ABSTRACT:

Fish are considered rich sources of nutrients. Health care throughout its production chain aims to ensure quality, minimizing the risks of transmission of foodborne diseases. In order to evaluate the microbiological quality of trahira fish (H. malabaricus), 40 samples were analysed for Most Probable Number (MPN) of coliforms at 45ºC, counts of aerobic mesophilic bacteria and Staphylococcus spp., identification of E. coli, Salmonella spp. and Aeromonas spp.. Analyses were conducted according to official methods, procedures, and recommendations. Microbiological results showed coliform values at 45 °C ranging from <3 to > 1.1 × 10³ MPN/g, presence of E. coli in 14 (35%) samples, counts of mesophilic aerobic bacteria from 9 × 10² to 10⁹ CFU/g and absence of coagulase positive Staphylococcus aureus. Salmonella spp. was detected in 2 (5%) samples, which is in disagreement with the standards required by the RDC Nº 12 of January, 2001 (ANVISA) regarding Aeromonas spp. In total, 36 (90%) samples were contaminated, 7 (19.4%) by A. cavie and 29 (80.6%) by A. hidrophila. The results of this research showed unsatisfactory hygienic and sanitary conditions of fish from the municipality of São Bento (MA), exposing consumers to the risk of foodborne diseases.

KEYWORDS:
Aeromonas spp.; E. coli; Salmonella spp.; hygiene; sanity

RESUMO:

Os peixes são considerados fontes ricas de nutrientes. Cuidados sanitários durante toda a sua cadeia produtiva visam garantir a qualidade, minimizando os riscos de transmissão de doenças alimentares. Com objetivo de avaliar a qualidade microbiológica de peixes traíra (H. malabaricus), foram analisadas 40 amostras quanto à determinação do Número Mais Provável (NPM) de coliformes a 45 ºC, contagem de bactérias aeróbias mesófilas e Staphylococcus spp. e identificação de E. coli; Salmonella spp. e Aeromonas spp. As análises foram processadas conforme métodos, procedimentos e recomendações oficiais. Os resultados microbiológicos mostraram valores de coliformes a 45 ºC variando de < 3 a > 1,1 × 10³ NMP/g, presença de E. coli em 14 (35%) amostras, contagens de bactérias aeróbias mesófilas de 9 × 10² a 10⁹ UFC/g e ausência de Staphylococcus aureus coagulase positivo. Detectou-se Salmonella spp. em 2 (5%) amostras, portanto, em desacordo com os padrões exigidos pela RDC Nº 12 de janeiro de 2001 (ANVISA) e para Aeromonas spp. No total, 36 (90%) amostras estavam contaminadas, sendo 7 (19,4%) por A. cavie e 29 (80,6%) por A. hidrophila. Os resultados da pesquisa demonstram que os peixes provenientes do município de São Bento (MA) apresentaram condições higiênico-sanitárias insatisfatórias, expondo os consumidores a risco de doenças veiculadas por alimentos.

PALAVRAS-CHAVE:
Aeromonas spp.; E. coli; Salmonella spp.; higiene; sanidade

INTRODUCTION

Among the native species from the wetlands of Baixada Maranhense, Hoplias malabaricus, Bloch, 1794, stands out; popularly known as “trahira”, this is a neotropical fish belonging to the Erythrinidae family, which is world-wide distributed (FOWLER, 1950FOWLER, H.W. Os peixes da água doce do Brasil. Arquivos de Zoologia do Estado de São Paulo, v.6, n.2, p.205-404, 1950.; NELSON, 1994NELSON, J.S. Fishes of the world. 3ª ed. New York: John Wiley & Sons, 1994. 600p.). For human nutrition, fish is a source of proteins of high biological value; however, being a highly perishable food, it requires much care in handling all the way: from the capture process and storage on fishing boats to commercialization (FAO, 2012FOOD AND AGRICULTURE ORGANIZATION (FAO). The state of world fisheries and aquaculture. Rome: FAO, 2012. 209p.; LEITÃO et al., 1997LEITÃO, M.F.F.; RIOS, D.P.F.A.; GUIMARÃES, J.G.L.; BALDINI, V.L.S.; MAINADES PINTO, C.S.R. Alterações químicas e microbiológicas em pacu (Piaractus mesopotamicus) armazenado sob refrigeração a 5ºC. Ciência e Tecnologia de Alimentos, v.17, n.2, p.160-166, 1997.).

