Microbiological quality of salmon (Salmo salar) sold in cities of the state of São Paulo, Brazil

Nespolo, Natália Maramarque; Martineli, Thaís Mioto; Rossi Jr., Oswaldo Durival

doi:10.1590/S1517-83822012000400021

Abstract

The present paper evaluated the microbiology of salmon by quantifying mesophilic heterotrophic microorganisms, total and thermotolerant coliforms, and the presence of Vibrio parahaemolyticus, Staphylococcus aureus, Salmonella sp., Escherichia coli and Aeromonas sp. in the meat. This study can provide technical support for the suggestion of a new regulation of a Brazilian legislation through specific microbiological standards concerning the consumption of raw fish. A number of 31 (16 cooled and 15 frozen) samples of salmon were collected in the retail market network of a few cities in the State of São Paulo, Brazil. Results presented populations of mesophilic heterotrophic microorganisms ranging from 1.0 x 10 and 3.9 x 10(6) CFU/g, total and thermotolerant coliforms in 32.25% and 19.35% of the samples, respectively, and Aeromonas sp. in 41.95% of the samples with a populational variation ranging from 2.0 x 10² to 8.0 x 10³ CFU/g. Staphylococcus aureus was found in one sample whereas Vibrio parahaemolyticus, Salmonella sp. and Escherichia coli were not found. These results demonstrated the presence of potencially pathogenic microorganisms in fresh fish consumed in Brazil, highlighting the necessity of control measures to avoid public health problems related to the consumption of raw fish.

fish; salmon; microbiology; pathogenic bacteria

FOOD MICROBIOLOGY

Microbiological quality of salmon (Salmo salar) sold in cities of the state of São Paulo, Brazil

Natália Maramarque NespoloI, ^* * Corresponding Author. Mailing address: Departamento de Medicina Veterinária Preventiva, Universidade Estadual Paulista, Via de Acesso Prof. Paulo Donato Castellane, s/n, Bairro Rural, 14884-900, Jaboticabal, SP, Brasil.; Tel.: (+5516) 3209-2646.; E-mail: natinespolo@yahoo.com.br ; Thaís Mioto Martineli^I; Oswaldo Durival Rossi Jr.^II

^IPrograma de Pós-Graduação em Medicina Veterinária Preventiva, Universidade Estadual Paulista, Jaboticabal, SP, Brasil

^IIDepartamento de Medicina Veterinária Preventiva, Universidade Estadual Paulista, Jaboticabal, SP, Brasil

ABSTRACT

The present paper evaluated the microbiology of salmon by quantifying mesophilic heterotrophic microorganisms, total and thermotolerant coliforms, and the presence of Vibrio parahaemolyticus, Staphylococcus aureus, Salmonella sp., Escherichia coli and Aeromonas sp. in the meat. This study can provide technical support for the suggestion of a new regulation of a Brazilian legislation through specific microbiological standards concerning the consumption of raw fish. A number of 31 (16 cooled and 15 frozen) samples of salmon were collected in the retail market network of a few cities in the State of São Paulo, Brazil. Results presented populations of mesophilic heterotrophic microorganisms ranging from 1.0 x 10 and 3.9 x 10⁶ CFU/g, total and thermotolerant coliforms in 32.25% and 19.35% of the samples, respectively, and Aeromonas sp. in 41.95% of the samples with a populational variation ranging from 2.0 x 10² to 8.0 x 10³ CFU/g. Staphylococcus aureus was found in one sample whereas Vibrio parahaemolyticus, Salmonella sp. and Escherichia coli were not found. These results demonstrated the presence of potencially pathogenic microorganisms in fresh fish consumed in Brazil, highlighting the necessity of control measures to avoid public health problems related to the consumption of raw fish.

Key words: fish, salmon, microbiology, pathogenic bacteria.

INTRODUCTION

Fish is a highly digestible food and a source of high biological value and polyunsaturated fatty acids. It can be consumed by people at any age as well as by convalescent patients. It also plays an important role for fetuses and newborns regarding brain cell development. On the other hand, such outstanding protein compound with its high water content turns fish into an excellent substratum for microbial development (22, 34, 35).

