 C). During the same months in which wild fish were sampled, pond-reared rainbow trout (Oncorhynchus mykiss, 30 specimens) were obtained from two aquaculture farms located on river (Duerna and Órbigo, respectively) systems within the geographic area of the study. On each sampling day, three rainbow trout were caught by net in the ponds; the water temperature of the ponds ranged from 7 to 11 C. samples (200 ml) were aseptically collected at the same locations from which the fish were harvested, from a depth of about 50 cm. Iced fish (in plastic boxes) and refrigerated water samples were transported to the laboratory within6h. Microbiological analysis. From each fish specimen, skin samples were taken from two to three locations on the surface (by scraping and rising) using a cylinder (with a 6.5 cm 2 open surface) and 10 ml of 0.1% (wt/vol) peptone water. In addition, we excised enough of the gills to obtain about 10 g as well as the whole gut; excised samples were placed in sterile bags containing enough 0.1% (wt/vol) peptone water to make a 1:10 dilution, and they were then blended for 2 min in a Stomacher 400 Lab Blender (Seward Medical, London, UK). Fish and water samples were then serially diluted, spread plated onto tryptone soya agar (TSA; Oxoid, Basingstoke, UK) plates (two plates per dilution and incubation temperature), and incubated at 30 and 7 C for 2 and 10 days, respectively. Five colonies from each set of counts were randomly selected, streaked to purity on TSA, and stored on slants of the same medium for further examination. Ten-fold dilutions were also utilized for Enterobacteriaceae counts, which were determined on violet red bile glucose agar (Oxoid) after incubation at 37 C for 24 h (16); enterococci counts, which were estimated by using kanamycin aesculin azide broth (Oxoid); and the most probable number method (27). For the total anaerobic counts, reinforced clostridial broth (Oxoid) was used in the preparation of homogenates and 10-fold dilutions; 0.1 ml of each dilution was spread onto duplicate plates of reinforced clostridial agar (Oxoid); and counts were recorded after 5 days of anaerobic incubation at 37 C (5). Detection of P. shigelloides was carried out on inositol brilliant-green bile agar (36). Presumptive P. shigelloides isolates were identified according to the criteria of Miller and Koburger (25), and serotyping of the confirmed strains was kindly performed by Dr. Aldova (Czech Republic National Institute for Human Health). In order to detect L. monocytogenes, samples (10 g of fish samples or 10 ml of water) were mixed with 90 ml of a selective enrichment broth (trypticase soy broth supplemented with yeast extract, acriflavin, nalidixic acid, and cycloheximide; (24)), and after 48 h of incubation at 30 C, 0.1 ml of these cultures was streaked onto Oxford agar (Oxoid) plates. Suspect listerial colonies were subjected to identification tests (34, 37). Hydrophobic grid-membrane filters (Iso-Grid, M-Tech Diagnostics Ltd., Warrington, UK) were used for detection of Salmonella (9). The selective enrichment was performed in Rappaport Vassiliadis enrichment broth (Oxoid), and, upon completion of filtration, filters were transferred to EF-18 agar plates (Iso-Grid, M-Tech Diagnostics). After incubation for 24 h at 42 C, fluorescent colonies were detected using an ultraviolet lamp (UV light; 365 nm). In order to aid in the detection of weak fluorescent colonies, MU- CAP-test reactive (Biolife, Milan, Italy) was employed. Presumptive salmonellae that provided typical reactions on triple-sugar iron agar (Oxoid) and lysine iron agar (Oxoid) slants were inoculated into API 20E strips (biomérieux España, S.A.). Final confirmation and serotyping of isolated samples were performed at the National Salmonella Reference Laboratory (Centro Nacional de Microbiología, Virología e Inmunología Sanitarias, Majadahonda, Madrid, Spain). The 979 strains picked from TSA plates were examined for Gram reaction (12), cell morphology (41), presence of spores (41), motility (7), oxidase (7), and catalase reactions (7) and for Oxidation/Fermentation test (7). They were then grouped together according to the criteria of Mossel et al. (26), further tested for the most relevant characteristics of each group, and identified to the genus level by the simple key schemes of the above authors. Antimicrobial susceptibility testing. Identified isolates of P. shigelloides and Salmonella were examined for antimicrobial resistance using the disc diffusion method. Mueller-Hinton agar (Oxoid) and the following antimicrobial discs (Mast Diagnostics, Mast Laboratories Ltd., Merseyside, UK) were employed: ampicillin (25 g), chloramphenicol (150 g), colistin (100 g), gentamicin (10 g), kanamycin (30 g), nalidixic acid (30 g), nitrofurantoin (50 g), streptomycin (25 g), tetracycline (100 g), ticarcillin (75 g), trimethoprim (2.5 g), and sulphamethoxazole (50 g). Tests were conducted as described by Krumperman (22), and the multiple antibiotic resistance (MAR) index was also calculated according to Krumperman (22). The MAR index is defined as a b, where a represents the number of antibiotics to which the isolate was resistant and where b represents the number of antibiotics to which the isolate was exposed. Statistical analysis. The Statistica for Windows Release 4.5 software (Statsoft Ltd., Chicago, Ill.) was used to determine the correlation between total counts performed at 30 and 7 C as well as to evaluate statistical differences (by a Student s t test analysis) between means of microbial counts (after log 10 transformation). RESULTS AND DISCUSSION Bacterial counts. Bacterial levels for each species of fish (surfaces, gills, and intestinal contents) and those for waters from which the fish were caught are given in Table 1. Although it is widely accepted that the initial microbial load of freshwater fish varies depending on water conditions and temperature, wild and farmed specimens taken from cold and relatively unpolluted waters generally yield aerobic counts (mesophiles and psychrotrophs) of 10 2 to 10 5 CFU/cm 2 of skin surface and of 10 3 to 10 6 CFU/g of gills. Numbers in the intestinal contents of striped bass and salmonids may be high ( 10 8 CFU/g), but most of the available literature on different freshwater fish species (tilapia, striped bass, white amur, rainbow trout, catfish, etc.) reports bacterial counts of 10 4 to CFU/g (1, 11, 29 33, 42, 43, 46, 48). In this study (Table 1), mesophilic (AMC) and psychrotrophic counts for skin, gills, and intestines were in the range found in earlier studies of wild and aquacultured freshwater fish, with surface AMCs lower than the m value ( CFU/cm 2 ) recommended by the International Commission on Microbiological Specifications for Foods (ICMSF) (17) for whole marine and freshwater fish. With only two exceptions, counts at 7 C were higher than those at 30 C, but the differences were not statistically significant (P 0.05). The relationship between counts of

3 1272 GONZÁLEZ ET AL. J. Food Prot., Vol. 62, No. 11 TABLE 1. Counts from the skin, gills, intestinal contents, and water of freshly caught wild and farmed freshwater fish Variable Brown trout (wild) (mean standard error) a Pike (wild) (mean standard error) Rainbow trout (farmed) (mean standard error) Mesophilic counts b Psychrotrophs Anaerobes Enterobacteriaceae Enterococci A c AB A A A AB A A A A A A A A A A A A A A B B A A B B A A B B B A A A A A ND d A A A AB A B A B A B A B A B A A B B A ND A ND A a CFU/cm 2, CFU/g, or CFU/ml. b Aerobic plate count at 30 C. c Means in the same row followed by the same letter are not significantly different (P 0.05). d ND, not detected. AMC and psychrotrophs (r 0.89) permitted us to develop an equation (counts at 7 C AMC) that could be used to estimate the psychrotrophic contamination of freshly caught freshwater fish. Aerobic counts for river and pond waters did not differ significantly and compared well with findings in other geographical environments. There were, however, significant differences between numbers on the surface of wild brown trout and pike. The lowest counts from gills and intestines were observed in reared fish, and statistically significant differences were found for some comparisons (Table 1). As was the case for our aerobic counts, numbers of anaerobes, Enterobacteriaceae, and enterococci were not significantly different between water samples. For the skin and gut, anaerobic counts were significantly higher in brown trout, whereas Enterobacteriaceae counts were significantly lower in samples from gills and guts of farmed fish. The incidence and numbers of enterococci detected in water and in some samples were lower than those reported by others (14, 23). The mucus film that covers the pike s surface is thicker than that of the brown trout. This characteristic might have influenced the skin counts (AMCs, psychrotrophs, and anaerobes) for both fish species. properties, such as antibacterial substances in the mucus and the thickness of the slime layer, could potentially influence the bacterial load and shelf life of certain fish species (13, 15, 28). In wild specimens, bacterial numbers in the gut were much higher (by 3 log units) than were those in the surrounding water. This observation, which agrees with data reported by others, indicates that fish intestines provide a favorable ecological niche for some bacteria (15, 47). There is evidence that the use of antimicrobials in aquaculture at both therapeutic and subtherapeutic levels may exert an effect on fish bacterial populations, especially within the digestive tract (4). Unfortunately, data on the use of these compounds were not provided by the sampled facilities; however, it seems likely that the consistently lower counts found in reared rainbow trout could be related to this practice. Types of bacteria. The main bacterial groups detected among the 979 isolates randomly selected from TSA plates incubated at 30 and 7 C were (i) gram-negative aerobic motile rods (Alcaligenes, Pseudomonas, and Shewanella); (ii) gram-negative aerobic coccobacilli and rods (Acinetobacter, Moraxella, and Psychrobacter); (iii) Vibrionaceae (Aeromonas and Vibrio); (iv) Enterobacteriaceae (Ci-