Fish also play an important role in transmission of foodborne diseases. Therefore, for the control and prevention of these diseases, indicators of environmental and fecal contamination, through enumeration of coliforms and research of pathogens such as Salmonella spp. and Staphylococcus aureus, are of great importance for public health, and such investigation is essential to determine the quality of the product (LIMA; REIS, 2002LIMA, M.G.; REIS, R.B. Incidência de Salmonella spp.: comparação entre metodologias de detecção em amostras de Pacu (Piaractus mesopotamicus) de rio e cultivado comercializadas no município de Cuiabá - MT. Higiene Alimentar, v.16, n.101, p.43-49, 2002.).

The presence of these microorganisms in the raw material may be related to their environment of development, contamination during landing, processing or storage, which contributes to a poor-quality product (DIAS et al., 2010DIAS, V.L.N.; FERREIRA, E.F.; CORÉIA, G. de A.; SILVA, E.C.R. da; OLIVEIRA, I. de N.; MOUCHEK FILHO, V.E.; LOPES, J.M.; CARVALHO, N.C.C. Avaliação da qualidade de peixe comercializado em Imperatriz, MA. Revista Higiene Alimentar, v.24, n.186/187, p.109-112, 2010.). Contamination may also occur in improper handling and processing, or by the use of contaminated equipment and utensils, also important factors related to presence of bacteria in fish. The most common types are Aeromonas, Salmonella spp., Escherichia coli, S. aureus, Bacillus cereus, Clostridium perfringens and Clostridium botulinum (MURATORI, 2000MURATORI, M.C.S. Consórcio suíno peixe: riscos ambiental e sanitário. Proposta alternativa para descontaminação. 71f. Thesis (Doutorado em Ciência Animal) - Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, 2000.; VIEIRA, 2004VIEIRA, R.H.S.F. Microbiologia, higiene e qualidade do pescado: teoria e prática. São Paulo: Varela , 2004.).

Other pathogenic microorganisms found in fish are bacteria of the genus Aeromonas, which can be found in soil, fresh and salt water, chlorinated water, and animal feces (SOUZA, 2003SOUZA, A.T.S. Certificação da Qualidade de Pescados. Biológico, São Paulo, v.65, n.1-2, p.11-13, 2003.). These bacteria carry several virulence factors, which justify their status of threat as a human pathogen, since they are responsible for diarrhea, extra-intestinal infections, and, in immunocompromised patients, septicemia, meningitis, and death (YAMADA et al., 1997YAMADA, S.; MATSUSHITA, S.; DEJSIRILERT, S.; KUDOH, Y. Incidence and clinical symptoms of Aeromonas-associated travellers’ diarrhoea in Tokyo. Epidemiology and Infection, v.119, n.2, p.121-126, 1997.). Gastroenteritis is the most common form of human infection caused by Aeromonas. In fish, the disease can show a variety of clinical signs, such as exophthalmos, fin erosion and hemorrhagic septicemia, leading to death. Aeromonas are associated with opportunistic infections in humans, as well as in homeothermic animals and in fish, and may be resistant to multiple drugs (JANDA; ABBOTT, 1998JANDA, J.M.; ABBOTT, S.L. Evolving concepts regarding the genus Aeromonas: an expanding panorama of species, disease presentations, and unanswered questions. Clinical Infectious Diseases, v.27, n.2, p.332-344, 1998.; PALU et al., 2006PALU, A.P.; GOMES, L.M.; MIGUEL, M.A.; BALASSIANO, I.T.; QUEIROZ, M.L.; FREITAS-ALMEIDA, A.C.; OLIVEIRA, S.S. Antimicrobial resistance in food and clinical Aeromonas isolates. Food Microbiology, v.23, n.5, p.504-509, 2006. ).