Among the potentially pathogenic microorganism that can be conveyed through fish are Salmonella sp., Vibrio parahaemolyticus, Escherichia coli, Staphylococcus aureus, and Aeromonas sp., which reach fish through environmental contamination during food processing processes, ranging from capture to its preparation for consumption (9, 12, 16).

Fish consumption - including fresh raw fish - has increased due to the findings provided by nutrition and food science. Salmon (Salmo salar) is among the fish whose consumption as a fresh raw food has gradually increased, mainly presented as sushi and sashimi. As a result, hygienic-sanitary quality should be a matter of greater concern for this kind of food consumption as exposing consumers to different pathogenic microorganisms might lead to simple gastroenteritis and even death (16, 32).

Several studies reveal the importance of keeping fish in quality hygienic-sanitary conditions as well as the concern with pathogenic microorganisms that might affect the human being (5, 6, 9, 10, 15, 20, 23, 24, 36, 37, 38).

Therefore, the present study was developed towards an evaluation of salmon microbiology through quantifying mesophilic heterotrophic microorganisms, total and thermotolerant coliforms, in addition to the risk of infection by Vibrio parahaemolyticus, Staphylococcus aureus, Salmonella sp., Escherichia coli and Aeromonas sp. that might be found in the fish.

MATERIALS AND METHODS

From June 2007 to April 2008, a number of 31 samples of salmon (Salmo salar) were collected from the local retail market in the cities of Araraquara, Jaboticabal, Ribeirão Preto and São Carlos, in the Northeast of the State of São Paulo. The samples were taken in fillets or portions weighing approximately 500 g. From the total amount of samples, 16 were cooled whereas 15 were frozen samples. The samples were properly packed in ice, placed inside isothermal boxes. Next, they were taken to the laboratory to be analyzed. According to the APHA methodology (3), 50 g of each sample were immersed in 450mL of 0.1% peptone water (Himedia, Mumbai, India). After a 1-minute homogenization process, an initial 10^-1 dilution was obtained. Next, decimal dilutions were prepared up to 10^-5 using 0,1 mL of the dilution used before. The standard count in mesophilic heterotrophic microorganism plate counts was performed after the dilutions as well as the determination of the most probable number (MPN) for total coliforms/g and thermotolerant coliforms/g, test forEscherichia coli, Staphylococcus sp. count, Staphylococcus coagulase-positive count and test for Staphylococcus aureus, test for Salmonella sp. and determination of MPN for Vibrio parahaemolyticus (3).

For Aeromonas sp. isolation, 25 g of each sample were immersed in 225 mL of tryptone soya broth (TSB) (Himedia, Mumbai, India) with ampicillin (30 mg/L) (Sigma, Steinheim, Germany) under incubation at 28ºC for 24 hours. After that, an aliquot of this broth was streaked in phenol red-starch ampicillin agar (19, 25) and ampicillin-dextrin agar (13), both containing antimicrobial at a 10 mg/L concentration. The incubation was performed in BOD incubator (Tecnal-390, Brasil) at 28ºC for 24 hours. From both medium, five colonies suggestive of the genus were taken and streaked in tubes with tryptone soya agar (TSA) (Himedia, Mumbai, India), which were incubated at a temperature of 28ºC for 24 hours. After checking morphology and staining through the Gram's method, straight and short rod-shaped bacteria, in pairs, isolated or in short chains and Gram-negative were replicated in triple sugar iron agar (TSI) (Oxoid, Basingstoke, UK) (30). After incubation, cultures presenting acid reaction both in the butt and in the slant, either with or without gas formation, were inoculated in TSA slants and incubated at a temperature of 28ºC for 24 hours. As the oxidase test was performed, the positive cultures were considered as pertaining to the genus known as Aeromonas. The scheme proposed by Popoff (29) and updated by Furuwatari et al. (11) was used for species characterization, supplemented by a few tests recommended by Abott et al. (1), which is made up by performing the following tests: indol production; esculin and arginine hydrolysis; lysine decarboxylase and ornithine decarboxylase; inositol, salicin, sucrose, mannitol, and arabinose fermentation; acetoin production (VP) and gas production from glucose, growth in nutrient broth at 37ºC with 0%, 3%, and 6% of sodium chloride and nitrate reduction (18). For the counting of Aeromonas, phenol red-starch ampicillin agar plate counts were used, prepared as previously described in this paper. The streak was made in the surface in volumes of 0.1 mL of each of five dilutions and incubated in BOD at 28ºC for 24 hours. The counting of colonies presenting features of the gender Aeromonas followed. Up to five characteristic colonies were isolated to confirm the genus. The colonies underwent identification tests as previously presented. The final result for the Aeromonas count was obtained upon the result of the gender identification tests in proportion to the number of colonies found in the plate count, multiplied by ten and by the dilution factor. The results referring to the mesophilic heterotrophic microorganism population underwent the Student's t -test (31) and the results from the other determinations were analyzed through the nonparametric chi-square test (8).

