Abstract Listeria monocytogenes is a gram-positive bacterium that can survive in food production environments and food products. This microorganism is associated with listeriosis, a serious infection caused by the consumption of contaminated food. The aim of this study was to conduct an integrative literature review on the regulation of L. monocytogenes in food in Brazil, its occurrence and methodologies used for identification in studies published from 2001 to 2021, and the epidemiological surveillance of cases of infection. The current regulations for L. monocytogenes prohibit ready-to-eat foods of more than 102 per gram or milliliter. Officially, only two cases of outbreaks have been identified in the Epidemiological Surveillance of Foodborne Diseases system; however, the circulation of L. monocytogenes was observed in foods of different origins, with occurrence ranging from 3.1 to 48.7. The most commonly used identification method was ISO 11290. The isolation and identification methods for this pathogen are expensive and laborious, making it difficult to implement these methodologies in many laboratories. This scenario contributes to the underreporting of cases of listeriosis and therefore represents a risk for the population that is exposed to potentially contaminated food.

Abstract Conventional culture media are expensive owing to their constituents. Thus, several studies have sought to develop and evaluate the efficacy of alternative, low-cost culture media, in most cases, using natural and easily accessible raw materials. The present study is a literature review, observing various formulations of culture media based on products of plant origin for the growth of microorganisms and production of microbial compounds of industrial interest. In most formulations, vegetable substrates, such as soy, certain beans, corn, and rice, were used in addition to hortofruticultural products. Compared to conventional media, the alternative culture media often present satisfactory results in terms of microbial growth efficiency and production cost.

Abstract The psychrotrophic bacteria count and the profile of Gram-negative bacteria present in commercial Brazilian organic dairy products (27 samples, pasteurized whole milk, Minas Frescal cheese, and yoghurt, equally distributed) as well as the biofilm-producing capacity and the production of deteriorating enzymes were investigated. Most of the samples (59%) presented psychrotrophic bacteria counts higher than 4 log CFU/g, indicating problems in the microbiological quality of the products. The identification of Gram-negative bacteria revealed the presence of micro-organisms that can be potentially harmful (39.4% Acinetobacter baumanii/calcoaceticus, 23.2% Burkoderia pseudomallei, 10.1% Halfnia alvei) and/or deteriorating micro-organisms (6.1% Pseudomonas aeruginosa) with the capacity of producing proteases (36.4%), lecithinases (41.4%), and lipases (4%). Roughly, 68.7% of micro-organisms were non-producers of biofilms, 28.3% were medium biofilm producers, and 3.0% were high biofilm producers. The findings suggest there are problems related to the good practices of obtaining organic milk and in the manufacture of dairy products.

Abstract Although quite controversial from the point of view of food safety, a considerable number of people across different age and socioeconomic groups consume raw goat’s milk. Even if refrigerated after milking, raw goat's milk may still be susceptible to the lipolytic and proteolytic actions of psychrotrophic microorganisms, such as Pseudomonas. In this study, Pseudomonas sp. from 21 refrigerated raw goat’s milk samples were characterized. All samples were freely commercialized, without any type of inspection, by small producers and markets from different regions of Rio de Janeiro, Brazil. A total of 136 isolates were identified, most of which belonged to the species, P. putida, P. koreensis, P. monteilii, and P. fluorescens. The production of protease and lipase varied according to the incubation temperature. We found that 91.4% of the Pseudomonas isolates were susceptible to all antibiotics tested. None of the isolates exhibited a multidrug-resistance phenotype. None of the raw goat’s milk samples contained detectable antibiotic residues, which may explain the very low frequency of resistant bacteria. Although Pseudomonas is not considered a typical food-related pathogen, its presence indicate that the determination of microbiological standards for raw goat's milk and greater control of the commercialization of this product, are necessary.

RESUMO: O leite de cabra tem sido sugerido como alternativa ao leite de vaca, sendo uma opção com melhor digestibilidade e menor alergenicidade. No entanto, a presença de bactérias contaminantes pode afetar significativamente a segurança do produto. Neste trabalho, relatamos o isolamento e caracterização de Acinetobacter spp. isolados de amostras de leite cru de cabra adquiridas no Estado do Rio de Janeiro, Brasil. Vinte e uma amostras foram analisadas e dez isolados de Acinetobacter spp. foram obtidos, sendo seis identificados como A. guillouiae, três como A. ursingii e um como A. bereziniae. Estes isolados foram caracterizados e oito apresentaram atividade proteolítica, sete mostraram atividade lipolítica e cinco foram capazes de produzir ambas as enzimas. Nenhum dos isolados foi produtor de biofilme. No entanto, todos os isolados apresentaram fenótipo ESBL-positivo, enquanto oito (80%) foram positivos para KPC. Este trabalho demonstra, portanto, que o leite de cabra cru também pode ser uma fonte de Acinetobacter spp., que podem produzir enzimas deteriorantes termoestáveis e desempenhar, também, um papel de reservatórios de genes de resistência a antibióticos.

