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Hygiene conditions of mussels Perna perna captured in Niterói, RJ, Brazil: thermal intervention and microbiological evaluation

Abstract

Bivalve molluscs are important fishing resources for human consumption around the world. However, for safe consumption, good hygiene conditions must be ensured throughout the production chain. Eating bivalve molluscs, particularly raw, can pose a significant health risk to consumers because they can act as vectors of infectious pathogens. This study aimed to evaluate the microbiological quality of mussels from Niterói, RJ and the use of cooking to ensure food safety, as well as assessing the susceptibility of Escherichia coli and coagulase-positive Staphylococcus strains to different antimicrobials. Therefore, microbiological analyzes were performed on mussels Perna perna before and after thermal processing, the impact of cooking on the microorganisms was verified. Cooking the mussels in water at 95°C for 1 (one) minute reduced product contamination. The use of the time x temperature binomial was sufficient to reduce pathogenic microorganisms to acceptable levels according to Brazilian legislation, although the pathogens were only completely eliminated after five minutes. The bacteria investigated had a high antibiotic resistance index: 66% of E. coli strains and 34.78% of coagulase-positive Staphylococcus strains were found to be resistant to multiple drugs.

Keywords:
bivalve molluscs; thermal processing; cooking; pathogenic microorganisms; antimicrobials

1 Introduction

Bivalve molluscs are important sources of food and income for fishing communities, playing an important socioeconomic and cultural role (Legat et al., 2010Legat, J. F. A., Puchnick-Legat, A., Pereira, A. M. L., Fogaça, F. H. S., & Colin, F. N. (2010). Levantamento das principais espécies de moluscos bivalves capturadas no Estado do Piauí. In Associação Brasileira de Oceanografia – AOCEANO (Org.), Anais do 3° Congresso Brasileiro de Oceanografia. Rio Grande: AOCEANO.). They are considered a prominent commodity of high nutritional value and are widely used for human consumption (Daltro et al., 2013Daltro, A. C. S., Silva, I. P., Souza, J. S., Saraiva, M. A. F., & Evangelista-Barreto, N. S. (2013). Enterococcus faecalis em amostras de sururu processado comercializado no mercado municipal de São Francisco do Conde - Bahia. Revista de Educação Continuada em Medicina Veterinária e Zootecnia do CRMV-SP, 11(1), 73.).

These organisms are responsible for several epidemic outbreaks (Daltro et al., 2013Daltro, A. C. S., Silva, I. P., Souza, J. S., Saraiva, M. A. F., & Evangelista-Barreto, N. S. (2013). Enterococcus faecalis em amostras de sururu processado comercializado no mercado municipal de São Francisco do Conde - Bahia. Revista de Educação Continuada em Medicina Veterinária e Zootecnia do CRMV-SP, 11(1), 73.), as they may bioaccumulate in their tissues several microorganisms present in the water (Brasil, 2014Brasil. Ministério da Pesca e Aquicultura. (2014). Manual do MPA para o Programa Nacional de Controle Higiênico-Sanitário de Moluscos Bivalves - PNCMB (2. ed.). Brasília: MPA.; Ballesteros et al., 2018Ballesteros, E. R., Andrade, V. da C., Barbieri, E., Pinto, A. B., Oliveira, R. S., & Oliveira, A. J. F. C. (2018). Qualidade microbiológica de ostras (Crassostrea sp.) e de águas coletadas em cultivos e em bancos naturais de Cananéia (SP). Boletim do Instituto de Pesca, 42(1), 134-144.). However, consumers' perception of food risks is multifactorial, ranging from social, cultural, psychological, ethical to moral aspects. In addition to technical-scientific knowledge, subjective issues contribute to the perception of risks, considering the benefits that some food provides (Rembischevski & Caldas, 2020Rembischevski, P., & Caldas, E. D. (2020). Risk perception related to food. Food Science and Technology, 40(4), 779-785. http://dx.doi.org/10.1590/fst.28219.
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).

Thus, the lack of basic sanitation, as well as poor hygiene during food processing can represent a risk to the health of consumers (Mafra et al., 2016Mafra, J. F., Marques, V. F., Carneiro, C. S., Oliveira, T. A. S., & Evangelista-Barreto, N. S. (2016). Avaliação da qualidade microbiológica de moluscos bivalves processados e comercializados em Maragogipe, estado da Bahia, Brasil/Microbiological quality evaluation of bivalve mollusks processed and commercialized in Maragogipe, Bahia State, Brazil. Acta of Fisheries and Aquatic Resources, 4(2), 39-43.). Moreover, genetic alterations in pathogenic microorganisms in fish are vectors for the emergence of multi-drug resistant microorganisms and a possible consequence of the spreading of their waste around ocean currents (Cordeiro et al., 2020Cordeiro, K. S., Galeno, L. S., Mendonça, C. D. J. S., Carvalho, I. A., & Costa, F. N. (2020). Ocorrência de bactérias patogênicas e deteriorantes em sashimi de salmão: avaliação de histamina e de susceptibilidade a antimicrobianos. Brazilian Journal of Food Technology, 23, e2019085. http://dx.doi.org/10.1590/1981-6723.08519.
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).

High antibiotic resistance indexes in foodborne bacteria pose a significant health risk to consumers, as lower susceptibility to these drugs implies that there are fewer therapies available to treat possible infections caused by eating contaminated food (Cordeiro et al., 2020Cordeiro, K. S., Galeno, L. S., Mendonça, C. D. J. S., Carvalho, I. A., & Costa, F. N. (2020). Ocorrência de bactérias patogênicas e deteriorantes em sashimi de salmão: avaliação de histamina e de susceptibilidade a antimicrobianos. Brazilian Journal of Food Technology, 23, e2019085. http://dx.doi.org/10.1590/1981-6723.08519.
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).

