Microbiota of freshwater catfish species , Filhote ( Brachyplatystoma filamentosum ) and Dourada ( Brachyplatystoma rousseauxii ) from the Amazon Region ( Belém-Pará-Brazil )

This paper identified the bacterial flora composition and its influence on the microbiological spoilage process in economically important fish species: Filhote (Brachyplatystoma filamentosum) and Dourada (Brachyplatystoma rousseauxii) in the Amazon region. Microbiological characterization was performed: counts of total mesophilic aerobic bacteria, psychrotrophic bacteria and coliforms at 35 and 45°C, respectively. Bacteria were also isolated through seeding in agar surface using Violet Red Bile Glucose (VRBG) for enterobacteria strains and Baird-Parker Agar with Egg-Yolk Tellurite for Staphylococcus species, both with incubation at 36°C for 48 h. The bacteria isolated were identified using the API 20E kit (Enterobacteria), and Gram-positive bacteria with API Staph (Staphylococci). Finally, the lag phase for strain growth was measured using spectrophotometry readings (620 nm) at different temperatures (10, 15 and 37°C). The mesophilic aerobic bacteria counts for fresh fish samples ranged from 5.21 to 7.64. The count ranges of psychrotrophic aerobic bacteria found were 5.11 – 6.91 log CFU/g. They also had an average score above 10 3 MPN/g for total coliforms. The most predominant were Hafnia alvei, Pseudomonas luteola, Staphylococcus xylosus and Staphylococcus lugdunensis. When subjected to temperature of 10°C, the strains achieved growth after 6 h (p<0.05).


INTRODUCTION
Fish is one of the most nutritionally complete foods due to the availability of large amounts of essential nutrients such as high-biological-value protein, vitamins-especially A and D, lipid fraction, high unsaturated fatty acids content and low cholesterol concentration (Córser et al., 2000;Koffi-Nevri et al., 2011).These nutrients present in fish provide a good medium for microbial growth responsible for the overt spoilage and thereby establishes product shelf life (Oliveira et al., 2008;Gram, 1995;Gram and Huss, 1996).
The spoilage of fresh fish by microbial activity is usually due to its microbiota located mainly in the outer surfaces (skin and gills) and in the intestines of live and newly caught fish (Amaral and Freitas, 2013).It can also be the consequence of fish cross-contamination associated with inappropriate handling and storage (Cruz-Romero et al., 2008).It is known that the flora in tropical fish often carries a slightly higher load of Gram-positive and enteric bacteria than fish from temperate waters.But also, it can be similar to that flora dominated by psychrotrophic Gram-negative, rod-shaped bacteria belonging to the genera Pseudomonas, Moraxella, Acinetobacter, Shewanella, Flavobacterium, Vibrionaceae, Aeroemonadaceae and to a lesser degree, Bacillus, Micrococcus, Clostridium, Lactobacillus and Corynebacterium (Liston, 1980;Apun et al., 1999;Austin, 2002;ICMSF, 2005).
In the Amazon River (Brazil), fish species represent one of the most important resources and significantly contribute to the local economy, playing a vital role in the local diet as one of the primary sources of protein for the majority of the population (Angelini et al., 2006).The catfish including filhote and dourada constitute a valuable resource and are exported to several countries (Angelini et al., 2006).
Filhote or piraiba (Brachyplatystoma filamentosum) is one of the most consumed fish species in the Amazon region.It holds great commercial value for its good taste and high yield in the filleting process.It can reach up to 2.50 m in length and 300 kg in weight.It is also considered the largest species of catfish from South America and one of the world's largest (Petrere Junior et al., 2004).
Brachyplatystoma rousseauxii is a large (>1.5 m) migratory catfish of the family Pimelodidae, commonly known as dorado in Bolivia and dourada in Brazil (Carvajal-Vallejos et al., 2014).It is one of the most emblematic species of the Amazon basin, owing to its economic importance and exceptional life cycle, which involves the largest known freshwater migration (Barthem and Goulding, 1997;Alonso, 2002).
The specific microbiota of dourada and filhote are not yet known.The objective of this paper was to identify microbiota and to determine the lag phase of isolated microorganisms to improve the refrigeration temperature conditions in the fish species filhote (Brachyplatystoma

Fish samples
Successive commercial-sized fish samples (n=4) (Filhote and Dourada) were collected between March and May 2014.At the time of collection, the samples were placed in sterile bags kept under refrigeration (around 10°C) and transported to the Laboratory of Food Microbiology (Federal University of Pará -UFPA) for further analysis.