4 J. Food Prot., Vol. 62, No. 11 BACTERIAL MICROFLORA OF TROUT AND PIKE 1273 trobacter, Enterobacter, Edwardsiella, Hafnia, Klebsiella, Proteus, and Providencia); (v) Micrococcaceae (Kocuria, Macrococcus, Micrococcus, Planococcus. and Staphylococcus); (vi) lactic acid bacteria (Carnobacterium, Enterococcus, Lactobacillus, and Lactococcus); (vii) gram-positive bacteria not included within the two latter groups (Bacillus and coryneforms); and (viii) unidentified gram-negative bacteria. Overall, the predominant bacterial groups in brown trout were groups i, ii, iv, and vi, with representatives of group v on their skin. Groups i, ii, v, and vi were dominant in rainbow trout and pond water, whereas groups iii and iv were the most prevalent in pike and the in water in which these fish were caught. Percent distribution of these bacterial groups and genera in each fish species and surrounding waters is given in Tables 2 and 3 for isolates from TSA plates incubated at 7 and 30 C, respectively. Motile and nonmotile aerobic gram-negative bacteria together with a varied mixture of Enterobacteriaceae were detected in all water samples, where they accounted for 50 to 70% of the psychrotrophic isolates. In river water, motile aeromonads were readily isolated from the environment surrounding pike specimens (ca. 40% of psychrotrophs; Table 2), but they were not detected in those waters in which brown trout were caught. The incidence and types of grampositive bacteria were also different. Carnobacterium and coryneforms accounted for nearly 30% of psychrotrophs isolated from brown trout environments, whereas Kocuria accounted for only ca. 10% of the psychrotrophic isolates from pike environments (Table 2). Flowing waters are complex ecosystems, and consequently, microbiological communities, like other biological communities, may vary not only between rivers of the same regional, geological, and climactic conditions but also from site to site and along the length of a river. Overall, the taxonomic composition of the microflora that we found in water from rivers and ponds is characteristic of freshwater environments and, as generally recognized, is dominated by gram-negative aquatic bacteria. Enterobacteriaceae, Micrococcus, Bacillus, lactic acid bacteria, and coryneforms are also frequently reported in varying proportions (3, 4, 15). Our findings do not support the generally accepted thesis about the influence of the environment on the bacterial flora of fish. Thus, it was common that microorganisms that predominated or that were a significant part of the microflora recovered from the outer surfaces of fish were not detected among water isolates. For instance, the skin of brown trout and the gills of rainbow trout carried significant numbers of psychrotrophic Micrococcaceae, which were absent in the water samples; similarly, psychrotrophic coryneforms present in both the skin and gills of pike were not isolated from the surrounding water (Table 2). Acuff et al. (1) reported that the distribution of microbial types of water and tilapia differed, whereas in aquacultured striped bass, Nedohula and Westhoff (30) found a close relationship between the water, skin, and gill isolates. The variety of microorganisms in the intestinal tract has been related to the environment as well as to the quantity and origin of food consumed by fish (3, 15, 18). It should be noted that the diet of brown trout largely consists of insects, molluscae, and crustaceans (10), whereas pike is a piscivorous fish (6). Furthermore, it has been suggested that the indigenous gut microflora may play a role in the digestion of macromolecules and in the prevention of colonization by pathogenic bacteria through the production of certain enzymes and antibacterial compounds, respectively (39, 47). In this study, Aeromonas represented nearly 65% of the psychrotrophic gut isolates from pike, and Carnobacterium were dominant (nearly 40% of psychrotrophs) in brown trout intestinal samples, the latter genus also being present (ca. 30%; Table 2) in rainbow trout gut. A significant number of aeromonads produce amylases and proteases (39, 40), and the ability of lactic acid bacteria, in general, and of Carnobacterium, in particular, to produce bacteriocins and other inhibitory compounds is well known (38). The intestines of reared fish yielded high numbers of psychrotrophic Bacillus and coryneforms (ca. 45%; Table 2). This combined with the absence of Enterobacteriaceae in all