Therefore, the consumption of undercooked bivalve molluscs can cause diseases, especially when processed in poor hygiene conditions or when they come from contaminated environments (Barros et al., 2005Barros, L. M. O., Theophilo, G. N. D., Costa, R. G., Rodrigues, D. P., & Vieira, R. H. S. F. (2005). Contaminante fecal da ostra Crassostrea rhizophorae comercializada na Praia do Futuro, Fortaleza-Ceará. Ciência Agronômica, 36(3), 285-289.). Thermal treatment at 100 °C for approximately 15 minutes is effective to reduce microbial load on mussels (Pereira et al., 2007Pereira, C. S., Possas, C. D. A., Viana, C. M., & Rodrigues, D. D. P. (2007). Características de Vibrio parahaemolyticus isolados de mexilhões (Perna perna) comercializados em Niterói, Rio de Janeiro. Revista da Sociedade Brasileira de Medicina Tropical, 40(1), 56-59. http://dx.doi.org/10.1590/S0037-86822007000100011. PMid:17486255.
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).

This study aimed to evaluate the microbiological quality of mussels from the city of Niterói, RJ and the use of cooking to ensure food safety, as well as assessing the susceptibility of Escherichia coli and coagulase-positive Staphylococcus strains to different antimicrobials.

2 Material and methods

Mussels (Perna perna) were collected in Baía da Guanabara (22° 52’13’’ S 43°9’53’’ W) by a community from Niterói, Rio de Janeiro. Nine (9) collections were carried out, with 1 kg of precooked packaged mussels and 60 animals (males and females) that were still alive collected (American Public Health Association, 2001American Public Health Association – APHA, American Water Works Association – AWWA, Water Environment Federation – WEF. (2001). Standard methods for the examination of water and wastewater (20th ed.). Washington: APHA.).

The mussels were refrigerated in clean isothermal boxes with recyclable ice and taken to the Laboratory for Microbiological Control of Animal Origin Products (CMPOA), of Universidade Federal Fluminense. The still alive mussels were washed under running water, with a brush, air dried after the process and sent for microbiological analysis (American Public Health Association, 2001American Public Health Association – APHA, American Water Works Association – AWWA, Water Environment Federation – WEF. (2001). Standard methods for the examination of water and wastewater (20th ed.). Washington: APHA.).

In addition to the group of fresh animals, the mussels were divided into five groups of animals weighing approximately 200 g: control (precooked by shellfish collectors) and four thermal treatments. The groups that underwent thermal processing in the laboratory were placed in water heated to a temperature of 95 °C ± 1 °C, measured with a food thermometer (XT-1234, XTRAD®). They were divided into four groups and cooked for different times: 1 min (T1), 3 min (T3), 5 min (T5) and 7 min (T7).

The temperature of 95 °C aimed to simulate cooking conditions in homes where the formation of small bubbles indicate the boiling point of water was reached and the mussels can be added to the pot.

Mussel analyzes included count of Aerobic Mesophilic Heterotrophic Bacteria (CAMHB), search for Salmonella spp. and count of coagulase-positive Staphylococci, according to the APHA methodology described in the “Compendium of Methods for the Microbiological Examinations of Foods” (Salfinger & Tortorello, 2015Salfinger, Y., & Tortorello, M. L. (2015). Compendium of methods for the Microbiological Examinations of Foods. (5th ed). Washington, DC: APHA.).

For the analysis of coliforms and E. coli, a fast miniaturized technique in multiple tubes, according to Merck Microbiology (2000)Merck Microbiology. (2000). Microbiology manual (407p). Berlin: Merck., modified by Franco & Mantilla (2004)Franco, R. M., & Mantilla, S. P. M. (2004). Escherichia coli em corte de carne bovina: Avaliação da metodologia aplicada e sensibilidade antimicrobiana dos sorovares predominantes. In W. Lilenbaum (Eds.), Anais do 14º Seminário de Iniciação Científica e Prêmio UFF Vasconcellos Torres de Ciência e Tecnologia. Niterói: UFF. was used, and Fluorocult broth was the culture medium (Himedia®, code M1453).

The results were compared with the provisions of IN 60 (Normative Instruction. 60, of December 23, 2019, of ANVISA), based on the classification of fish species, since although mussels are bivalve molluscs, they are not consumed live (raw) (Brasil, 2019Brasil. Ministério da Saúde, Agência Nacional de Vigilância Sanitária. (2019, December 23). Estabelece as listas de padrões microbiológicos para alimentos (Instrução normativa nº 60, de 23 de dezembro de 2019). Diário Oficial da União.).

E. coli and coagulase-positive Staphylococcus strains were tested for susceptibility to antimicrobials, according to the recommendations of Clinical and Laboratory Standards Institute (2021)Clinical and Laboratory Standards Institute – CLSI. (2021). Performance standards for antimicrobial susceptibility testing - CLSI supplement M100 (31st ed.). Wayne: Clinical and Laboratory Standards Institute.. The bacterial suspension was standardized according to the Kirby-Bauer method (Bauer et al., 1966Bauer, A. W., Kirby, W. M. M., Sherris, J. C., & Turck, M. (1966). Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology, 45(4), 493-496. http://dx.doi.org/10.1093/ajcp/45.4_ts.493. PMid:5325707.
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), with turbidity adjusted with sterile saline solution, corresponding to 1.0 Mc Farland standard, in a total of 3x108 bact/mL of medium.

The inoculum was seeded on plates containing Müeller Hinton Agar with the aid of a sterilized swab. With previously flamed and cooled forceps, Sensifar vet sensitivity discs (Cefar® brand) were applied on the culture medium containing the inoculum. The following antimicrobials were tested: tetracycline (TET - 30 mcg), gentamicin (GEN - 10 mcg), chloramphenicol (CLO - 30 mcg), ciprofloxacin (CIP - 5 mcg), norfloxacin (NOR - 10 mcg) and nitrofurantoin (NIT - 300 mcg) for both bacteria.

For E. coli, ampicillin (AMP - 10 mcg), cefuroxime (CRX - 30 mcg), cephalexin (CFE - 30 mcg) and cephalothin (CFL - 30 mcg) were also tested. Also, clindamycin (CLI - 2 mcg) and erythromycin (ERI - 15 mcg) were chosen exclusively for coagulase-positive Staphylococcus.

The plates were incubated at 35-37 °C for 18-24 hours and inhibition halos were observed, which were measured with a halometer (millimeters). Standard measures of the inhibition zones of each antimicrobial were used in the interpretation of the test, according to a table established by Clinical and Laboratory Standards Institute (2021)Clinical and Laboratory Standards Institute – CLSI. (2021). Performance standards for antimicrobial susceptibility testing - CLSI supplement M100 (31st ed.). Wayne: Clinical and Laboratory Standards Institute. that classifies each strain as resistant, intermediate or susceptible.

