Hygienic-sanitary quality of ready-to-eat salmon sashimi (Salmo Salar) Qualidade higiênico-sanitária de sashimi de salmão (Salmo salar) pronto para o consumo Calidad higiénico-sanitaria del sashimi de salmón (Salmo salar) listo para consumir

Fifty sashimi samples from 5 restaurants were characterized for enumeration of Aerobic Psychrotrophic Heterotrophic Bacteria (APHB) and Aerobic Mesophilic Heterotrophic Bacteria (AMHB), Enterobacteriaceae, coliforms at 45oC (thermotolerant coliforms), coagulase-positive Staphylococci, presence of Vibrio parahaemolyticus and Salmonella sp., and determination of hydrogen potential (pH) and total volatile basic nitrogen (TVB-N). The bacterial isolates were evaluated for their resistance profile to the antimicrobial agents Penicillin, Ampicillin, Cefoxitin, Cefotaxime, Amikacin, Gentamicin, Tetracycline, and Trimethropim-sulfamethoxazole. The results showed that APHB, AMHB and coagulase-positive Staphylococci counts and the determination of pH and TVB-N were in accordance with national and international standards adopted as safe limits for consumption. In contrast, the Enterobacteriaceae and thermotolerant coliforms counts and presence of Salmonella sp. and V. parahaemolyticus were in disagreement with those standards, raising concern about the hygienic-sanitary quality of sashimi. The Staphylococcus aureus and V. parahaemolyticus isolates showed resistance to Penicillin, Ampicillin, Cefoxitin, Cefotaxime, Tetracycline, Gentamicin, and Amikacin, while the Salmonella sp. isolate showed no resistance to all the antimicrobial agents studied. The results showed that 48% of the samples were fit for consumption while 52% had unsatisfactory hygienicsanitary quality for the parameters evaluated.


Introduction
The general public's view of the modern diet and human health has led to new eating habits in recent years. Fish consumption has increased worldwide, due to its high nutritional value and intrinsic characteristics. However, this food matrix is very susceptible to microbiological contamination, requiring care from capture to consumption, such as hygiene, cold storage, and heat treatment.
Although an adequate cooking process represents food safety, the globalization of Japanese cuisine has led to the consumption of fish in the form of sashimi, which consists of thin slices of raw fish served with sauces, and not subjected to cooking steps. Therefore, it is essential to apply quality control tools, such as the adoption of good manufacturing practices, with temperature control in all stages after the capture and use of ice made with treated water 50 for ensuring hygienic-sanitary quality.
Food safety, one of the general food hygiene principles of the Codex Alimentarius, is defined as the assurance that food will not cause harm to the consumer during preparation and consumption (FAO, 1998). Failures in the hygienic-sanitary aspects can contribute to foodborne illness occurrences, with risks to consumers' health (WHO, 2018;Brasil, 2019). In this context, raw fish stand out for being more susceptible to deterioration and microbial contamination.
National and international microbiological guidelines have been adopted to ensure food safety and quality, including those of the National Health Surveillance Agency (ANVISA) (Brasil, 2001), International Commission on Microbiological Specifications for Foods (ICMSF, 1986), Center for Food Safety (CFS, 2014) and Ryser and Schuman (2015), as well as the Regulation of the Industrial and Sanitary Inspection of Products of Animal Origin (RIISPOA), which has established values for the hydrogen potential (pH) and total volatile basic nitrogen of fresh fish (Brasil, 2017).
The emergence of bacteria resistant to antimicrobial agents is another trouble related to fish consumption, which has been a concern of international health agencies and several interrelated institutions, in addition to consumers, once antimicrobial resistance is one of the most serious global public health threats. The resistance of bacteria increases in places with intense transit, such as hospitals, food animal production and aquaculture Cabello e Godfrey, 2016; Venter, Henning e Begg, 2017). Research, Society andDevelopment, v. 10, n. 12, e573101220900, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i12.20900 3 Therefore, this study aimed to investigate the hygienic-sanitary quality of salmon (Salmo salar) sashimi ready for consumption in the city of Cuiaba, Mato Grosso, Brazil, through the microbiological, physicochemical, and antimicrobial resistance profile, and the potential risks to consumers' health.