The region of Baixada Maranhense is an complex ecosystem in which human beings play an important role for management, use, and conservation of several of its components. As a consequence of abundant regional water resources, fishing is possibly the most important socioeconomic activity (ARAÚJO; PINHEIRO, 2008ARAÚJO, N.A.; PINHEIRO, C.U.B. Avaliação socioeconômica da pesca artesanal e do potencial aquícola na região lacustre de Penalva - APA da Baixada Maranhense. Boletim do Laboratório de Hidrobiologia, v.21, n.1, 2008.). Considering the economic importance of the artisanal fishing activity in the region, the great availability of fish resulting from floodwaters in the region and the availability of the product for sale in local markets, this research aimed to evaluate the hygienic sanitary conditions and microbiological aspects of trahira fish (H.malabaricus) found in the municipality of São Bento (MA).

MATERIAL AND METHODS

Fish were slaughtered and not eviscerated, acquired in fairs and markets of the municipality of São Bento (MA), between July and December, 2011. Four samples of trahira (H. malabaricus) were collected, each sample consisting of three units (average weight of 250 g per piece) from the same supplier. The samples were separated into sterile polyethylene bags, identified, and packaged in isothermal boxes, then transported to the Laboratory of Microbiology of Food and Water of the Veterinary Medicine Course at Universidade Estadual do Maranhão (UEMA), where they were analyzed according to the American Public Health Association (APHA, 2001AMERICAN PUBLIC HEALTH ASSOCIATION (APHA). Compendium of methods for the microbiological examination of foods. Washington, D.C.: APHA, 2001.), (VANDERZANT; SPLITTSTOESSER, 1992VANDERZANT, C.; SPLITTSTOESSER, D.F. Compendium of methods for the microbiological examination of foods. 3ª ed. Washington, D.C.: American Public Health Association (APHA), 1992. 121p.; SILVA et al., 2001SILVA, N.D.A.; JUNQUEIRA, V.C.A.; SILVEIRA, N.F.A. Manual de Métodos de Análise Microbiológica de Alimentos. 2ª ed. São Paulo: Varela, 2001. 295p.).

Dilution of samples

For the dilution of samples, 25g of product were aseptically weighed and then added to bottles containing 225 mL of 0.1% peptone water, constituting the dilution 10^-1. Then, 1mL of the first dilution was pipetted into tubes containing 9mL of the same diluent, obtaining successive dilutions up to 10^-6.

Determination of Most Probable Number of Coliforms at 45 ºC per gram (MPN/g)

Initially, 1mL of the previously prepared dilutions was withdrawn and transferred to three tubes containing 10 mL of Tryptose Lauryl Sulfate Broth (TLS), which were incubated at 35 ºC/48 h. Aliquots were withdrawn from the gas-formed tubes, which were then inoculated into tubes containing Brilliant Green-bile 2% broth (BG). BG-positive cultures were transferred to tubes with Escherichia coli Broth (EC) and incubated at 44,5-45 ºC/24 h for detection of coliforms at 45 ºC. The results were interpreted using the table of Most Probable Number (MPN). Positive cultures in EC medium were seeded in Eosin-Methylene Blue Agar (EMB) at 35-37 ºC/24 h. Colonies with blackened centers and metallic green sheen were submitted to biochemical tests (Indol production, voges-proskaer test, red methyl test and citrate test) for identification of E. coli, abiding by the American Public Health Association (APHA) standards, as described by SILVA et al. (2001SILVA, N.D.A.; JUNQUEIRA, V.C.A.; SILVEIRA, N.F.A. Manual de Métodos de Análise Microbiológica de Alimentos. 2ª ed. São Paulo: Varela, 2001. 295p.).