Exploratory analysis of the variables was performed in statistical analysis, and Friedman and Wilcoxon nonparametric tests were used, both for paired samples. The global significance level adopted was 5% and, for multiple comparisons (comparisons of 2 groups) between the time intervals assessed, a significance level of 0.4% was used in each assessment, according to Bonferroni's correction proposal.

3 Results and discussion

The results of the analyzes of fresh and processed mussels submitted to different cooking times are described in Table 1.

Table 1
Mean bacteriological count values of mussels Perna perna in the different groups fresh, precooked (control), T1 (cooking 1 min), T3 (cooking 3 min), T5 (cooking 5 min) and T7 (cooking 7 min).

CAMHB ranged from 1.2 x 103 to 7.4 x 104 CFU/g in fresh mussels and from 7.7 x 103 to 5.1 x 106 CFU/g in precooked mussels (control). A significant difference (p < 0.001) was found between the intervals of time evaluated regarding CAMHB. The most significant differences were found between the fresh or precooked product (control) and the products observed after three or more minutes of cooking. This is consistent with Nascimento et al. (2011)Nascimento, V. A., Santos, R. C. A., Mittaraquis, A. S. P., Travália, B. M., & de Aquino, L. C. L. (2011). Qualidade microbiológica de moluscos bivalves-sururu e ostras submetidos a tratamento térmico e estocagem congelada. Scientia Plena, 7(4), 1-5., who reported values ranging from 103 to 107 CFU/g for the fresh product. The authors found similar values, of 106 CFU/g in the precooked product.

Increase in CAMHB after processing was expected, as according to Daltro et al. (2013)Daltro, A. C. S., Silva, I. P., Souza, J. S., Saraiva, M. A. F., & Evangelista-Barreto, N. S. (2013). Enterococcus faecalis em amostras de sururu processado comercializado no mercado municipal de São Francisco do Conde - Bahia. Revista de Educação Continuada em Medicina Veterinária e Zootecnia do CRMV-SP, 11(1), 73., appropriate hygiene measures are not observed by the people who extract product, and handling after capture directly influences the microbiological quality of the end product.

After cooking by immersion in water at 95 °C for one minute, CAMHB ranged from 1.0 x 102 to 8.9 x 104 CFU/g, showing that proper thermal treatment can reduce microbial load on these products. The count ranged from 7.1 x 10 to 1.7 x 103 CFU/g after three minutes. At five minutes, it remained between 1 x 10 to 1.3 x 103 CFU/g and between 1x 10 to 1.2 x 103 CFU/g after seven minutes. After three minutes of cooking, the mean was practically constant, in 102 CFU/g. Despite a slight reduction in CAMHB, extra cooking time is not necessary at a temperature of 95 °C.

Lower counts were obtained by Nascimento et al. (2011)Nascimento, V. A., Santos, R. C. A., Mittaraquis, A. S. P., Travália, B. M., & de Aquino, L. C. L. (2011). Qualidade microbiológica de moluscos bivalves-sururu e ostras submetidos a tratamento térmico e estocagem congelada. Scientia Plena, 7(4), 1-5. who submitted Charru mussels and oysters to steam precooking or immersion in water at 95 °C for 05, 10 and 15 minutes after opening the shells and found CAMHB values < 10 CFU/g. However, in this case, the bivalves were in the shells and the precooking process observed good hygiene practices, unlike the present study where the mussels sampled had been previously precooked by the shellfish collectors.

Analysis of the samples based on the referred IN 60 (Brasil, 2019Brasil. Ministério da Saúde, Agência Nacional de Vigilância Sanitária. (2019, December 23). Estabelece as listas de padrões microbiológicos para alimentos (Instrução normativa nº 60, de 23 de dezembro de 2019). Diário Oficial da União.) regarding coagulase-positive Staphylococci count for fresh and precooked mussels (control) is described in Figure 1.

Figure 1
Judgment of mussel samples according to IN 60 (Brasil, 2019Brasil. Ministério da Saúde, Agência Nacional de Vigilância Sanitária. (2019, December 23). Estabelece as listas de padrões microbiológicos para alimentos (Instrução normativa nº 60, de 23 de dezembro de 2019). Diário Oficial da União.), based on the coagulase-positive Staphylococci count for fresh (A) and precooked (control) (B) mussels.

In fresh animals, the coagulase-positive Staphylococci count ranged from 1.7 x 102 CFU/g to 3.4 x 105 CFU/g. There was a significant difference (p < 0.001) between the intervals of time evaluated for this count. The most significant differences were observed between the fresh or precooked product (control) and the products observed after cooking. Similar results were reported by Silva et al. (2021)Silva, B. R. D., Menegardo, S. B., Aride, P. H. R., Lavander, H. D., Spago, F. R., & Souza, T. B. D. (2021). Qualidade microbiológica da água e dos mexilhões Perna perna (Linnaeus, 1758) cultivados em Piúma, Espírito Santo, Brasil. Engenharia Sanitaria e Ambiental, 26(1), 89-95. http://dx.doi.org/10.1590/s1413-415220180169.
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in Piúma (ES), who found a density of S. aureus ranging from 1.28 x 102 to 4.35 x 103 CFU/g in mussels Perna perna.

According to IN 60, a maximum value of 103 CFU/g in fish is recommended for this microorganism. The quality of a product with values ranging between 102 to 103 CFU/g is classified as intermediate (Brasil, 2019Brasil. Ministério da Saúde, Agência Nacional de Vigilância Sanitária. (2019, December 23). Estabelece as listas de padrões microbiológicos para alimentos (Instrução normativa nº 60, de 23 de dezembro de 2019). Diário Oficial da União.).

Therefore, 44.46% of fresh mussel samples were unacceptable and 55.6% had intermediate quality. The number of unacceptable samples increased, with 88.9% (8) in precooked (control) mussels and counts ranging from 5 x 102 CFU/g to 4.8 x 106 CFU/g. The levels of CFU/g obtained were higher than those described by Freitas et al. (2015)Freitas, F., Santos, M. L., Neiva, G. S., Silva, I. M. M., Amor, A. L. M., & Assunção, R. J. (2015). Qualidade sanitária de sururu (Mytella guyanensis) beneficiado por comunidade quilombola. Boletim do Centro de Pesquisa e Processamento de Alimentos, 33(2), 9-18. http://dx.doi.org/10.5380/cep.v33i2.47233.
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, who reported that 33.33% of Charru mussels precooked by shellfish collectors was contaminated with S. aureus at levels above those permitted by law, and that the tools were also contaminated by E. coli in 11.11% of the samples.