Methodology
A survey of all Japanese cuisine restaurants in the city of Cuiaba, Mato Grosso, Brazil (n = 28) was carried out. Five restaurants were randomly selected for sample collection, four of which specialized in Japanese cuisine. The samples consisted of portions of approximately 200 grams of salmon sashimi, and 10 samples were collected in each restaurant (R1, R2, R3, R4, and R5), totaling 50 samples. The samples were ordered and taken from each restaurant at the beginning of the working day.
The sashimi portions were packed in the packages offered by the establishment, which were immediately placed in a thermal box with ice packs to maintain the temperature during transport to the Food Microbiology Laboratory of the Federal Institute of Education, Science and Technology of Mato Grosso (IFMT) Cuiaba -Bela Vista Campus, for analysis. The time interval between collecting the samples and starting the analysis did not exceed one hour. This quantitative study used a completely randomized design (Pereira, 2018).

Microbiological characterization
The microbiological analyses consisted of the enumeration of Aerobic Psychrotrophic Heterotrophic Bacteria (APHB)  (Silva et al., 2017). The microbiological assays were performed in triplicate, with the results expressed in Log CFU/g.

Physicochemical characterization
The samples were characterized for hydrogen potential (pH) according to ISO 2917ISO (1999 with direct reading on bench pH and total volatile basic nitrogen (TVB-N) according to Brazil (1981), and the results expressed in mg TVB-N/100 g.
All determinations were performed in triplicate.

Antimicrobial resistance profile
After biochemical confirmation (Silva et al., 2017), the bacterial strains isolated from salmon sashimi were identified and kept refrigerated until analysis. The antimicrobial resistance profile was performed according to the antibiotic disk diffusion method (Bauer et al., 1966).

Statistical analysis
The results of the physicochemical and microbiological characterization were analyzed by analysis of variance (ANOVA) at a 5% level of significance (p <0.05). The Kolmogorov-Smirnov normality test was applied to all data. The normally distributed data were analyzed through a completely randomized design (CRD) using the Tukey's test (pH, TVB-N, APHB, Enterobacteriaceae). The Kruskal-Wallis and the Wilcoxon mean comparison tests were applied to data that presented no normality (AMHB, coliforms at 45 °C, coagulase-positive staphylococci). The microbiological counts (APHB, AMHB, Enterobacteriaceae, coliforms at 45 °C, coagulase-positive staphylococci) were transformed into Log10 by the statistical program. The chi-square test was used for the results of the presence of Salmonella sp. and Vibrio parahaemolyticus, to determine whether the results were homogeneous concerning the five restaurants. Principal Component Analysis (PCA) was also performed to correlate the physicochemical and microbiological data between restaurants, using a correlation matrix. To determine the hygienic-sanitary quality of the samples, the results were compared with legislation standards and international references, determining the number of satisfactory and unsatisfactory samples, in percentage. The statistical program R version 3.6.1 was used (R Core Team, 2019).