Standard Count on Mesophilic Aerobic Bacteria Plate

Aliquots of 1 mL of each dilution were pipetted into sterile Petri plates containing Standard Agar for Counting (SAC) incubated at 35-37 ºC/48 h. For counting, plates of the same dilution presenting 30 to 300 colonies were considered; the result was expressed in colony-forming units (CFU)/mL (SILVA et al., 2001SILVA, N.D.A.; JUNQUEIRA, V.C.A.; SILVEIRA, N.F.A. Manual de Métodos de Análise Microbiológica de Alimentos. 2ª ed. São Paulo: Varela, 2001. 295p.).

Staphylococcus spp. count

From the initial up to 10^-3 dilutions, 0,1 mL were inoculated onto Baird-Parker Agar plates, incubated at 35-37 ºC for 45-48 h. Soon after, the typical colonies were counted and confirmed by the coagulase test. The results were expressed in CFU/g (SILVA et al., 2001SILVA, N.D.A.; JUNQUEIRA, V.C.A.; SILVEIRA, N.F.A. Manual de Métodos de Análise Microbiológica de Alimentos. 2ª ed. São Paulo: Varela, 2001. 295p.).

Research of Salmonella spp.

25 g of the sample unit were weighed, homogenized in 225 mL of peptone water and incubated at 37 ± 1ºC / 18 ± 2 h. Subsequently, 0.1 mL of the sample was transferred to 10 mL of Rappaport-Vassilidis Soy Broth (RVS) and 1mL to 10 mL of Cystine Selenite Broth. Incubation occurred at 37 ºC/24 h. From each culture, Xylose Lysine Deoxycholate Agar (XLD), Hectoen Enteric Agar (HE) and Brilliant Green-bile (BG) broths were prepared. The XLD, HE and BG plates were incubated upside down at 37 ± 1ºC/24 ± 3 h. After incubation, colonies typical of Salmonella spp. were seen, which were later inoculated in Triple Sugar Iron Agar (TSI) and Lysine Iron Agar (LIA) for biochemical proof. Confirmation was made by serological tests, using polyvalent anti-Salmonella sp. antigen, according to ICMSF (1988INTERNATIONAL COMMISSION ON MICROBIOLOGICAL SPECIFICATIONS FOR FOODS (ICMSF). Microrganisms in food: their significance and methods of enumeration. 2 ed. Toronto: Toronto University Press, 1988. 436p.).

Identification of Aeromonas spp.

For pre-enrichment, Tripticase Soy Broth (TSB) and ampicillin (30 mg/L) were used. Aliquots of the cultures grown in TSB were stretched onto plates containing Phenol Red Agar amide-ampicillin (PALUMBO et al. 1985PALUMBO, S.A.; MAXINO, F.; WILLIAMS, A.C.; BUCHANAN, R.L.; THAYER, D.W. Starch-ampicillin agar for the quantitative detection of Aeromonas hydrophila. Applied and Environmental Microbiology, v.50, n.4, p.1027-1030, 1985.; MAJEED et al. 1990MAJEED, K.N.; EGAN, A.F.; MAC RAE, I.C. Production of exotoxins by Aeromonas spp. at 5ºC. Journal of Applied Bacteriology, v.69, n.3, p.332-337, 1990. ) and Dextrin-ampicillin Agar, as suggested by HAVELAAR; VONK (1988HAVELAAR, A.H.; VONK, M. The preparation of ampicillin dextrin agar for the enumeration of Aeromonas in water. Letters in Applied Microbiology, v.7, p.169-171, 1988.), added of ampicillin (10 mg/L) and incubated at 28 ºC for 24 h. Then, colonies were seeded in Trypticase Soy Agar (TSA) and incubated at 28 ºC for 24 h. Gram staining and replication were performed on Triple Sugar Iron Agar (TSI) (SAAD et al., 1995SAAD, S.M.I.; IARIA, S.T.; FURLANETTO, S.M.P. Motile Aeromonas spp in retail vegetables from São Paulo, Brazil. Revista de Microbiologia, v.26, n.1, p.22-27, 1995.). Positive cultures were submitted to oxidase tests, catalase test and resistance to O/129 vibriostatic agent (2,4-diamino-6,7-diisopropylpteridine phosphate). Strains characterized as belonging to the genus Aeromonas were seeded in TSA so the colonies could be kept viable for subsequent analysis by biochemical methods, according to Aerokey II classification key (CARNAHAN et al., 1991CARNAHAN, A.M.; BEHMAN, S.; JOSEF, S.W. Aerokey II: A flexible key for identifying clinical Aeromonas species. Journal of Clinical Microbiology, v.29, n.12, p.2843-2849, 1991.).