According to Freitas et al. (2015)Freitas, F., Santos, M. L., Neiva, G. S., Silva, I. M. M., Amor, A. L. M., & Assunção, R. J. (2015). Qualidade sanitária de sururu (Mytella guyanensis) beneficiado por comunidade quilombola. Boletim do Centro de Pesquisa e Processamento de Alimentos, 33(2), 9-18. http://dx.doi.org/10.5380/cep.v33i2.47233.
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, the main cause of contamination of processed bivalves is the lack of proper handwashing by shellfish collectors and improper cleaning of tools, which can cause contamination especially by coliforms and S. aureus. This is consistent with the present study where greater contamination of mussels was detected after their processing by fishermen, indicating that their hands and/or the tools used by the workers can be sources of recontamination of the product.

The judgment of mussels based on IN 60 (Brasil, 2019Brasil. Ministério da Saúde, Agência Nacional de Vigilância Sanitária. (2019, December 23). Estabelece as listas de padrões microbiológicos para alimentos (Instrução normativa nº 60, de 23 de dezembro de 2019). Diário Oficial da União.), regarding the count of coagulase-positive Staphylococci after thermal treatments in the laboratory is described in Figure 2.

Figure 2
Judgment of mussel samples based on the count of coagulase-positive Staphylococci after thermal treatment for 1 minute (A), 3 minutes (B), 5 minutes (C) and 7 minutes (D), according to IN 60 (Brasil, 2019Brasil. Ministério da Saúde, Agência Nacional de Vigilância Sanitária. (2019, December 23). Estabelece as listas de padrões microbiológicos para alimentos (Instrução normativa nº 60, de 23 de dezembro de 2019). Diário Oficial da União.).

After the one-minute thermal treatment, 77.8% (7) of the samples became acceptable, while 22.2% (2) showed intermediate quality, with a maximum count of 3 x 102 CFU/g. After three minutes of treatment, 88.9% (8) of the samples were acceptable, and 11.1% (1) had intermediate quality, with a maximum count of 1 x 102 CFU/g. However, despite the lower counts, it is still desirable to improve the quality, in order to include all the bivalves classified as acceptable according to the legislation.

After five and seven minutes, 100% (9) of the samples were acceptable, with values lower than 100 CFU/g. Thus, thermal treatment at 95°C from 5 (five) minutes ensures a safe product regarding the presence of coagulase-positive Staphylococci.

The number of total coliforms ranged from 9 x 10 to 4.6 x 104 MPN/g in fresh mussels. In precooked mussels (control), it ranged from < 3 to 1.1 x 104 MPN/g. A significant difference (p < 0.001) was found between the time intervals evaluated considering this count. However, it was merely found that coliform counts in fresh mussels differed from those observed after at least three minutes of cooking. Although high, the number of total coliforms is not a parameter in Brazilian legislation. However, coliforms are considered an indicator of hygiene conditions in food processing: a high number of coliforms is associated with poor handling practices.

The judgment of fresh and precooked (control) mussels based on IN 60 (Brasil, 2019Brasil. Ministério da Saúde, Agência Nacional de Vigilância Sanitária. (2019, December 23). Estabelece as listas de padrões microbiológicos para alimentos (Instrução normativa nº 60, de 23 de dezembro de 2019). Diário Oficial da União.) according to the Most Probable Number (MPN) of E. coli is described in Figure 3.

Figure 3
Results of the judgment of fresh (A) and precooked (B) mussels Perna perna, according to IN 60 (Brasil, 2019Brasil. Ministério da Saúde, Agência Nacional de Vigilância Sanitária. (2019, December 23). Estabelece as listas de padrões microbiológicos para alimentos (Instrução normativa nº 60, de 23 de dezembro de 2019). Diário Oficial da União.) based on E. coli MPN.

For fish not consumed raw, E. coli parameters range from 50 to 5x102 MPN/g according to the Brazilian legislation (Brasil, 2019Brasil. Ministério da Saúde, Agência Nacional de Vigilância Sanitária. (2019, December 23). Estabelece as listas de padrões microbiológicos para alimentos (Instrução normativa nº 60, de 23 de dezembro de 2019). Diário Oficial da União.). Thus, in fresh mussels, only one sample (11.1%) was acceptable. The others (88.9%) had intermediate quality, ranging from 9 x 10 to 4.3 x 102 MPN/g. A significant difference (p < 0.001) was found between the time intervals evaluated regarding E. coli counts. However, although higher counts were noticed in fresh and peeled products, no significant differences were detected between the different time intervals in multiple comparisons (comparisons of 2 groups).

Less significant figures were described by Silva et al. (2021)Silva, B. R. D., Menegardo, S. B., Aride, P. H. R., Lavander, H. D., Spago, F. R., & Souza, T. B. D. (2021). Qualidade microbiológica da água e dos mexilhões Perna perna (Linnaeus, 1758) cultivados em Piúma, Espírito Santo, Brasil. Engenharia Sanitaria e Ambiental, 26(1), 89-95. http://dx.doi.org/10.1590/s1413-415220180169.
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in their evaluation of the microbiological quality of mussels Perna perna cultivated in Píuma, Espírito Santo. The authors found that the amount of total coliforms ranged from 28 to >1,600 MPN/g and the number of thermotolerant coliforms ranged from <2 to 21.5 MPN/g.

However, for Charru mussels extracted from Sepetiba Bay, Palmeira et al. (2018)Palmeira, K. R., Calixto, F. A. A., Keller, L. A. M., Holanda, M. C. K. D., & Mesquita, E. F. M. (2018). Qualidade microbiológica do sururu (Mytella charruana) comercializado por catadoras da Baía de Sepetiba, Rio de Janeiro, Brasil. Revista Brasileira de Ciência Veterinária, 25(3/4), 93-97. http://dx.doi.org/10.4322/rbcv.2018.018.
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reported a higher count of thermotolerant coliforms. A count of 3 x 105 MPN/g was found for fresh Charru mussels and 7.5 x 103 MPN/g for precooked Charru mussels, above the limits established by the legislation.