Hygienic-sanitary quality
APHB, also known as spoilage bacteria, grow in refrigerated products (0 -7 °C) leading to food deterioration due to the activity of proteolytic and lipolytic enzymes (Lanzarin et al., 2011). Some authors have reported counts above 7, 4.59, and 6.18 Log CFU/g in sashimi (Muscolino et al., 2014, Miguéis et al., 2015, Miguéis et. al, 2016. In the present study, as shown in while R1 and R5 presented the highest counts with values of 5.06 and 5.14 Log CFU/g, respectively (p> 0.05). These microorganisms are considered indicators of food quality, demonstrating hygienic conditions during the manufacturing process, in addition to contributing to the determination of the shelf life. The present counts were satisfactory, with microbial counts lower than 6 log CFU/g, according to the classification of the Center for Food Safety (2014) and Ryser and Schuman (2015). Several authors have found different mesophilic bacteria counts in sushi and sashimi, with values of 5.5 Log CFU/g; 5.3 Log CFU/g; 5.11 Log CFU/g; and 7.0 Log CFU/g (Liang et al., 2016;Kim et al., 2016;Miguéis et al., 2015;Muscolino et al., 2014). Studies with salmon and tuna sashimi found Enterobacteriaceae counts of 3.39; 3.43; and 3.25 Log CFU/g, respectively (Miguéis, et al., 2015;Miguéis, et al., 2016) corroborating the findings of this study.  (2021).
The highest count was found in R1 with 3.49 Log CFU/g (2.94 to 4.03 Log CFU/g), followed by R5 with 2.11 Log CFU/g (0 to 3.76 Log CFU/g) (p <0.05). All samples (100%) from the restaurant R1 and half (50%) of the samples from R5 had counts higher than those established by the Brazilian legislation (2 Log CFU/g) (Brasil, 2001), corroborating the results of Enterobacteriaceae and showing concern with hygienic-sanitary quality since this group of microorganisms may contain potentially pathogenic and FBD-causing strains. In this study, although Escherichia coli was not detected, other bacteria from the coliform group were detected, such as Enterobacter sp., Klebsiella sp., and Pantoea sp.
Regarding the enumeration of positive coagulase staphylococci (STA), all samples were classified as satisfactory, with counts lower than 3.69 Log CFU/g (Brasil, 2001), with the lowest values observed for R1, R2, R3, and R4, with 0; 0.13; 0.38; 0.13 Log CFU/g, respectively (p> 0.05) ( Table 1) (counts ranging from 0; 0 to 1.30; 0 to 1.43; and 0 to 1.30 Log CFU/g for R1, R2, R3, and R4, respectively). In contrast, the restaurant R5 presented the highest count, 1.38 Log CFU/g, (p <0.05) (Table 1), ranging from 0 to 2.58 Log CFU/g. Although the samples presented satisfactory counts, this group of microorganisms stands out as is enterotoxins producers and the contamination may be due to the lack of hygiene during handling or processing since this bacterium is natural in the animal and human microbiota. From 2009 to 2018, this agent was considered one of the main bacteria responsible for foodborne outbreaks in Brazil, being the third etiological agent with 9.5% of cases (Brasil, 2019). In the present study, Staphylococcus aureus, a species with great potential for toxin production, was found in the restaurants R2, R3, R4, and R5. Staphylococci are popularly identified in sashimi, with recent studies in Italy (Muscolino et al., 2014), Portugal (Miguéis et al., 2015;Miguéis et al., 2016) and Malaysia (Puah et al., 2017). They are also found in oysters, shrimp, sushi, and a variety of fresh marine fish in several countries such as Turkey (Onmaz et al., 2015), Switzerland (Boss et al., 2016), Iran (Arfatahery et al., 2016), and Denmark (Li et al., 2019).
The presence of Salmonella sp. in food is a worrying factor, as it is the main cause of food-borne illnesses with tens of millions of cases per year worldwide (WHO, 2018). In Brazil, from 2000 to 2017, 12,503 outbreaks were reported by etiologic agents, with 236,403 sick people registered with the Ministry of Health, and Salmonella sp. was the most common bacterial cause, with 30% of cases (Brasil, 2018). As can be seen in Table 1, the presence of Salmonella sp. was detected in a sample from the restaurant R1, which is in disagreement with the legislation (Brasil, 2001) and considered inappropriate for human Research, Society and Development, v. 10, n. 12, e573101220900, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i12.20900 consumption, becoming an alert factor with potential pathogenic risk. Prevention requires care in all stages of the food chain, from production to consumption (WHO, 2018). Puah et al. (2017) found Salmonella enterica in 21.66% (13/60) of retail sashimi samples in Valley Klang, Malaysia, which was also observed by other authors in fresh fish in Nigeria (Beshiru et al., 2019), Jordan (Obdait e Salman, 2017) and Turkey (Onmaz et al., 2015).
Vibrio parahaemolyticus is a marine bacterium responsible for illness of gastroenteritis after consumption of raw, inadequately cooked, or croos-contamined seafood (Sakazaki, 2003). The present study found the presence of Vibrio parahaemolyticus in 24% of the samples studied, consisting of twelve samples (6 samples in R1, 5 samples in R4, and 1 sample in R5), which is of concern, as this bacterium is related to cases of gastroenteritis evolving to septicemia in immunocompromised individuals, even leading to death. The presence of this agent in the samples of this study may be due to the origin of the raw material, suggesting that it comes from contaminated water and different producers. Other Vibrio sp. species were also verified in this study, such as V. mimicus, V. metchnikovii, which serves as a warning for the occurrence of unknown pathogenic strains that can be risk factors associated with outbreaks (FDA, 2012). Some authors have reported Vibrio sp. in sashimi, with counts ≥ 2 and ≥ 3 Log CFU/g (Muscolino et al., 2015;Kim et al., 2016). V. parahaemolyticus has also been found in several fresh seafood worldwide, such as in Ecuador (Sperling et al., 2015), China (Xu et al., 2016), Vietnam (Vu et al., 2016), Poland (Lopatek et al., 2018), Brazil (Silva et al., 2018), and Korea (Kang et al., 2019, Ryu et al., 2019. Some compounds are produced from bacterial metabolism and fish decomposition, which can be indicative of fish quality, such as total volatile basic nitrogen (TVB-N) and hydrogen potential (pH) (Gonçalves, 2011 respectively, when compared to R5, which presented 18.98 mg TVB-N/100g. The restaurants R1 and R4 presented TVB-N levels similar to those observed for the restaurants R2, R3, and R5 (p> 0.05). The determination of volatile basic nitrogen (TVB-N) are used to characterize the freshness of fish since the formation of nitrogen compounds results from the enzymatic and bacterial deterioration, forming products from the decomposition of the amino acids such as dimethylamine, trimethylamine, ethylamine, monomethylamine, ammonia, putrescine, cadaverine, and spermidine (Ogawa e Maia, 1999;Howgate, 2010;Nollet e Toldrá, 2010;Gonçalves, 2011). Therefore, the TVB-N levels can be indicative of fish conservation, especially regarding the stage of deterioration.
However, some species can present higher levels of these compounds, even though they are not under the decomposition process, such as the Salmo salar species for example, which has a higher level up to 35 mg N/100g, according to the Annex I of the European Communities (1995). For the Brazilian legislation, fish, in general, must have total volatile bases levels lower than 30 mg of N/100g of muscle tissue (Brasil, 2017). All samples analyzed in this study met this parameter.
The pH of the samples ranged from 6.20 to 6.36; 6.19 to 6.27; 6.18 to 6.25; 6.22 to 6.35; and 6.25 to 6.30, for R1, R2, R3, R4, and R5, respectively, with the lowest pH found in the restaurants R3 and R4 (6.22 and 6.24, respectively), and the highest pH in the restaurants R1, R2, and R5 (6.28, 6.30, and 6.28, respectively) as shown in Table 1 (p> 0.05). As reported by Ogawa and Maia (1999), the pH in salmon can range from 6.1 to 6.3. According to the Brazilian legislation, the pH of fish should be lower than 7.0 to be considered fresh and acceptable for consumption (BRASIL, 2017). All samples of this study were within the limit established by law. From the physicochemical point of view, the pH value is directly related to the quality and conservation of fish, and the bacterial load is consistent with the variation of pH. Changes in the concentration of hydrogen ions, due to the hydrolytic, oxidative, or fermentative decomposition of the muscle can lead to higher pH values, thus leading to an increase in the bacterial activity. Fish with a pH below 5.6 has a longer shelf life when compared to those with pH from 6.2 and 6.6 due to the greater availability of bacterial changes (Ogawa e Maia, 1999;Gonçalves, 2011). Research, Society andDevelopment, v. 10, n. 12, e573101220900, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i12.20900 7 The hygienic-sanitary quality of the salmon sashimi samples was determined by comparing the microbiological and physicochemical results of each restaurant with national and international standards. Regarding the samples from the restaurant R1, 10% were unsatisfactory for Enterobacteriaceae, 100% for thermotolerant coliforms, 1% exhibited the presence of Salmonella sp. and 6% Vibrio parahaemolyticus, while in R4, 40% were unsatisfactory for Enterobacteriaceae and 5% were contaminated with V. parahaemolyticus. The samples from the restaurant R5 were 100% unsatisfactory for Enterobacteriaceae, 50% for thermotolerant coliforms and 1% exhibited the presence of V. parahaemolyticus. Finally, the samples from the restaurants R2 and R3 were 100% satisfactory (Table 2). Principal Component Analysis (PCA) was used to synthesize the quantitative results considered as variables, aimed to understand the correlation between variables (Figure 1). The principal components 1 and 2, called Dimension 1 (Dim1) and Dimension 2 (Dim 2), are the main explanations of this statistical tool. The two principal dimensions were responsible for 78.1% of the total variation on the contamination rate of sashimi, and Dim 1 explained 55.2% and Dim 2 explained 22.9%.
PCA (Figure 1) showed that both R2 and R3 were clustered together, once both dimensions Dim 1 and Dim 2 decrease together. The restaurants R1 and R4 were located in another group, with an increase in Dim 2 with an increase in Dim 1. The restaurant R5 was located alone, not forming a cluster with the others, and it was more distant from the origin, showing greater discrepancy; while Dim 1 was positive and increasing, dimension Dim 2 was negative and decreasing. Source: Authors (2021) For a better interpretation of the graph, the angle between variables was measured (APHB, AMHB, pH, TVB-N, etc).
Dim 1 showed a strong positive correlation between the variables TVB-N and AMHB, thus it is possible to state that AMHB increased with an increase in TVB-N, as they formed an acute angle and the vectors had very close sizes. Concerning Dim 2, the variables thermotolerant coliforms, pH, and APHB presented quite strong correlations, as they form acute angles to each other, that is, the increase in pH was affected by the increase in thermotolerant coliforms, and the APHB growth was the most responsible for increasing the pH. There was a strong correlation between R1 and R4 for the variable APHB, with fast growth in R1. A strong correlation between the variables S. aureus and Enterobacteriaceae was observed, with a strong correlation with R5. In contrast, R2 and R3 were located in opposite directions to all variables, indicating a strong negative correlation, that is, there was no interaction between the variables of these restaurants, thus they can be classified as the best quality restaurants according to the PCA, confirming the results of microbiological and physicochemical characterization.