RESULTS AND DISCUSSION

As presented in Table 1, the population of mesophilic heterotrophic microorganisms varied from 10 to 3.9 x 10⁶ CFU/g, with 16.13% of the samples presenting populations above 10⁵ CFU/g. The population varied from 1.1 x 10³ to 3.9 x 10⁶ CFU/g for the cooled product and from 10 to 4.2 x 10⁴ CFU/g for the frozen product.

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While studying frozen freshwater fish, Aquino et al. (5) found higher values pointing to a variation from 3.0 x 10³ to 2.5 x 10⁷ CFU/g. In comparison to the results obtained in the present study, such variations indicate a reasonable condition presented by frozen salmon as well as the importance of such conservation method in order to maintain the initial microbiological features of the product so as to avoid deterioration. Populations above 10⁵ CFU/g have also been studied by Muratori et al. (23) in 55.9% of fresh samples taken from the so-called branquinha fish (Curimatus ciliatus).

The mesophilic population average among the cooled and frozen samples amounted respectively to 5.0 x 10⁵ CFU/g and 4.4 x 10³ CFU/g, leading to a statistically significant difference (p<0.01), i.e., freezing was more efficient to retard the growing of microbiological population than cooling. The importance of low temperatures for product conservation was also observed by Soares & Germano (33) in salmon samples used in sashimi and by Lourenço et al. (17) while examining the meat of the crab known as caranguejo-uçá in two localities in the State of Pará, Brazil.

In the absence of parameters in the Brazilian law concerning mesophiles in ready-to-use meals based on fresh raw fish (4), the creation of a standard is recommended for fresh raw fish, helping the authorities with hygienic-sanitary inspection either for seafood or seafood-derived meals.

Table 2 shows that the total coliform population variation ranged from < 3.0 to 1.1 x 10³ MPN/g while thermotolerant coliforms ranged from < 3.0 to 4.6 x 10² MPN/g. The results point to the good hygienic-sanitary quality of the raw material referring to fecal contamination as values < 3.0 MPN/g were observed in 80.64% of the samples for thermotolerant coliforms. The conservation method influenced microorganism population as well as product conservation: among 21 samples presenting a total coliform population < 3.0 MPN/g, 8 were cooled and 13 were frozen. Among 25 samples with the same thermotolerant coliform population, 11 were cooled and 14 were frozen.

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Fang et al. (10) also found low total coliform populations ranging from 2.3 to 6.1 CFU/g in 80.0% of seafood samples and, unlike the present study, the authors found E. coli in 6.0% of the samples.

Soares & Germano (33) evaluated the hygienic-sanitary quality in sashimi sold in shopping malls. Total coliform populations ranged from 1.25 to 7.0 x 10⁴ CFU/g and thermotolerant coliforms varied from < 10 to 4.0 x 10³ CFU/g, where 66.4% of the samples presented thermotolerant coliforms, unlike the present study, where such group was not found in 80.64% of the samples. In another study, the same authors found total coliform populations ranging from 35 to 1.1 x 10⁶ CFU/g in fresh salmon used in sashimi (34), unlike the present study, where this group reached a maximum 1.1 x 10³ MPN/g.