In the control group (precooked mussels), only one sample had intermediate quality (11.1%) regarding E. coli and the others (88.9%) were acceptable. Such finding contrasts with those of Leôncio et al. (2020)Leôncio, G. G., Ferreira, E. M., Lopes, I. S., Costa, L. E. P., Areco, A. E. T., Barbosa, K. F. D., & Alves, L. M. C. (2020). Aspectos higiênico-sanitários e químicos do Sururu (Mytella falcata) desconchado comercializado em feiras e mercados públicos de São Luis–MA / Hygienic-health and chemical aspects of Sururu (Mytella falcata) discharge marked in fairs and public markets in São Luís-MA. Brazilian Journal of Development, 6(2), 5848-5858. http://dx.doi.org/10.34117/bjdv6n2-041.
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in São Luís (MA) who detected the presence of E. coli in 50% of the samples of Charru mussels, values above the levels acceptable by the legislation, suggesting poor conditions of preparation, conservation and marketing at room temperature.

In Maceió, Alagoas, Silva et al. (2015)Silva, A. A. V., Costa, A. F. M., Freitas, R. M. S., Santos, M. B. S. V., Lourenço, A. L. N., Malta, A. S., Fireman, A. L., Ferreir, A. S., Rohrig, L., Santos, S. C., Noé, P. V. R., Silva, C. H. B., Souza, E. C., & Rocha, T. J. M. (2015). Qualidade parasitológica e condições higiênico-sanitárias de sururu (Mytella charruana) e alface (Lactuca sativa) comercializados em um mercado público de Maceió-AL. Revista de Ciências Farmacêuticas Básica e Aplicada, 36(4), 525-529. found total and thermotolerant coliform counts above 1100 MPN/g in samples of Charru mussels, which may be related to the selling of the products in outdoor markets without meeting the minimum hygiene standards. In the present study, the precooked product (control) had the lowest E. coli count, with MPN ranging from < 3 to 9 x 10 MPN/g. The values found can be associated with the type of display of the product, sold in plastic packaging, and therefore not fully exposed during selling.

All thermal treatments at 95 °C for 1 (one) minute or longer were efficient to reduce E. coli counts to less than 3 MPN/g, making these foods acceptable to be consumed according to the Brazilian legislation (Brasil, 2019Brasil. Ministério da Saúde, Agência Nacional de Vigilância Sanitária. (2019, December 23). Estabelece as listas de padrões microbiológicos para alimentos (Instrução normativa nº 60, de 23 de dezembro de 2019). Diário Oficial da União.).

Araújo et al. (2009)Araújo, A. R. R., Silva, F. D., Santana, R. F., & Lopes, D. F. C. (2009). Gestão da pesca de Mytella charruana (D’ORBIGNY, 1846) no litoral do estado de Sergipe: indicadores de sustentabilidade. Revista Brasileira de Engenharia de Pesca, 4(2), 56-72. also reported the efficiency of the thermal treatment when they observed the absence of E. coli in precooked Charru mussels from the village of Taiçoca de Fora, Sergipe. On the other hand, pathogens were found in fresh Charru mussels.

According to Nascimento et al. (2011)Nascimento, V. A., Santos, R. C. A., Mittaraquis, A. S. P., Travália, B. M., & de Aquino, L. C. L. (2011). Qualidade microbiológica de moluscos bivalves-sururu e ostras submetidos a tratamento térmico e estocagem congelada. Scientia Plena, 7(4), 1-5., precooking of bivalve molluscs by steam or by immersion in water at a temperature of 95 °C for 5, 10 and 15 minutes is considered an efficient thermal treatment. The authors reported that no Salmonella spp was detected after these treatments, total thermotolerant coliforms were less than 3.0 MPN/g and CAMHB was less than 10 CFU/g, which is consistent with the present study where total coliforms and E. coli counts were reduced to less than 3 MPN/g after thermal treatment.

Salmonella spp was not found in any sample of the analyzed products. Guimarães et al. (2019)Guimarães, J. L. B., Calixto, F. A. A., Keller, L. A. M., Silva, L. E., Furtado, Â. A. L., & Mesquita, E. F. M. (2019). Development of a low commercial value fish-sausage from the fish trawling “mix” category. Food Science and Technology, 39(Suppl. 1), 115-121. http://dx.doi.org/10.1590/fst.38317.
http://dx.doi.org/10.1590/fst.38317...
developed fish sausages from Brazilian flathead (Percophis brasiliensis) captured in Niterói City and also verified that according to Brazilian legislation they had absence of this bacteria in the analyzed products.

However, different results in the microbiological analysis for bivalve molluscs were found by others authors. In California, Shapiro et al. (2018)Shapiro, K., Silver, M., Byrne, B. A., Berardi, T., Aguilar, B., Melli, A., & Smith, W. A. (2018). Fecal indicator bacteria and zoonotic pathogens in marine snow and California mussels (Mytilus californianus). FEMS Microbiology Ecology, 94(11), 1-11. http://dx.doi.org/10.1093/femsec/fiy172. PMid:30239730.
http://dx.doi.org/10.1093/femsec/fiy172...
described contamination of mussels (Mytilus californianus) by serovars Enteritidis and Infantis. In Baía de Santos, Passos et. al. (2011)Passos, E. D. C., Mello, A. R. P. D., Sousa, C. V. D., Oliveira, M. A. D., Casarini, L. M., & Motta, N. D. S. (2011). Detecção de Salmonella spp. em mexilhões Perna perna dos bancos naturais de baía densamente urbanizada. Revista do Instituto Adolfo Lutz, 70(4), 631-636. detected the presence of Salmonella spp. in 20.7% of the samples of mussels Perna perna analyzed at different times of the year. The dominant serotype was S. Livingstone, and other serotypes were also isolated, such as S. Infantis, S. enterica subsp. salamae and S. Albany.