sampling sites could again be related to the use of antimicrobials in aquaculture (4). Pathogenic bacteria. L. monocytogenes was not found in any of the examined samples. L. monocytogenes is a widespread microorganism that may be found in surface waters and in freshwater fish (19, 23, 29, 30, 44, 45). In farmed fish, its prevalence is usually low, though a combination of circumstances can lead to a high incidence of this organism (19). In a number of surveys (30, 32, 33), as in ours, no isolations were made from striped bass, rainbow trout, pacu, and tilapia grown in different production systems. It seems likely that the occurrence of L. monocytogenes in water and fish varies according to a number of factors, including major water contamination (sewage, drainage from abattoirs, wild birds, etc.), aquaculture system, and rearing and management practices (18, 19). One isolate of P. shigelloides (strain 313) was recovered from the intestinal content of a pike and another one (strain 314) was recovered from the surrounding habitat. Samples yielding Plesiomonas strains were collected in summertime. Strain 313 belonged to the serovar O38:H unknown (International Antigenic Scheme), whereas strain 314 belonged to the serovar O29:nonmotile (Schubert s Scheme). Both P. shigelloides strains shared resistance to ampicillin, streptomycin, and ticarcillin as well as sensitivity to the rest of the tested antimicrobial compounds, with the exception of sulphametoxazole. Whereas strain 314 was resistant to this last compound, strain 313 showed no conclusive result. MAR indexes were 0.27 (strain 313) and 0.35 (strain 314). P. shigelloides is usually found in aquatic environments and may also be isolated from farmed and wild fish (21). This organism appears to be of interest in aquaculture because it may be a significant component of the intestinal microflora in fish from ponds and flow-through tanks (29, 30). In temperate climates, there is a marked seasonal variation in the numbers of P. shigelloides isolated from water, which may be related to the inability of most strains to

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7 1276 GONZÁLEZ ET AL. J. Food Prot., Vol. 62, No. 11 grow at temperatures below 8 C (20, 21). As mentioned previously, our plesiomonads strains were recovered in summertime from the gut of a pike specimen and from the pike s surrounding habitat. Rather surprisingly, this is the first report on the recovery of plesiomonads from Spanish inland waters and freshwater fish. One of the serovars found (O38), which accounted for 3.8% of the isolates obtained by Aldova (2) from different samples, has been involved in episodes of diarrhea (E. Aldova, personal communication). The antibiotic-resistance patterns and MAR indices of both strains indicate that they likely came from high-risk environments (22). Two identical strains of Salmonella enterica subsp. enterica I serotype Typhimurium 4,5,12:i:1,2 and lysotype 104b were detected in pond-water samples taken from the same facility on different sampling days. Both strains were only resistant to ampicillin, streptomycin, tetracycline, ticarcillin, and sulphametoxazole (with no conclusive results related to nitrophurantoin); their MAR index was Salmonella can occur in both the growing waters and in the fresh fish from rearing ponds, but low incidence and even absence is not uncommon (29, 31, 32, 33). Although the presence of Salmonella in water depends on many factors (44), and although feed should never be overlooked, we suspect that pollution from a nearby poultry farm could be the potential source of the two identical Salmonella isolates that were obtained from pond-water samples collected on different days. Furthermore, the MAR index (0.45) suggests that these strains originated from a source of contamination, such as poultry, in which antibiotics are often used. The presence of multiple antibiotic-resistant pathogenic bacteria in surface and pond waters as well as in fish is of serious concern because it may hamper the efficacy of medically important drugs. The fact that resistance characteristics can be transferred to nonresistant recipient cells adds yet another dimension to this problem. ACKNOWLEDGMENTS The authors thank Mr. Carlos Bayón for providing us with the brown trout specimens and the Department of Animal Biology (University of León, Spain) for providing us with pike. This work was supported by a grant from the Spanish Comisión Interministerial de Ciencia y Tecnología (project no. ALI ). Dr. González-Serrano was the beneficiary of a fellowship from the Spanish Ministerio de Educación y Ciencia. REFERENCES 1. Acuff, G., A. L. Izat, and G. Finne Microbial flora of pondreared tilapia (Tilapia aurea) held on ice. J. Food Prot. 47: Aldova, E Plesiomonas shigelloides in the Czech and Slovak Republics. Alpe Adria Microbiol. 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