Microbiological analysis
To analysis, 25 g of each sample (ventral part of the filet) were aseptically collected and added to 225 mL of 0.1% sterile peptone water (SPW), thus obtaining 1:10 dilution, which were homogenized in a stomacher (STOMACHER 400 CIRCULATOR SEWARD) at 2,300 rpm for 30 s. Next, counts of total mesophilic aerobic bacteria, psychrotrophic bacteria and coliform at 35 and 45°C were performed according to Brazil (2003).The total mesophilic aerobic and psychrotrophic bacteria counts were carried out in pour plate using plate count agar followed by incubation at 35°C/48 h for mesophilic and 7°C/10 days for psychrotrophic bacteria.Coliforms at 35 and 45°C were counted through the most probable number (MPN), with three sets of three tubes.Lauryl sulfate tryptose broth (LST) was used as a presumptive medium and incubated at 35°C for 24-48 h.The positive tubes were transferred to brilliant green bile broth 2% (GB) and Escherichia coli (EC) broth.The former was incubated at 35°C/24 -48 h for confirmation of total coliforms and EC broth tubes were incubated in a water bath at 45.5°C/24 h for confirmation of thermo-tolerant coliforms.

Bacteria isolation
The homogenized matter used for microbiological characterization was subsequently used for bacteria isolation.Colonies were isolated from VRBG and Baird-Parker with egg-yolk Tellurite plates after incubation for 48 h at 36°C.Next, one plate was selected for each medium and 5-10 colonies per plate were randomly chosen.The selected colonies were sub-cultured in VRBG or Baird-Parker agar plates.After incubation at 36°C/48 h, one colony was transferred from each plate to brain heart infusion (BHI) with 10% glycerol and stored in a freezer to be used for further tests.

Bacterial strain identification
The bacteria isolated were previously identified with Gram stain tests.Next, Gram-negative strains were identified using the API20E kit (Enterobacteria), and Gram-positive strains with API Staph Table 1.Mean values of microbiological characterization in fresh fish.(Staphylococci).The procedure was in accordance with the manufacturer's recommendations (Biomérieux, France) (Harrigan, 1998).

Statistical analysis
Tukey's test was applied to evaluate the difference of means among microorganism groups (mesophilic and psychrotrophic bacteria, total and thermotolerant coliforms) found in different fishes.The optical density (lag phase determination) data were subjected to ANOVA considering different groups of isolated microorganisms.The software Statistica 8.0 was applied considering a 95% level of significance.

