Evaluation of minimum ripening time of artisanal mountain cheese from Santa Catarina prior to selling / Avaliação do período mínimo de maturação para comercialização do queijo artesanal serrano de Santa Catarina

Artisanal mountain cheese (queijo artesanal serrano; AMS) is produced from raw milk, which makes it a suitable medium for the multiplication of pathogenic microorganisms. The ripening process involves physical, chemical and microbiological factors unfavorable to pathogen development. For this reason, the Brazilian legislation requires a minimum ripening time of 60 days. However, according to Normative Instruction (NI) no 30, of August 7, 2013, and Decree no. 9.013, of March 29, 2017, this time can be reduced provided that scientific studies show that there is no alteration in the safety of the product. This study aimed to evaluate the microbiological quality of AMS in 19 cheese factories in the Serra Catarinense region, at 14, 21, 28 and 35 days of ripening, and to determine which period would meet the microbiological limits. A total of 76 samples of AMS, 19 samples of water and 19 samples of raw milk were studied. The microorganisms were investigated following Ordinance no. 146, of March 7, 1996. The investigation and count of coliforms at 30 °C, E. coli and coagulase-positive Staphylococcus followed protocols by Silva et al. (2010), using Petrifilm™. The investigation of Salmonella spp. followed Silva et al. (2010), whereas the investigation of Listeria monocytogenese and count of mesophilic aerobes followed NI no. 30, of June 26, 2018. Water potability was evaluated by the Most Probable Number (MPN) technique, in accordance with NI no. 30/2018 and Consolidation Ordinance no. 5, of September 28, 2017. Results were tested by Analysis of Variance and Linear Regression using the SAS statistical package with significance determined at p < 0.05. No safe ripening period for sale could be determined. At 35 days of ripening, 14 farms still showed counts above the limit for the microorganisms described in the legislation. On one of the farms, the presence of L. monocytogenes was detected in the four evaluated periods, demonstrating that ripening was not efficient to eliminate the pathogen. The microbiological quality of the milk was not satisfactory, with 63.16% of the samples not conforming to the standards. As regards the potability of the water used in the cheese factories, 52.63% of the analyzed samples showed unsatisfactory results. The research revealed heterogeneity in the hygienic-sanitary standards adopted by the cheese factories, indicating that there is a need for training in Good Agricultural Practices and Good Manufacturing Practices, support from institutions, periodic inspection and commitment of producers to standardize the process.(AU)

O queijo artesanal serrano (QAS) é produzido a partir de leite cru, tornando-se um meio propício para a multiplicação de micro-organismos patogênicos. O processo de maturação envolve fatores físicos, químicos e microbiológicos que desfavorecem o desenvolvimento de patógenos. Diante disso, a legislação exige um período mínimo de 60 dias de maturação, porém, de acordo com a IN nº 30, de 7 de agosto de 2013 e com o Decreto nº 9.013, de 29 de março de 2017, pode-se considerar a possibilidade de reduzir esse tempo, desde que estudos científicos comprovem que não há alteração da inocuidade do produto. O objetivo do trabalho foi avaliar a qualidade microbiológica de QAS, em 19 queijarias da Serra Catarinense, com 14, 21, 28 e 35 dias de maturação e verificar qual período atenderia aos limites microbiológicos. Foram objetos de estudo 76 amostras de QAS, 19 amostras de água e 19 amostras de leite cru. Os micro-organismos pesquisados seguiram a Portaria nº 146, de 07 de março de 1996. A pesquisa e quantificação de coliformes a 30°C, E. coli e Staphylococcus coagulase-positiva, seguiram protocolos de Silva et al. (2010), utilizando Petrifilm™. A pesquisa de Salmonella spp. seguiu Silva et al. (2010) e a pesquisa de Listeria monocytogenese e a quantificação de aeróbios mesófilos seguiram a Instrução Normativa nº 30, de 26 de junho de 2018. Para avaliação da potabilidade da água foi realizada a técnica do Número Mais Provável (NMP), seguindo a IN nº 30/2018 e a Portaria de Consolidação nº 5, de 28 de setembro de 2017. Os resultados passaram por teste de Análise de Variância e Regressão Linear do pacote estatístico SAS com significância (p < 0.05). Nesse estudo, não foi possível determinar um período de maturação seguro para comercialização. Aos 35 dias de maturação, 14 propriedades ainda apresentaram quantificações acima do limite para os micro-organismos descritos na legislação. Em uma das propriedades, foi detectada a presença de L. monocytogenes nos quatro períodos avaliados, demonstrando que a maturação não foi eficiente para eliminar o patógeno. A qualidade microbiológica do leite não foi satisfatória, sendo que 63,16% das amostras estavam fora dos padrões. Com relação à potabilidade da água das queijarias, 52,63% das amostras analisadas apresentaram resultados insatisfatórios. A pesquisa indicou heterogeneidade nos padrões higiênico-sanitários das queijarias, demonstrando que são necessárias capacitações em Boas Práticas Agropecuárias e Boas Práticas de Fabricação, apoio de instituições, fiscalização periódica e comprometimento dos produtores para padronização do processo.(AU)