RESULTS

All 40 samples of trahira analyzed had coliform values at 45 ºC, ranging from < 3 to > 1.1 × 10³ MPN/g. Regarding E. coli identification, 14 (36.9%) samples were contaminated by this pathogen, which is concerning from the sanitary point of view. In 2 (5%) samples Salmonella spp. was detected, which is in disagreement with standards established by RDC Nº 12, January 200, Agência Nacional de Vigilância Sanitária (ANVISA). Aerobic mesophilic microorganisms counting ranged from 9 × 10² to 10⁹ CFU/g (Table 1).

Staphylococcus spp. was present in 18 (45%) samples, with population ranging from 10² to 1.8 × 10⁸ CFU/g, being all negative upon coagulase test (Table 2).

Research of Aeromonas spp showed 36 (90%) samples of trahiras contaminated: 7 (19.4%) by Aeromonas cavie and 29 (80.6%) by Aeromonas hydrophilic (Table 3).

DISCUSSION

Isolation of pathogens and/or indicator organisms contributes to evaluate the quality and safety of food, allowing sanitary control. The RD Nº 12 of January, 2001, ANVISA, determines absence of Salmonella spp. in 25 g and values of up to 10² MPN/g for coliforms at 45ºC as the standard for fish, the latter only for raw consume (BRASIL, 2001BRASIL. Agência Nacional de Vigilância Sanitária (ANVISA). Resolução RDC nº 12, de 02 de janeiro de 2001. Brasília, 2001.). The high density of coliforms at 45 ºC and the presence of E. coli in 14 trahira samples (35%) reflect poor hygiene conditions and displays the degree of contamination this food is exposed to. This contributes to food deterioration and represents a risk to consumer’s health, since five E. coli groups can lead to gastroenteritis in humans (enterotoxigenic E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroinvasive E. coli, and enteroaggregative E. coli) (VILA et al., 2009VILA, J.; ÁLVAREZ-MARTÍNEZ, M.J.; BUESA, J.; CASTILLO, J. Diagnóstico microbiológico de las infecciones gastrointestinales. Enfermedades Infecciosas y Microbiología Clínica, v.27, n.7, p.406-411, 2009.). The results obtained from these indicators can be attributed to fishing activities in polluted areas, exposure to unsanitary conditions such as sources of sewage, increasing the risk of contamination and interfering in the quality of products commercialized. ALVES et al. (2001ALVES, L.M.C.; COSTA, F.N.; SILVA, M.I.; SALES, S.S.; CORREA, M.R. Toxinfecção alimentar por Salmonella enteretidis: relato de um surto ocorrido em São Luís - MA. Higiene Alimentar, v.15, n.80-81, p.57-58, 2001.) consider that the high density may indicate contamination, inefficient heat treatment or multiplication during storage.