Antimicrobial resistance profile
Antimicrobial susceptibility tests were applied according to the classification of the Clinical and Laboratory Standards Institute (CLSI, 2010(CLSI, , 2016(CLSI, e 2018, for drugs of first, second, and third choice for the treatment of pathogenic bacteria isolated in this study. The antimicrobial resistance profile is shown in Table 3, for the isolates Vibrio parahaemolyticus (n = 12) Staphylococcus aureus (n = 12) and Salmonella sp. (n = 1).
Knowledge about the sensitivity of Salmonella isolates to the antimicrobial agents used in this study is important for public health, as it allows the use of these agents in therapies during outbreaks since Salmonella outbreaks have been reported as the first causes of ATD in Brazil and in the world (Brasil, 2019;WHO, 2018).
Although the V. parahaemolyticus isolates were resistant to all the antimicrobials used, better results were observed for Trimethropim-sulfamethoxazole, with sensitivity for the isolates from two restaurants (R4 and R5). According to CLSI (2010), Halophilic Vibrio sp. is normally resistant to sulfonamides, penicillins, and other cellelaphosporins. In this study, resistance to aminoglycosides (Amikacin, Gentamicin), Tetracycline, and semi-synthetic penicillin (Ampicillin) was also observed.
The microorganism V. parahaemolyticus is frequently associated with diseases transmitted by seafood, being of concern in fish, mollusks, shrimp and consequently to human health, due to its multi antimicrobial resistance. This microorganism has shown higher resistance rates to Ampicillin (91%), demonstrating the ineffective action of this antibiotic, not being an alternative to combat this species, as also reported by some authors in Korea (Ryu et al., 2019;Kang et al., 2018), Brasil (Silva et al., 2018), Poland (Lopatek et al., 2018), Vietnam (Vu et al., 2016), China (Xu et al., 2016) e no Ecuador (Sperling et al., 2015). Studies have shown that Tetracycline, which exhibited 91% resistance in this study, presented great sensitivity for these bacteria in Korea, Brazil, Poland, and China. Cefotaxime, defined as a first-choice drug by CLSI (2010), showed a significant resistance (58.3%), indicating low in vitro activity. Some authors have also reported a strong sensitivity to Trimethropim-sulfamethoxazole, as observed in the present study (Millanao et al., 2018;Cabello e Godfrey, 2016).
In general, several factors are correlated with increased resistance of antimicrobials, such as overuse in aquaculture (XU et al., 2016) and genetically modified organisms in the aquatic environment. Although some antimicrobial agents behave similarly for different marine fish and seafood species, such comparison is still difficult, due to the different origin, the collection procedures, and the methodologies used, as observed by Lopatek et al. (2018).

Conclusion
It was concluded that 48% of sashimi samples were suitable for human consumption, while 52% presented unsatisfactory hygienic-sanitary quality for the parameters evaluated, which can be a risk to the consumers' health, such as the development of a Foodborne Disease (FBD). The restaurants R2 and R3 presented better hygienic-sanitary conditions when compared to R1, R4, and R5.
The antimicrobial resistance profile revealed that both the S. aureus and V. parahaemolyticus isolates showed sensitivity in vitro only to Trimethropim-sulfamethoxazole, which is a challenge related to the guidelines to assist with treatment choices in the case of foodborne illness.
Based on this study, it is suggested that similar work be carried out in several cities in the country for a broader survey of the quality of this food matrix, as well as scientific studies related to handling and raw materials.