Detection of virulence genes and antimicrobial resistance profiles of Escherichia coli isolates from raw milk and artisanal cheese in Southern Brazil / Detecção de genes de virulência e perfis de resistência antimicrobiana de Escherichia coli isoladas de leite cru e queijo artesanal no Sul do Brasil

The serrano artisanal cheese is a typical product from South region of Brazil, which is produced by skilled cheesemakers using raw milk. The contamination of this food by Escherichia coli has a great impact on public health, since it could threat the consumers health. The study evaluated the presence of virulence genes, antimicrobial susceptibility profiles and bofilm-production ability of Escherichia coli isolates obtained from raw milk and artisanal cheese produced in Southern Brazil. A total of 117 isolates of E. coli were characterized by multiplex PCR to detect the following virulence genes: eae for enteropatogenic E. coli (EPEC), lt and st for enterotoxigenic E. coli (ETEC), stx for shiga toxin-producing E. coli (STEC), stx and eae for enterohemorrhagic E. coli (EHEC), ipaH for enteroinvasive E. coli (EIEC) and aggR for enteroaggregative E. coli (EAEC). In addition, antimicrobial susceptibility profile to 22 antimicrobial agents was also performed by disk diffusion method, and we searched for extended-spectrum beta-lactamases (ESBL) and/or carbapenemase- producing isolates. Isolates that were positive for ESBL and carbapenemase were further investigated for the presence of the genes: bla(TEM), bla(SHV), bla(OXA), bla(CTX-M), for ESBL and bla(OXA-48) for carbapenemase. Further, isolates had their ability to form biofilms investigated by the red Congo agar method. Virulence genes of E. coli were identified in 21.37% of the tested isolates, which were classified as EPEC (the most prevalent pathotype) and ETEC or EAEC. Ten (8.55%) of the total studied E. coli isolates revealed a multidrug-resistant profile, since they were resistant to three or more antimicrobial classes; whereas four isolates (3.42%) were classified as ESBL-producers and showed the presence of bla(TEM) gene. None of the isolates exhibited carbapenemase activity nor did they carry carbapenemase genes. From the total of E. coli isolates, 79 (67.52%) were considered potential biofilm...(AU)