Despite the presence of other enterobacteria, such as E. coli and the absence of Salmonella spp. in mussels, Freitas et al. (2015)Freitas, F., Santos, M. L., Neiva, G. S., Silva, I. M. M., Amor, A. L. M., & Assunção, R. J. (2015). Qualidade sanitária de sururu (Mytella guyanensis) beneficiado por comunidade quilombola. Boletim do Centro de Pesquisa e Processamento de Alimentos, 33(2), 9-18. http://dx.doi.org/10.5380/cep.v33i2.47233.
http://dx.doi.org/10.5380/cep.v33i2.4723...
reported that water contamination by total coliforms and E. coli may not be sufficient to contaminate bivalves.

As for the antibiotic resistance index of the detected bacteria, susceptibility of E. coli to different antimicrobials is described in Table 2.

Table 2
Susceptibility of E. coli to antimicrobials.

Of the E. coli strains isolated from mussels, 58.34% were resistant to ampicillin. This result was expected, as according to Dias et al. (2010)Dias, M. T., Santos, P. C. R. F., Oliveira, L. A. T., & Marin, V. A. (2010). Avaliação da sensibilidade de cepas de Escherichia coli isoladas de mexilhões (Perna perna Linnaeus, 1758) à antimicrobianos. Food Science and Technology, 30(2), 319-324. http://dx.doi.org/10.1590/S0101-20612010000200005.
http://dx.doi.org/10.1590/S0101-20612010...
, E. coli is generally able to inactivate antibiotics of the penicillin group due to the action of β-lactamase enzymes.

In their evaluation of E. coli strains isolated from mussels P. perna, also in Niterói, Dias et al. (2010)Dias, M. T., Santos, P. C. R. F., Oliveira, L. A. T., & Marin, V. A. (2010). Avaliação da sensibilidade de cepas de Escherichia coli isoladas de mexilhões (Perna perna Linnaeus, 1758) à antimicrobianos. Food Science and Technology, 30(2), 319-324. http://dx.doi.org/10.1590/S0101-20612010000200005.
http://dx.doi.org/10.1590/S0101-20612010...
found that only 2.27% of the strains were resistant to ampicillin, a percentage lower than that found in this study. However, a similar result was obtained by Vieira et al. (2008)Vieira, R. H. S. F., Atayde, M. A., Carvalho, E. M. R., Carvalho, F. C. T., & Fonteles Filho, A. A. (2008). Contaminação fecal da ostra Crassostrea rhizophorae e da água de cultivo do estuário do Rio Pacoti (Eusébio, Estado do Ceará): isolamento e identificação de Escherichia coli e sua susceptibilidade a diferentes antimicrobianos. Brazilian Journal of Veterinary Research and Animal Science, 45(3), 180-189. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2008.26695.
http://dx.doi.org/10.11606/issn.1678-445...
, who found that 50% of the Crassostrea rhizophorae oysters analyzed were resistant to ampicillin.

Resistance to cephalexin was found in 41.67% of the strains and to cefuroxime and norfloxacin, in 25%. Oliveira (2016)Oliveira, R. S. (2016). Densidade e diversidade de fenótipos de resistência a antimicrobianos de Enterococcus sp., Escherichia coli e Aeromonas sp. isoladas de água, sedimento e mexilhão coletados em Santos e Itanhaém, São Paulo, Brasil (Masters dissertation). Universidade Estadual Paulista, São Vicente. isolated E. coli strains from mussels collected in Santos and found a similar percentage (20%) of resistance to norfloxacin. Miotto et al. (2019)Miotto, M., Ossai, S. A., Meredith, J. E., Barretta, C., Kist, A., Prudencio, E. S., Vieira, C. R. W., & Parveen, S. (2019). Genotypic and phenotypic characterization of Escherichia coli isolated from mollusks in Brazil and the United States. MicrobiologyOpen, 8(5), e00738. http://dx.doi.org/10.1002/mbo3.738. PMid:30311420.
http://dx.doi.org/10.1002/mbo3.738...
reported 96% of susceptibility to norfloxacin in bivalves cultivated in Santa Catarina, higher than in the present study where 75% of E. coli strains were susceptible to the antimicrobial.

Of the isolated strains, 8.34% were resistant to gentamicin, which is consistent with the findings of Jayme et al. (2014)Jayme, M. M., Matos, J. G. C. N., Sales, A., Nunes-Carvalho, M. C., Queiroz, M. L. P., Freitas-Almeida, A. C., & Araújo, D. F. V. (2014). Avaliação microbiológica, detecção e susceptibilidade aos antimicrobianos de potenciais enteropatógenos das famílias enterobacteriaceae e vibrionaceae em mexilhões Perna perna coletados na praia de Itaipu, Niterói-RJ. In B. Franco, M. Landgraf, M. Taniwaki & M. T. Destro (Eds.), Anais do 12º Congresso Latino-americano de Microbiologia e Higiene de Alimentos - MICROAL 2014. São Paulo: Editora Blucher., who obtained 7.8% in the analysis of mussels isolated from Itaipu, Niterói.

The percentage of strains resistant to antimicrobials tetracycline and ciprofloxacin was 33.33%, higher than that reported by Jayme et al. (2014)Jayme, M. M., Matos, J. G. C. N., Sales, A., Nunes-Carvalho, M. C., Queiroz, M. L. P., Freitas-Almeida, A. C., & Araújo, D. F. V. (2014). Avaliação microbiológica, detecção e susceptibilidade aos antimicrobianos de potenciais enteropatógenos das famílias enterobacteriaceae e vibrionaceae em mexilhões Perna perna coletados na praia de Itaipu, Niterói-RJ. In B. Franco, M. Landgraf, M. Taniwaki & M. T. Destro (Eds.), Anais do 12º Congresso Latino-americano de Microbiologia e Higiene de Alimentos - MICROAL 2014. São Paulo: Editora Blucher., who found 10.3% resistance to tetracycline and 1.3% to ciprofloxacin. Vieira et al. (2008)Vieira, R. H. S. F., Atayde, M. A., Carvalho, E. M. R., Carvalho, F. C. T., & Fonteles Filho, A. A. (2008). Contaminação fecal da ostra Crassostrea rhizophorae e da água de cultivo do estuário do Rio Pacoti (Eusébio, Estado do Ceará): isolamento e identificação de Escherichia coli e sua susceptibilidade a diferentes antimicrobianos. Brazilian Journal of Veterinary Research and Animal Science, 45(3), 180-189. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2008.26695.
http://dx.doi.org/10.11606/issn.1678-445...
found similar percentages for tetracycline in oysters, with 25% resistance of the strains, but no resistance of E. coli to ciprofloxacin was reported.