Microbiological characterization
The mesophilic and psychrotrophic aerobic bacteria count, as well as total and thermotolerant coliform values, showed variation considering 95% significance level.The mesophilic aerobic bacteria counts for fresh fish samples ranged from 5.21 to 7.49 log CFU/g for filhote and 5.14 to 7.64 log CFU/g for dourada (Table 1).Brazil (2001) does not establish microbiological standards for mesophilic bacteria count in fresh fish.However, the International Commission on Microbiological Specifications for Foods (ICMSF, 1986) recommends the limits for mesophilic aerobic should not exceed values of 10 7 CFU/g or cm 2 in chilled fish samples for human consumption.Considering this limit, most of the samples were within the standards (except sample 1 for Filhote and sample 4 for Dourada).
Thus, it appears that mesophilic bacteria are relevant in characterizing the food handling conditions, so it is very important that the current legislation set limits for these microorganisms in fresh fish in order to ensure higher quality (Lopes et al., 2012).
The count of psychrotrophic aerobic bacteria ranged from 5.11 to 6.91 log CFU/g for filhote and from 5.17 to 5.65 log CFU/g for dourada (Table 1).The Brazilian legislation establishes no maximum limit for psychrotrophic microorganisms in fish for human consumption.But considering the ICMSF (1986), filhote and dourada species showed values above the maximum limit (10 7 CFU/g).
In refrigerated fish, the psychrophilic and psychrotrophic bacteria play direct roles in fish deterioration because they multiply well in these conditions (Franco et al., 1996).Bal'a et al. (2000) found psychrotrophic counts of about 10 3 -10 7 in fresh fillets of channel catfish (Ictalurus punctatus).Lazarin et al. (2011) found psychrotrophic values of 6.54 log CFU/g in pintado (P.coruscans) fillets.Rodrigues et al. (2008), while studying the quality of tilapia (Oreochromis niloticus), observed heterotrophic aerobic psychrotrophic microorganism counts between 0 and 7.90 log CFU/g in skinned muscle.Although, the current legislation does not establish limits, the ability of these microorganisms to deteriorate fish through proteolytic processes, even at freezing temperatures, is widely known, which would reduce the product's shelf life (Santos et al., 2008).
The Filhote and Dourada collected had an average score above 1,100 MPN/g for total coliforms (Table 1).Although, this does not indicate the presence of pathogens, total coliforms are important indicators of potential product deterioration and its mean shelf life (Agnese et al., 2001).Brazil (1997) and Brazil (2001) set the value of 10 2 MPN/g as the maximum acceptable standard for thermotolerant coliforms in fish and fishery products.Except for samples 4 for Filhote and 1 and 4 for Dourada, the others showed values that exceeded those established by the Brazilian legislation.Lopes et al. (2012) found total and thermotolerant coliform values ranging from 3.0 to 93 MPN/g in brackish-water grey snapper (Cynoscion acoupa).Araújo et al. (2012) observed values of 2,400 MPN/g and 11 to 150 MPN/g for total and fecal coliforms, respectively, in freshwater tambaqui (Colossoma macropomum).Oku and Amakoromo (2013) found values of 15 to 43 MNP/g for thermo-tolerant coliforms in the freshwater fish species C. angularis, C. obscura and C. auratus.
Total and thermo-tolerant coliforms are indicators of hygienic quality, not representing direct contact of the product with human or animal feces, but showing the degree of microbial pollution to which the food has been exposed.This score thus indirectly reflects the quality of production practices.