O queijo artesanal serrano é um produto típico da região sul do Brasil e se caracteriza por ser produzido a partir de leite cru. A contaminação desse alimento por Escherichia coli assume grande relevância para a saúde pública, pois oferece risco a saúde dos consumidores. Esse estudo avaliou a presença de genes de virulência, os perfis de susceptibilidade antimicrobiana e a capacidade de produção de biofilme de isolados de E. coli obtidos a partir de leite cru e queijo artesanal produzido no Sul do Brasil. Um total de 117 isolados de E. coli foram caracterizados por multiplex PCR para detecção dos seguintes genes de virulência: eae para E. coli enteropatogênica (EPEC), lt e st para E. coli enterotoxigênica (ETEC), stx para E. coli produtora da toxina shiga (STEC), stx e eae para E. coli enterohemorrágica (EHEC), ipaH para E. coli enteroinvasiva (EIEC) e aggR para E. coli enteroagregativa (EAEC). Adicionalmente, o perfil de susceptibilidade antimicrobiana a 22 agentes antimicrobianos foi determinado pelo método de disco difusão e a busca de isolados produtores de beta-lactamases de espectro estendido (ESBL) e carbapenemase foram realizadas. Os isolados positivos para ESBL e carbapenemase foram investigados quanto a presença dos genes: bla(TEM), bla(SHV), bla(OXA), bla(CTX-M) para ESBL e bla(OXA-48) para carbapenemase. Além disso, a potencial capacidade dos isolados de E. coli em produzir biofilme foi determinada pela técnica do ágar vermelho Congo. Os genes de virulência de E. coli foram identificados em 21,37% dos isolados testados, que foram classificados como EPEC (patótipo mais prevalente), ETEC ou EAEC. Dez (8,55%) isolados de E. coli apresentaram perfil de multirresistência, pois foram resistentes a três ou mais classes de antimicrobianos; enquanto que quatro isolados (3,42%) foram classificados como produtores de ESBL, sendo identificado o gene blaTEM. Nenhum isolado foi classificado como produtor de carbapenemase. Do total de...(AU)

Ocorrência de Campylobacter spp. em carcaças resfriadas de frangos abatidos na região oeste de Santa Catarina / Ocurrence of Campylobacter spp. in chilled chicken carcasses slaughtered in the west region of Santa Catarina, Brazil

Background: The thermophilic bacteria of the genus Campylobacter are important agents of alimentary gastroenteritis, called campylobacteriosis. These microorganisms multiply in temperatures ranging from 25ºC to 46ºC, however, low temperatures are incompatible with their multiplication. For this reason, the seasons of the year may interfere with the level of contamination by Campylobacter sp. The main sources of transmission are contaminated meat and giblets from poultry during poorly conducted slaughter operations. The disease may present itself with a different range of forms of disease: from mild signs of gastrointestinal infection to more severe cases, such as the Guillain-Barré syndrome. Material, Methods & Results: Due to the great importance of western Santa Catarina to the poultry industry, it was necessary to verify the occurrence of the pathogen in cold carcasses of broilers slaughtered in this region, and its variation through the seasons of the year. From January 2013 to February 2015 broiler carcasses were collected weekly, after the water cooling process, in slaughterhouses under Federal Inspection of the three largest microregions of western Santa Catarina in terms of number of broilers slaughtered, totaling 808 samples. The assessment of thermophilic Campylobacter was performed according to the methodology recommended by ISO 10272-1: 2006. Of the 808 samples analyzed, the frequency of isolation of thermophilic Campylobacter was 1.82% (8/440) in microregion 1, 4.95% (10/202) in microregion 2 and 13.86% (23/166) in the microregion 3, totaling 5.07% of positive samples (41/808). Comparing the microregions, it was verified that there was no significant difference (P > 0.05) between the isolation frequencies of microregions 1 and 2. However, there was a significant difference (P < 0.05) between the isolation rates of Microregion 3 in relation to microregions 1 and 2.[...]

Ocorrência de Campylobacter spp. em carcaças resfriadas de frangos abatidos na região oeste de Santa Catarina / Ocurrence of Campylobacter spp. in chilled chicken carcasses slaughtered in the west region of Santa Catarina, Brazil

Background: The thermophilic bacteria of the genus Campylobacter are important agents of alimentary gastroenteritis, called campylobacteriosis. These microorganisms multiply in temperatures ranging from 25ºC to 46ºC, however, low temperatures are incompatible with their multiplication. For this reason, the seasons of the year may interfere with the level of contamination by Campylobacter sp. The main sources of transmission are contaminated meat and giblets from poultry during poorly conducted slaughter operations. The disease may present itself with a different range of forms of disease: from mild signs of gastrointestinal infection to more severe cases, such as the Guillain-Barré syndrome. Material, Methods & Results: Due to the great importance of western Santa Catarina to the poultry industry, it was necessary to verify the occurrence of the pathogen in cold carcasses of broilers slaughtered in this region, and its variation through the seasons of the year. From January 2013 to February 2015 broiler carcasses were collected weekly, after the water cooling process, in slaughterhouses under Federal Inspection of the three largest microregions of western Santa Catarina in terms of number of broilers slaughtered, totaling 808 samples. The assessment of thermophilic Campylobacter was performed according to the methodology recommended by ISO 10272-1: 2006. Of the 808 samples analyzed, the frequency of isolation of thermophilic Campylobacter was 1.82% (8/440) in microregion 1, 4.95% (10/202) in microregion 2 and 13.86% (23/166) in the microregion 3, totaling 5.07% of positive samples (41/808). Comparing the microregions, it was verified that there was no significant difference (P > 0.05) between the isolation frequencies of microregions 1 and 2. However, there was a significant difference (P < 0.05) between the isolation rates of Microregion 3 in relation to microregions 1 and 2.[...](AU)