Although the high resistance to ciprofloxacin detected differed from that described by other authors, according to Oliveira (2016)Oliveira, R. S. (2016). Densidade e diversidade de fenótipos de resistência a antimicrobianos de Enterococcus sp., Escherichia coli e Aeromonas sp. isoladas de água, sedimento e mexilhão coletados em Santos e Itanhaém, São Paulo, Brasil (Masters dissertation). Universidade Estadual Paulista, São Vicente., the resistance profiles in E. coli obtained from aquatic environments vary significantly. One explanation is the impact of effluent emissions directly into rivers and seas, contaminating waters and conveying resistant microorganisms that can pose health risks.

This is consistent with the study of Vieira et al. (2008)Vieira, R. H. S. F., Atayde, M. A., Carvalho, E. M. R., Carvalho, F. C. T., & Fonteles Filho, A. A. (2008). Contaminação fecal da ostra Crassostrea rhizophorae e da água de cultivo do estuário do Rio Pacoti (Eusébio, Estado do Ceará): isolamento e identificação de Escherichia coli e sua susceptibilidade a diferentes antimicrobianos. Brazilian Journal of Veterinary Research and Animal Science, 45(3), 180-189. http://dx.doi.org/10.11606/issn.1678-4456.bjvras.2008.26695.
http://dx.doi.org/10.11606/issn.1678-445...
who despite obtaining susceptibility in 100% of E. coli samples isolated from oysters to ciprofloxacin, it did not occur in water. The authors found resistance in 8% of the isolates from water.

There was also resistance of 33.33% of the strains to nitrofurantoin, higher than that of Miotto et al. (2019)Miotto, M., Ossai, S. A., Meredith, J. E., Barretta, C., Kist, A., Prudencio, E. S., Vieira, C. R. W., & Parveen, S. (2019). Genotypic and phenotypic characterization of Escherichia coli isolated from mollusks in Brazil and the United States. MicrobiologyOpen, 8(5), e00738. http://dx.doi.org/10.1002/mbo3.738. PMid:30311420.
http://dx.doi.org/10.1002/mbo3.738...
who found 12% of resistant strains.

A percentage of 58.33% of resistance was found for cephalotin. This is similar to the findings of Rodrigues (2019)Rodrigues, L. S. Jr. (2019). Qualidade microbiológica da águia, sedimento e peixe da Laguna de Jansen, São Luís - MA (Masters dissertation). Universidade Federal do Maranhão, São Luís. who assessed the quality of a lake in an urban perimeter and detected a resistance of 51% of E. coli strains. A lower percentage was found by Evangelista-Barreto et al. (2012)Evangelista-Barreto, N. S. E., Moura, F. D. C. M., Teixeira, J. A., Assim, D. A., & Miranda, P. C. (2012). Avaliação das condições higiênico-sanitárias do pescado comercializado no município de Cruz das Almas, Bahia. Revista Caatinga, 25(3), 86-95., who reported 12.5% of the strains as resistant, although 62.5% were classified as having intermediate resistance.

Resistance to chloramphenicol was 83.33%, higher than the results of other authors. Rodrigues (2019)Rodrigues, L. S. Jr. (2019). Qualidade microbiológica da águia, sedimento e peixe da Laguna de Jansen, São Luís - MA (Masters dissertation). Universidade Federal do Maranhão, São Luís. reported only 12% resistance, and Evangelista-Barreto et al. (2012)Evangelista-Barreto, N. S. E., Moura, F. D. C. M., Teixeira, J. A., Assim, D. A., & Miranda, P. C. (2012). Avaliação das condições higiênico-sanitárias do pescado comercializado no município de Cruz das Almas, Bahia. Revista Caatinga, 25(3), 86-95. did not report E. coli resistance to chloramphenicol in fish marketed in Cruz das Almas, Bahia.

The results concerning coagulase-positive Staphylococci are described in Table 3.

Table 3
Susceptibility of coagulase-positive Staphylococci to antimicrobials.

Most antimicrobials were effective, with 86.36% of the strains susceptible to gentamicin and chloramphenicol; 81.82% to norfloxacin; 77.27% to tetracycline and nitrofurantoin; 68.18% to ciprofloxacin and 54.55% to clindamycin. Costa et al. (2008)Costa, M. M. D., Peixoto, R. D. M., Boijink, C. D. L., Castagna, L., Meurer, F., & Vargas, A. C. D. (2008). Sensibilidade antimicrobiana de bactérias isoladas de Jundiá (Rhamdia quelen). Pesquisa Veterinária Brasileira, 28(10), 477-480. http://dx.doi.org/10.1590/S0100-736X2008001000006.
http://dx.doi.org/10.1590/S0100-736X2008...
found a resistance of 18.18% to tetracycline, similar to this study (9.09%).

All isolates (100%) were susceptible to norfloxacin and 89% to ciprofloxacin in Voloski (2018)Voloski, F. L. S. (2018). Staphylococcus aureus isolados de Merluccius merluccius durante o processamento do pescado salgado: caracterização fenotípica e genotípica (Doctoral dissertation). Universidade Federal de Pelotas, Pelotas. studies. Costa (2016)Costa, T. D. (2016). Qualidade microbiológica e perfil de sensibilidade antimicrobiana dos isolados de tilápias (Oreochromis spp.) de pesque-pague da microrregião do estado de São Paulo (Masters dissertation). Universidade Estadual Paulista, Jaboticabal. also found high susceptibility to ciprofloxacin, with 91.66% of susceptible strains. In the present study, the percentage was lower, with 81.82% of susceptibility to norfloxacin and 68.18% to ciprofloxacin.

Only 18.18% of the strains were resistant to nitrofurantoin, contrasting with Voloski (2018)Voloski, F. L. S. (2018). Staphylococcus aureus isolados de Merluccius merluccius durante o processamento do pescado salgado: caracterização fenotípica e genotípica (Doctoral dissertation). Universidade Federal de Pelotas, Pelotas. regarding the resistance profile of Staphylococcus aureus in merluccid hake (Merluccius merluccius). The author found that 44% of the strains were resistant to nitrofurantoin.