Bacterial strain identification
Among the 51 isolates, it was observed that 52.94% were Gram negative and the other 47.06% were Gram positive.From the results of the Gram stain test, the kits were selected to identify the strains.The data show that Gram-negative bacteria belonged to 15 different species, described in Figures 1 and 2 luteola for Filhote (11.1%).In addition, the incidence of Serratia marcencens, Klebsiella oxytoca, Stenotrophomonas maltophilia and Aeromonas hidrophyla was observed, which are opportunistic pathogens but can also cause fish spoilage (Holt et al., 1994).
H. alvei is widely distributed in nature and has been found in a variety of mammals, fish and birds, and also in soil, sewage, freshwater and a number of foods such as meat and dairy products (Lindberg et al., 1998, Vivas et al., 2008).The pathogenicity of H. alvei is important in the food industry and infection outbreaks have been reported leading to septicemia in commercial laying hens, pullets and rainbow trout (Janda and Abbott, 2006;Liu et al., 2007;Crandall et al., 2006).In humans, H. alvei has been shown to be predominantly associated with several intestinal disorders, including gastroenteritis.Outbreaks or case reports of Hafnia associated with enteric infections have been chiefly reported (Hernandez-Milan and Nenendez-Rivas, 1998;Laguna et al., 1992;Orden and Franco, 1994;Reina et al., 1993;Reina and Borrell, 1991;Seral et al., 2001).
The genus Pseudomonas is known to change many protein-rich foods such as milk, eggs, meat, marinebased foods such as fish and shrimp, and vegetables.According to Franco and Landgraf (2006), pseudomonas are important in food due to their intense metabolic activity, being able to use a wide variety of organic compounds and produce water-soluble pigment and proteolytic and lipolytic enzymes.
P. luteola has been identified as a cause of infection in patients with underlying medical disorders (Otto et al., 2013;Anzai et al., 1997;Kiska andGilligan, 1999, Connor et al., 1987;Hawkins et al., 1991;Rohav et al., 1995).The normal habitat of P. luteola is unclear; it is frequently found in soil, on plants, and in aqueous and damp environments (Freney et al., 1988;Silver et al., 1985;Hawkins et al., 1991).Pseudomonas aeruginosa is recognized as belonging to the normal flora of plant surfaces, human skin and animals, and can form biofilms on some surfaces or substrates (Maia et al., 2009).The P. aeruginosa species is the most important opportunistic pathogen in humans (Massaguer, 2006;Maia et al., 2009).
The genus Staphylococcus contains 41 validly described species (DSMZ, 2008) that are traditionally grouped into coagulase-positive (CPS) and coagulasenegative staphylococci (CNS).The CNS S. xylosus strains play a significant role in food production.They can be used as starter cultures for the production of fermented meat products such as fermented sausages (Hammes and Hertel, 1998;Chajecka-Wierzchowska et al., 2015).They can also degrade biogenic amines in vitro (Martuscelli et al., 2000).S. xylosus have been identified in high numbers in dried salted cod (Vilhelmsson et al., 1997;Doe and Heruwati, 1988) and are rarely associated with human or animal infections (Kloos and Schleifer, 1986).S. lugdunensis is a common human skin commensal (Bellamy and Barkham, 2002;Vandenesch et al., 1995;Van der Mee-Marquet, 2003).These bacteria display pathogen characteristics, although they do not belong to this group, and exhibit pathogenicity similar to Staphylococcus aureus, with high associated morbidity and mortality (Cercenado, 2009;Frank and Patel, 2008;Poutanen and Baron, 2001).Considering that S. lugdunensis is not part of the normal fish microbiota, its presence in fish means there have been contamination from human sources.

Lag phase
Among the different 23 strains isolated, 13 bacteria were selected to determine the lag phase under different temperature conditions (Figures 4, 5 and 6).There was a significant variation (p<0.05) in optical density of the species studied at temperatures of 37, 15 and 10°C (Tables 2, 3 and 4   The lag phase of the majority of the bacteria studied (except E. sakazakii) at 37°C was approximately 2 h (Figure 4).At 15°C, the lag phase was also at least 2 h  This means that a safe condition of refrigeration temperature/time to prevent the multiplication of these microorganisms is about 10°C/6 h.Damasceno et al. (2015) noted that 16 strains isolated from Piramutaba (Brachyplatystoma vailantii) and Butterfly peacock bass (Cichla ocellaris) did not achieve growth for 6 h at 10°C.This result is consistent with the FDA (2011), which suggests that raw fish should be kept at 10°C throughout processing to inhibit the growth and toxin production of pathogenic bacteria.Thus, the adoption of correct measures in the fish industry, such as appropriate conservation through cold treatment and maintaining hygienic practices, tends to reduce the risk of transmitting the disease-causing agents and is able to produce a quality product at the end of the production chain (Lopes et al., 2012).

Conclusion
The microbiological assessment of filhote and dourada suggests these Amazon fish species sold in the Ver-o-Peso market have mesophilic and psychrotrophic bacteria counts within the limits set by the Brazilian legislation.The microorganisms selected after isolation underwent a significant variation (p<0.05) when they were subjected to different refrigeration temperatures for up to 10 h, which yields the optimal refrigeration temperature/time condition (10°C/6 h) to prevent the multiplication of these microorganisms.

*
Different letters in each column indicate difference at 95% level of significance.
and at 4 h, E. clocae, E. sakazakii and K. oxytoca were already in log phase.When they were exposed to temperatures of 10°C, the lag phase was at least 6 h.