Similar results were observed by LORENZON et al. (2010LORENZON, C.S.; GATTI JUNIOR, P.; NUNES, A.P.; PINTO, F.R.; SCHOLTEN, C.; HONDA, S.N.; AMARAL, L.A. Perfil microbiológico de peixes e água de cultivo em pesque-pagues situados na região nordeste do estado de São Paulo. Arquivos do Instituto Biológico, v.77, n.4, p.617-624, 2010. ) in a research involving fish culture, in which coliforms were found at 45 ºC, ranging from < 3 to 5.1 × 10³ MPN/g. An experiment carried out in Egypt, with investigation of pathogenic and potentially pathogenic organisms in tilapia units from the Nile detected E. coli in 7.9% of the samples (YOUSSEF et al., 1992YOUSSEF, H.; EL-TIMAWY, A.K.; AHMED, S. Role of aerobic intestinal pathogens of fresh water fish in transmission of human diseases. Journal of Food Protection, v.55, n.9, p.739-740, 1992.). FERREIRA et al. (2014FERREIRA, E.M.; LOPES, I.S.; PREREIRA, D.M.; RODRIGUES, L.C.; COSTA, F.N. Qualidade microbiológica de peixe serra (Scomberomerus brasiliensis) e do gelo utilizado na sua conservação. Arquivos do Instituto Biológico, v.81, n.1, p.49-54, 2014.) found inferior results (14%) by researching E. coli in sawfish and considered ice as the source of cross-contamination.

Salmonella spp. was detected in 2 (5%) samples, characterizing the product as improper for consumption and in disagreement with the current legislation, which request absence of these microorganisms in 25 g. Contamination by Salmonella spp. in fishery products may result from the contamination of the environment where the animals were reared or the lack of hygienic habits in handling and processing them (SANATH KUMAR et al., 2003SANATH KUMAR, H.; SUNIL, R.; VENUGOPAL, M.N.; KARUNASAGAR, I.; KARUNASAGAR, I. Detection of Salmonella spp. in tropical seafood by polymerase chain reaction. International Journal Food Microbiology, v.88, n.1, p.91-95, 2003.). Several authors consider the presence of Salmonella in the gastrointestinal tract of fish a concern, as they can contaminate meat during its handling. In Brazil, a study carried out in Botucatu, SP, analyzed intestinal contents of 77 freshwater fish from farmers and isolated different microorganisms from the group of enterobacteria, including Salmonella thyphimurium (2.28%), (LANGONI, 1999LANGONI, H. Flora microbiana intestinal aeróbica de peixes de diferentes hábitos alimentares. Boletim Técnico do CEPTA-IBAMA, Pirassununga, 1999.). According to MARTINS (2006MARTINS, F.O. Avaliação da qualidade higiênico-sanitária de preparações (sushi e sashimi) a base de pescado cru servidos em bufês na cidade de São Paulo. 105f. Dissertation (Mestrado em Saúde Pública) - Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, 2006.), cooking eliminates the risk of this pathogen, but in some cases, it takes very few Salmonella-infected cells (from 1 to 10 cells for some serotypes) to cause symptoms in humans (LINDER et al., 2011LINDER, C.E.; ROÇA, R.O.; PINTO, J.P.A.N.; SIGARINI, C.O. Salmonella spp. em sistema intensivo de criação de peixes tropicais de água doce. Higiene Alimentar , v.25, n.192-193, p.126-133, 2011.).

For mesophilic aerobic bacteria counting, the legislation does not determine a standard, however, excessive count contributes to a higher deterioration of the product, besides paving the way to pathogenic bacteria. In this research, the high counts did not determine macroscopic changes of the fish, since most animals had been freshly caught and showed characteristics consistent with the state of freshness. As stated by FURLAN et al. (2007FURLAN, E.F.; GALVÃO, J.A.; SALÁN, E.O.; YOKOYAMA, V.A.; OETTERER, M. Estabilidade físico-química e mercado do mexilhão (Perna perna) cultivado em Ubatuba - SP. Ciência e Tecnologia de Alimentos, v.27, n.3, p.516-523, 2007.), deteriorating microorganisms are responsible for reduced shelf life of the fish because of their proteolytic, pectinolytic, lipolytic capacity. A way to minimize high counts is the rapid cooling of the fish, since the mesophilic microbiota multiplies rapidly at room temperature. AGNESE et al. (2001AGNESE, A.P.; DE OLIVEIRA, V.M.; SILVA, P.P.O.; OLIVEIRA, G.A. Contagem de bactérias heterotrófica aeróbias mesófilas e numeração de coliformes totais e fecais, em peixes frescos comercializados no município de Seropédica - RJ. Higiene Alimentar, v.15, n.88, p.67-70, 2001.) Report that, values of mesophilic microorganisms higher than 10⁶ CFU/g in fish are considered critical to the freshness level. However, LIRA et al. (2001LIRA, G.M.; PEREIRA, W.D.; ATHAYDE, A.H. Avaliação da qualidade de peixes comercializados na cidade de Maceió, AL. Higiene Alimentar , v.15, n.84, p.67-74, 2001.) observed that some fish presenting more than 106 CFU/g had not alteration in its characteristics, whereas others with lower number were disqualified upon sensorial analysis, similar to what happened in the present study.