By relating the groups of total and thermotolerant coliforms with the storage and marketing temperatures, it was possible to notice that, for the cooled samples, the total coliform populations varied from < 3.0 to 1.1 x 10³ MPN/g, whereas lower values were found in the frozen samples, which varied from < 3.0 to 4.6 x 10² MPN/g. Considering samples with a result < 3.0 MPN/g as negative ones for total coliforms, the nonparametric chi-square test was applied to the averages that were found and such test revealed a significant difference (p<0.05) between the results, which confirms that freezing is the best conservation procedure for the product.

The population of thermotolerant coliforms in the cooled product varied from <3.0 to 1.5 x 10² MPN/g. In the frozen product, the variation ranged from < 3.0 to 4.6 x 10² MPN/g. In this group, the difference in the number of positive and negative samples between the frozen and cooled products did not prove to be statistically significant (p>0.01).

Pursuant to the results and considering the Resolution RDC no. 12 (4) issued by the Brazilian National Health Surveillance Agency (ANVISA), concerned with raw fish-based, ready-to-use meals, which allows the amount of 10² MPN/g for thermotolerant coliforms, specific legislation should be suggested in Brazil regarding the consumption of fresh raw fish with a more stringent standard for thermotolerant coliforms.

The importance of changing the current standards into specific ones has been also advocated by Martins (20) while evaluating the hygienic-sanitary quality of sushi and sashimi. Martins found thermotolerant coliform populations above 10² MPN/g in 50% of the samples and E. coli in 45%.

In a similar study, Soares & Germano (33) and Meldrum et al. (21) observed the absence of E. coli in the microbiological analysis of fish and fish-derived products. On the other hand, Taulo et al. (36) found E. coli in 49% of the samples of home-prepared fish in Malawi, where 5.0% presented E. coli O157:H7.

The Staphylococcus sp. population varied from < 50 to 1.3 x 10⁴ CFU/g (Table 3), with a variation of < 50 to 1.3 x 10⁴ CFU/g for the cooled product and amounting to < 50 a 2.6 x 10³ CFU/g for the frozen product, which presents freezing as the best conservation procedure. A single cooled sample with 5.5 x 10² CFU/g presented coagulase-positiveStaphylococcus, which shows the importance of practicing healthy habits during product manipulation in order to avoid contamination.

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Similar results such as < 10² and 3.0 x 10⁴ CFU/g have been found by Cunha Neto et al. (9) when analyzing fresh shrimp and processed food, including previously cooked fish. The authors quantified coagulase-positive Staphylococcus in a sample of cooked fish and in a sample of raw shrimp amounting to 9.5 x 10² CFU/g and 4.0 x 10² CFU/g, respectively, which is similar to the sample analyzed by the present paper.

Supporting the data presented by this study, one sample containing coagulase-positive Staphylococcus with a population of 80 CFU/g was found. Albuquerque et al. (2) also found coagulase-positive Staphylococcus in sushi sold in the State of Ceará, Brasil, with a population ranging from < 10² and 1.4 x 10⁶ CFU/g.

The presence of potentially pathogenic microorganisms in raw fish destinated to the human consumption suggests the necessity of a specific regulation for fresh raw fish. Such regulation should contains stringent microbiological standards because several foodborne microrganisms present very low infectious dose, while other pathogens can multiply in fresh fish exposed to abuse temperature.

Aeromonas sp. were identified in direct quantification in 11 samples with populations ranging from 2.0 x 10² and 8.0 x 10³ CFU/g - all of them under refrigeration. The population average was 3.2 x 10³ CFU/g and, among 11 samples, eight (72.73%) presented populations exceeding 10³ CFU/g, which means high populations that could endanger people´s health, mainly through the intake of the product in its natural state, now an increasing habit in Brazil. It was not possible to quantify Aeromonas in the frozen samples, which suggests the absence or a reduced population of such microorganism and shows this conservation procedure as the most adequate to preserve the product and impair contamination through meat consumption.

One analysis performed with fish sold in public markets identified Aeromonas sp. in 62% of 50 samples, with a population variation between 1.95 x 10² and 1.5 x 10⁷ CFU/g (14), unlike the present study, where 64.51% of the 31 salmon samples presented Aeromonas sp. with populations below 10² CFU/g.

Ullmann et al. (37) also found Aeromonas sp. in populations smaller than 10⁴ CFU/g in most (67.9%) of the 84 fish samples, including salmon sold in public markets of Berlin.