Avaliação da inocuidade e qualidade microbiológica do queijo artesanal serrano e sua relação com as variáveis físico químicas e o período de maturação / Evaluation of the Safety and Quality of Microbiological Handmade Cheese Serrano and its Relation to Physical and Chemical Variables the Period of Maturity

Background: The handmade cheese serrano is a typical product created on small farms in the states of Santa Catarina and Rio Grande do Sul. Its main feature is to be manufactured from raw. Due to the physico-chemical phenomena that occur during maturation, this process is the only way to eliminate the pathogenic microorganisms that may be present contaminating the cheese. The present study aimed to evaluate the safety of handmade cheeses by isolation and identification of Listeria monocytogenes and Salmonella spp. and evaluate the microbiological quality through the isolation and quantification of Staphylococcus aureus, Escherichia coli and total coliforms and relate the maturation time and variations physico-chemical with the contamination of handmade cheeses. Materials, Methods & Results: The experiment was conducted using 108 samples of handmade cheese Serrano from the towns that make up the mountainous region of SC, with maturation periods of 15, 30, 45 and 60 days. In each of the stages of maturation were analyzed twenty seven samples of cheeses, for the presence of Listeria monocytogenes by the method USDA/2009 and Salmonella spp. methodology according to ISO 6579/2007. The identifi cation and enumeration of Staphylococcus aureus, Escherichia coli and total coliform was performed in plates petrifilm™. Physicochemical analyzes consisted of determining moisture, acidity, fat and protein. In this study was evaluated a total of 108 samples, of these 63 (58.33%) were positive for total coliforms, 64 (59.26%) were positive for Escherichia coli and 36 (33.33%) showed counts of Staphylococcus aureus over 103 CFU / g. There was a decrease in the population of coliforms and Escherichia coli after 30 and 45 days of maturation, respectively. The enumeration of Staphylococcus aureus was not infl uenced by maturity period, however, there was a reduction in the count of this micro-organism from 15 to 45 days and after this period there was an increase in the population of the same. Salmonella spp. were not detected in any analyzed maturation periods. Listeria spp. were isolated in 21/108 (19.44%) of the samples analyzed, 18 (16.66%) were identified as Listeria innocua and three (2.77%) as Listeria monocytogenes. The isolation of Listeria spp. decreased from 30 days of ripening and Listeria monocytogenes isolated at 30 and 45 days of ripening. The physicochemical results found in this study are presented in a range of normal variation, the maturation period only influenced the fat content, where a significant decrease was observed from 15 to 30 days, and protein content which higher performed at the end of the period. Discussion: The presence of total coliforms, Escherichia coli and Staphylococcus aureus showed defi ciency in hygiene procedures during manufacturing or storage of the cheeses and the presence of Escherichia coli indicates fecal contamination of food, suggesting that other enteropathogens may be present. The presence of Listeria innocua suggests that Listeria monocytogenes may be present contaminating the product. Listeria monocytogenes makes handmade cheese unfit for human consumption. Thus, it was not possible to check a minimum maturation period of handmade cheese with no or low count of micro-organisms evaluated. The great variation in the composition of the cheeses analyzed demonstrates the lack of standardization in the production of handmade cheese.