Coagulase-positive Staphylococci were not detected in analyses, therefore, the samples met the standard required by the current legislation for these microorganisms. Nevertheless, coagulase-negative Staphylococci were seen in 18 (45%) samples, with counts ranging from 10² to 1.8 × 10⁸ CFU/g (Table 2). High values of coagulase-negative Staphylococci can be attributed to inadequate manipulation by fishermen and sellers bearing the bacterium on their mucosae and skin surface. Emphasis is placed on the implementation of good practices, guiding the seller regarding personal hygiene, thus improving product quality.

SANTOS (2003SANTOS, D.A. O papel do manipulador de alimentos em surtos de intoxicação alimentar causados por espécies de Staphylococcus ocorridos em quatro cidades do Estado de Minas Gerais, Brasil. 85f. Dissertation (Mestrado em Ciências Biológicas) - Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, 2003.) described the toxicity of 63.2% of Staphylococcus epidermidis strains and 84.6% of Staphylococcus cohnii strains isolated from food handlers involved in outbreaks of food poisoning. Current legislation does not establish a standard for coagulase negative Staphylococcus, although these bacteria produce enterotoxins responsible for food poisoning. LAMAITA (2003LAMAITA, H.C. Frequência de espécies de Staphylococcus, de TSST-1 e de enterotoxinas estafilocócicas em leite cru refrigerado em propriedades de Minas Gerais. 74f. Dissertation (Mestrado em Medicina Veterinária) - Escola de Veterinária, Universidade Federal de Minas Gerais, Belo Horizonte, 2003.) emphasizes that legal standards for food specify the presence of coagulase positive species only and reinforces the need of revision in the existing legislation of food hygiene by official services, including standards for coagulase-negative Staphylococci.

Aeromonas was found in 36 (90%) samples, of which 7 (19.4%) were identified as A. cavie and 29 (80.6%) as A. hydrophilic. The genus Aeromonas is autochthonous of the aquatic environment and the mobile and mesophilic species are found worldwide in fresh and brackish waters, both typical of the region under study. Besides being an important deterioration factor in fish, contamination by such species poses risk to the consumers’ health, since these are recognized as pathogenic to humans due to the production of cytotoxins, enterotoxins, and hemolysins, besides causing skin infections, mainly in immunocompromised patients. (GONZÁLEZ-SERRANO et al., 2002GONZÁLEZ-SERRANO, C.J.; SANTOS, J.A.; GARCIA-LOPEZ, M.L.; OTERO, A. Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case. Journal of Applied Microbiology, v.93, n.3, p.414-419, 2002.).