Pursuant to the results presented, the importance of seafood as a source of protein for adequate nourishment, mainly when eaten in its fresh, raw state, and the absence of parameters for quantification and research of Aeromonas, it is required to create specific laws.

After selective enrichment, 41.95% of the 31 samples presented Aeromonas sp., among which 76.90% were cooled and 23.10% were frozen. This reinforces the presence of bacteria at low temperatures and the importance of keeping the product frozen.

Azevedo et al. (6) obtained higher results revealing the presence of Aeromonas sp. in 87% of 26 fish samples and in 93% of 28 water samples, which represents risk of serious diseases for human beings.

Similar results for A. caviae (26.1%) and A. sobria (6.0%) were found by Ullmann et al. (37) in a study about the isolation and characterization of Aeromonas sp. in fish samples, including salmon.

Lower results for A. caviae (14.80%) and A. sobria (4.20%), and higher for A. trota (4.20%) were obtained by Pereira et al. (26) while evaluating the presence of emerging pathogens in mussels (Perna perna). The authors identified A. media, A. hydrophila, A. schubertii and A. jandaei as well. A result that surpasses the one found by the present study was also obtained by Azevedo et al. (6), who identified A. veronii, A. allosaccharophila and A. trota as the most frequent species isolated from fish samples. Herrera et al. (14) found similar results for A. sobria in fish samples commercialized in Spain, where 16% were A. caviae and 6.5% were A. sobria among the 31 positive samples for Aeromonas sp.

Considering that some Aeromonas species have been reported as emerging pathogens isolated from human and animal sources, it is extremely important to supervise their presence in seafood and evaluate their risks for Public Health.

None of the samples presented Salmonella and V. parahaemolyticus, which fits the standards set forth by Resolution RDC no.12 (4), regarding ready-to-use meals made from raw fish, in the absence of Salmonella and a maximum 10³ CFU/g of V. parahaemolyticus /g. In agreement with the present study, Aquino et al. (5), Basti et al. (7), Martins (20), and Pereira et a l. (27), while analyzing seafood, fish, food made from raw fish, and oysters, respectively, did not find Salmonella either. Basti et al. (7), Martins (20), and Pereira et al. (27) also researched V. parahaemolyticus, found only by Basti et al. (7) at high levels in salted fish and at lower levels in fresh fish. On the other hand, Lourenço et al. (17) identified Salmonella sp. in crab meat samples. Pereira et al. (28) noticed the presence of V. parahaemolyticus in 7.7% of mussel samples (Perna perna. collected in Rio de Janeiro.

Pursuant to these results and the increase in the consumption of fresh raw salmon, it is extremely important to adopt Good Manufacturing Practices (GMP) during every fish processing stage in order to minimize the risk of contamination by deteriorating or pathogenic bacteria that may cause harm to the health of consumers. Therefore, the participation of government entities through the creation of specific laws concerned with the fresh, raw-consumed fish and through the inspection of such products in every production stage is fundamental to improve the microbiological quality of the fish that is commercialized in Brazil.

ACKNOWLEDGEMENTS

Thanks to CNPq for a Master's scholarship and FAPESP for financial support.

Submitted: April 05, 2010

Returned to authors for corrections: January 30, 2012

Approved: June 07, 2012.

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*

Corresponding Author. Mailing address: Departamento de Medicina Veterinária Preventiva, Universidade Estadual Paulista, Via de Acesso Prof. Paulo Donato Castellane, s/n, Bairro Rural, 14884-900, Jaboticabal, SP, Brasil.; Tel.: (+5516) 3209-2646.; E-mail:

natinespolo@yahoo.com.br

Publication Dates

Publication in this collection
19 Feb 2013
Date of issue
Dec 2012

History

Received
05 Apr 2010
Accepted
07 June 2012
Reviewed
30 Jan 2012

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[1] 1. Abbott, S.L.; Cheung, W.K.W.; Janda, J.M. (2003). The genus Aeromonas biochemical characteristics, atypical reactions, and phenotypic identification schemes. J. Clin. Microbiol. 41 (6), 2348-2357.

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