Detection of virulence genes and antimicrobial resistance profiles of Escherichia coli isolates from raw milk and artisanal cheese in Southern Brazil / Detecção de genes de virulência e perfis de resistência antimicrobiana de Escherichia coli isoladas de leite cru e queijo artesanal no Sul do Brasil

The serrano artisanal cheese is a typical product from South region of Brazil, which is produced by skilled cheesemakers using raw milk. The contamination of this food by Escherichia coli has a great impact on public health, since it could threat the consumers health. The study evaluated the presence of virulence genes, antimicrobial susceptibility profiles and bofilm-production ability of Escherichia coli isolates obtained from raw milk and artisanal cheese produced in Southern Brazil. A total of 117 isolates of E. coli were characterized by multiplex PCR to detect the following virulence genes: eae for enteropatogenic E. coli (EPEC), lt and st for enterotoxigenic E. coli (ETEC), stx for shiga toxin-producing E. coli (STEC), stx and eae for enterohemorrhagic E. coli (EHEC), ipaH for enteroinvasive E. coli (EIEC) and aggR for enteroaggregative E. coli (EAEC). In addition, antimicrobial susceptibility profile to 22 antimicrobial agents was also performed by disk diffusion method, and we searched for extended-spectrum beta-lactamases (ESBL) and/or carbapenemase- producing isolates. Isolates that were positive for ESBL and carbapenemase were further investigated for the presence of the genes: bla(TEM), bla(SHV), bla(OXA), bla(CTX-M), for ESBL and bla(OXA-48) for carbapenemase. Further, isolates had their ability to form biofilms investigated by the red Congo agar method. Virulence genes of E. coli were identified in 21.37% of the tested isolates, which were classified as EPEC (the most prevalent pathotype) and ETEC or EAEC. Ten (8.55%) of the total studied E. coli isolates revealed a multidrug-resistant profile, since they were resistant to three or more antimicrobial classes; whereas four isolates (3.42%) were classified as ESBL-producers and showed the presence of bla(TEM) gene. None of the isolates exhibited carbapenemase activity nor did they carry carbapenemase genes. From the total of E. coli isolates, 79 (67.52%) were considered potential biofilm...

O queijo artesanal serrano é um produto típico da região sul do Brasil e se caracteriza por ser produzido a partir de leite cru. A contaminação desse alimento por Escherichia coli assume grande relevância para a saúde pública, pois oferece risco a saúde dos consumidores. Esse estudo avaliou a presença de genes de virulência, os perfis de susceptibilidade antimicrobiana e a capacidade de produção de biofilme de isolados de E. coli obtidos a partir de leite cru e queijo artesanal produzido no Sul do Brasil. Um total de 117 isolados de E. coli foram caracterizados por multiplex PCR para detecção dos seguintes genes de virulência: eae para E. coli enteropatogênica (EPEC), lt e st para E. coli enterotoxigênica (ETEC), stx para E. coli produtora da toxina shiga (STEC), stx e eae para E. coli enterohemorrágica (EHEC), ipaH para E. coli enteroinvasiva (EIEC) e aggR para E. coli enteroagregativa (EAEC). Adicionalmente, o perfil de susceptibilidade antimicrobiana a 22 agentes antimicrobianos foi determinado pelo método de disco difusão e a busca de isolados produtores de beta-lactamases de espectro estendido (ESBL) e carbapenemase foram realizadas. Os isolados positivos para ESBL e carbapenemase foram investigados quanto a presença dos genes: bla(TEM), bla(SHV), bla(OXA), bla(CTX-M) para ESBL e bla(OXA-48) para carbapenemase. Além disso, a potencial capacidade dos isolados de E. coli em produzir biofilme foi determinada pela técnica do ágar vermelho Congo. Os genes de virulência de E. coli foram identificados em 21,37% dos isolados testados, que foram classificados como EPEC (patótipo mais prevalente), ETEC ou EAEC. Dez (8,55%) isolados de E. coli apresentaram perfil de multirresistência, pois foram resistentes a três ou mais classes de antimicrobianos; enquanto que quatro isolados (3,42%) foram classificados como produtores de ESBL, sendo identificado o gene blaTEM. Nenhum isolado foi classificado como produtor de carbapenemase. Do total de...