Among the species of Aeromonas spp., A. caviae and A. veronii biovar sobria are more commonly associated with infections in humans and are responsible for more than 85% of all clinical cases related to this genus (AL-BENWAN et al., 2007AL-BENWAN, K.; ABBOTT, S.; JANDA, J.M.; HUYS, G.; ALBERT, M.J. Cystitis caused by Aeromonas caviae. Journal of Clinical Microbiology, v.45, n.7, p.2348-2350, 2007.; TSAI et al., 2009TSAI, Y.H.; HUANG, K.C.; HUANQ, T.J.; HSU, R.W. Case reports: fatal necrotizing fasciitis caused by Aeromonas sobria in two diabetic patients. Clinical Orthopaedics and Related Research, v.467, n.3, p.846-849, 2009.). In Brazil, there are reports of isolation of different species of Aeromonas spp. in clinical samples from cases of children diarrhea, hospital infection or gastroenteritis after fish, meat, and vegetables intake (MARQUES, 2011MARQUES, C.L.M. Caracterização molecular de cepas de Aeromonas spp. isoladas durante um surto de diarreia em São Bento do Una, PE, em 2004. 2011. 72f. Thesis (Doutorado em Saúde Pública) - Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, Recife, 2011.). A. hydrophila was reported as the agent causing diarrhea and pneumonia and further evolution to sepsis in a child exposed to contaminated water (RODRÍGUEZ et al., 2005RODRÍGUEZ, C.N.; CAMPOS, R.; PASTRAN, B.; JIMENEZ, I.; GARCIA, A.; MEIJOMIL, P.; RODRÍGUEZ-MORALES, A.J. Sepsis due to extended-spectrum b-lactamase-producing Aeromonas hydrophila in a pediatric patient with diarrhea and pneumonia. Clinical Infectious Diseases, v.41, p.421-422, 2005.). These data reinforce the need to implement new hygienic-sanitary measures for people directly dealing with food in order to reduce this problem.

SILVA (2010SILVA, R.M.L. Bactérias do gênero Aeromonas e indicadores de qualidade da água em pisciculturas da região da Baixada Ocidental Maranhense. 75f. Thesis (Doutorado em Medicina Veterinária) - Faculdade de Ciências Agrárias e Veterinárias, Universidade Estadual Paulista “Júlio de Mesquita Filho”, São Paulo, 2010.) evaluated the presence of Aeromonas in tambaqui (Colossoma macropomum), tambacu (C. macropomum x Piaractus mesopotamicus), tilapia (Oreochromis niloticus) and curimatá (Prochilodus lineatus) units from fish farms in the Baixada Maranhense region, and found out that these properties were an important natural reservoir of Aeromonas at the time of investigation, also posing risk to animals raised in the surroundings. The isolates of A. hydrophila found in this research were obtained from fish without clinical signs, which suggests that these specimens were carriers of these bacteria, and that intaking contaminated products could lead to gastroenteritis.

The results of A. hydrophila and A. cavie research corroborate the findings by RALL et al. (1998RALL, V.L.M.; IARIA, S.T.; HEIDTMANN, S.; PIMENTA, F.C.; GAMBA, R.C.; PEDROSO, D.M.M. Aeromonas species isolated from pintado fish (Pseudoplatystoma sp.): virulence factors and drug susceptibility. Revista de Microbiologia, v.29, n.3, p.222-227, 1998.), who found Aeromonas contamination in 48% of the samples of pintado (Pseudoplatystoma sp.) collected in supermarkets of the city of São Paulo, A. cavie being the most frequent (60%), followed by A. hydrophila (50%) and A. sobria (12%), emphasizing the relevance of these species in fish.

Fish units evaluated in this work were commercialized in fairs often under poor hygienic conditions, which represents a potential risk to consumers in case of contamination.

CONCLUSION

Trahiras from the municipality of São Bento (MA) have unsatisfactory hygienic-sanitary conditions and can carry Salmonella spp. and species of Aeromonas spp. for the consumers. A revision of RDC Nº 12, January 2001, ANVISA is necessary for the inclusion of parameters for coagulase-negative Staphylococci, since these may produce toxins that cause damage to human health.

ACKNOWLEDGEMENTS

The Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão (FAPEMA), for granting the scholarship. To the Universidade Estadual do Maranhão and its Laboratory of Microbiology of Food and Water, of the Veterinary Medicine Course, where the analyzes were carried out.

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