Most strains were resistant to erythromycin (54.54%) strains), followed by clindamycin (31.81%). Rosa et al. (2021)Rosa, G., Merlini, L. S., Bessi, W. H., Netto, A. P., Bezerra, K., Silva, F. G., & Santos, R. N. (2021). Resistance profile of Staphylococcus sp. isolates from tilapia fillet samples Oreochromis niloticus from the northwestern region of Paraná state, Brazil / Perfil de resistência de Staphylococcus sp. isolados de amostras de filete de tilápia Oreochromis niloticus da região noroeste do estado do Paraná, Brasil. Brazilian Journal of Development, 7(4), 41731-41742. http://dx.doi.org/10.34117/bjdv7n4-574.
http://dx.doi.org/10.34117/bjdv7n4-574...
found lower values for tilapia fillets, with 12% of strains resistant to erythromycin and 20% to clindamycin. However, in the analysis of antimicrobial susceptibility of bacteria isolated from Jundiá, Costa et al. (2008)Costa, M. M. D., Peixoto, R. D. M., Boijink, C. D. L., Castagna, L., Meurer, F., & Vargas, A. C. D. (2008). Sensibilidade antimicrobiana de bactérias isoladas de Jundiá (Rhamdia quelen). Pesquisa Veterinária Brasileira, 28(10), 477-480. http://dx.doi.org/10.1590/S0100-736X2008001000006.
http://dx.doi.org/10.1590/S0100-736X2008...
found a higher resistance to erythromycin (27.27%).

Also, 18.18% of the strains were resistant to nitrofurantoin and 13.64% to ciprofloxacin. Low values were found for tetracycline, chloramphenicol and norfloxacin (9.09%). Chloramphenicol efficacy was 86.36%. A similar result was found by Costa et al. (2008)Costa, M. M. D., Peixoto, R. D. M., Boijink, C. D. L., Castagna, L., Meurer, F., & Vargas, A. C. D. (2008). Sensibilidade antimicrobiana de bactérias isoladas de Jundiá (Rhamdia quelen). Pesquisa Veterinária Brasileira, 28(10), 477-480. http://dx.doi.org/10.1590/S0100-736X2008001000006.
http://dx.doi.org/10.1590/S0100-736X2008...
: 81.82% susceptibility of strains to this antimicrobial.

The most efficient antimicrobial was gentamicin, with only 4.54% of resistance and 86.36% of susceptibility. Rosa et al. (2021)Rosa, G., Merlini, L. S., Bessi, W. H., Netto, A. P., Bezerra, K., Silva, F. G., & Santos, R. N. (2021). Resistance profile of Staphylococcus sp. isolates from tilapia fillet samples Oreochromis niloticus from the northwestern region of Paraná state, Brazil / Perfil de resistência de Staphylococcus sp. isolados de amostras de filete de tilápia Oreochromis niloticus da região noroeste do estado do Paraná, Brasil. Brazilian Journal of Development, 7(4), 41731-41742. http://dx.doi.org/10.34117/bjdv7n4-574.
http://dx.doi.org/10.34117/bjdv7n4-574...
found slightly higher values for gentamicin, with 14% of resistant strains.

The high resistance of Staphylococcus spp. to antimicrobials was expected, given their high adaptability. This can be explained by several mechanisms, which include mutation (alteration of the antibiotic target site) or acquisition of resistance genes from other bacteria (mediated by plasmids and transposons) leading to destruction or inactivation of the antimicrobial (Cussolim et al., 2021Cussolim, P. A., Salvi Júnior, A., Melo, A. L., & Melo, A. (2021). Mecanismos de resistência do Staphylococcus aureus a antibióticos. Revista Faculdade do Saber, 6(12), 831-843.).

The occurrence of multidrug-resistant Staphylococcus spp. is common not only in fish and shellfish. Keyvan et al. (2020)Keyvan, E., Yurdakul, D., Demirtas, A., Yalcin, H., & Bilgen, N. (2020). Identification of Methicillin-Resistant Staphylococcus aureus in Bulk Tank Milk. Food Science and Technology, 40(1), 150-156. http://dx.doi.org/10.1590/fst.35818.
http://dx.doi.org/10.1590/fst.35818...
identified Staphylococcus aureus strains isolated from a milk tank and found resistance to most antimicrobials. Resistance to clindamycin was 67.9%.

Resistance of microorganisms is a growing concern. Of the pathogens investigated, 66% of E. coli strains and 34.78% of coagulase-positive Staphylococcus strains were resistant to multiple drugs. According to Maestri et al. (2020)Maestri, G., Schmeling, T. B., Valcarenghi, D., & Lemos, M. P. (2020). Quantificação de Staphylococcus aureus, Escherichia coli e mecanismos de resistência nas mãos de manipuladores de alimentos em UANS hospitalares em SC. Disciplinarum Scientia, 21(1), 91-105. http://dx.doi.org/10.37777/dscs.v21n1-009.
http://dx.doi.org/10.37777/dscs.v21n1-00...
, these pathogens are among the most frequent species in hospital-acquired infections due to their high adaptability and resistance to antimicrobials. The high rate of multiple drug-resistant strains poses a risk to public health, making treatment difficult and aggravating potentially curable clinical conditions.

4 Conclusions

Inadequate processing of mussels, disregarding hygiene and sanitary practices, is a source of contamination. Thus, the products must be cooked prior to consumption, to reduce contamination.

Although pathogenic microorganisms are eliminated only after 5 minutes of cooking, the binomial time x temperature (95 °C for 1 minute) was sufficient to reduce these microorganisms to acceptable levels according to Brazilian legislation, making it a safe food.

High resistance to multiple antimicrobials was found in the pathogens studied. Thus, there are various resistance profiles, especially in the aquatic environment.

  • Practical Application: Mussels are filtering organisms, widely used as a bioindicator. On the other hand, they are highly appreciated delicacies in cooking. Thus, it is important that the population feels safe when consuming it. The present study seeks to guide the population about the microbiological hazards and contributes to other studies so that the minimum cooking time required for mussels can be included in the labeling of this product.

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Publication Dates

  • Publication in this collection
    14 Mar 2022
  • Date of issue
    2022

History

  • Received
    12 Nov 2021
  • Accepted
    13 Dec 2021
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