ABSTRACT: Goat’s milk has been suggested as an alternative to cow’s milk, being a better digestible and hypoallergenic option. However, the presence of contaminating bacteria may significantly affect the safety of the product. In this research, we reported the isolation and characterization of Acinetobacter spp. isolates from raw goat milk samples purchased in the state of Rio de Janeiro, Brazil. Twenty-one samples were analyzed and ten isolates of Acinetobacter spp. were obtained. Six were identified as A. guillouiae, three as A. ursingii, and one as A. bereziniae. These isolates were characterized and eight showed proteolytic activity, seven showed lipolytic activity, and five isolates were able to produce both enzymes. None of the isolates was biofilm producer. However, when the production of antibiotic resistance enzymes KPC and ESBL were investigated, all isolates presented ESBL-positive phenotype, while eight (80%) were KPC-positive. This research, therefore, demonstrated that raw goat’s milk can also be a source of Acinetobacter spp., which can produce important thermostable deteriorating enzymes and may play a role of reservoirs of antibiotic resistance genes.

Neste trabalho, a produção de substâncias antimicrobianas por estirpes de Pseudomonas sp. isoladas de leite pasteurizado e seu potencial de ação contra bactérias associadas a alimentos foram investigados. As amostras de leite pasteurizado foram adquiridas em estabelecimentos comerciais, arbitrariamente escolhidos, da cidade do Rio de Janeiro-RJ, Brasil. Dentre as quatro amostras analisadas, três apresentaram várias colônias típicas de Pseudomonas. Cerca de cem colônias foram escolhidas e submetidas a testes bioquímicos para confirmação da identificação. Dezoito estirpes do gênero Pseudomonas foram identificadas e submetidas aos testes de produção de substâncias antimicrobianas. Doze estirpes (66,7%) foram identificadas como Pseudomonas fluorescens; quatro (22,2%) como P. aeruginosa; uma (5,5%) como P. mendocina, e uma (5,5%) como P. pseudoalcaligenes. Apenas duas estirpes de P. fluorescens não foram capazes de produzir qualquer substância antimicrobiana contra os microorganismos indicadores testados. A maioria das estirpes apresentou um amplo espectro de ação, inibindo estirpes de referência e associadas a alimentos, como Proteus vulgaris, Proteus mirabilis, Hafnia alvei, Yersinia enterocolitica, Escherichia coli e Salmonella Typhi. Cinco estirpes produtoras de substâncias antimicrobianas, que apresentaram o mais amplo espectro de ação, também foram testadas contra estirpes de referência de Staphylococcus aureus e contra 26 estirpes de Staphylococcus sp. isoladas a partir de alimentos, sendo, algumas delas, resistentes a antibióticos. As estirpes produtoras 8.1 e 8.3, ambas P. aeruginosa, foram capazes de inibir todas as estirpes de Staphylococcus testadas. As substâncias antimicrobianas produzidas pelas estirpes 8.1 e 8.3 não parecem ser bacteriocinas típicas, uma vez que foram resistentes às três enzimas proteolíticas testadas. Experimentos envolvendo a caracterização destas substâncias estão sendo realizados, a fim de avaliar a sua aplicação biotecnológica.

In this work, the production of antimicrobial substances by strains of Pseudomonas sp. isolated from pasteurized milk and their potential action against food-related bacteria were investigated. Samples of pasteurized milk were purchased from arbitrarily chosen commercial establishments in the city of Rio de Janeiro, Brazil. Of the four samples analyzed, three presented several typical colonies of Pseudomonas. About 100 colonies were chosen and subjected to biochemical tests for confirmation of their identity. Eighteen strains of the Pseudomonas genus were identified and submitted to tests for the production of antimicrobial substances. Twelve strains (66.7%) were identified as Pseudomonas fluorescens, four (22.2%) as P. aeruginosa, one (5.5%) as P. mendocina and one (5.5%) as P. pseudoalcaligenes. Only two P. fluorescens strains were unable to produce any antimicrobial substance against any of the indicator strains tested. Most of the strains presented a broad spectrum of action, inhibiting reference and food-related strains such as Proteus vulgaris, Proteus mirabilis, Hafnia alvei, Yersinia enterocolitica, Escherichia coli and Salmonella typhi. Five antimicrobial substance-producing strains, which presented the broadest spectrum of action, were also tested against Staphylococcus aureus reference strains and 26 Staphylococcus sp. strains isolated from foods, some of which were resistant to antibiotics. The producer strains 8.1 and 8.3, both P. aeruginosa, were able to inhibit all the staphylococcal strains tested. The antimicrobial substances produced by strains 8.1 and 8.3 did not seem to be typical bacteriocins, since they were resistant to the three proteolytic enzymes tested. Experiments involving the characterization of these substances are being carried out in order to evaluate their biotechnological application.

A NBR ISO/IEC 17025:2005, uma das normas mais utilizadas em laboratórios de ensaio, descreve como um de seus critérios para a garantia da qualidade dos resultados analíticos a participação periódica dos laboratórios em ensaios de proficiência (EP). Os analitos, utilizados nos EP são materiais de referência (MR) provenientes de um mesmo lote, e devem apresentar características de homogeneidade e estabilidade. O objetivo deste estudo foi produzir um MR qualitativo destinado ao ensaio de pesquisa de Listeria monocytogenes em matriz queijo pela técnica de liofilização. Para a produção do MR, foi utilizado como matriz o queijo Minas frescal (QMF) ultrafiltrado. A matriz foi distribuída em frascos, contaminada com a bactéria alvo e submetida à liofilização, tendo a sacarose como crioprotetor. O MR produzido foi considerado homogêneo e estável na temperatura de ≤-70°C durante todo o período estudado (10 meses). O material apresentou estabilidade a 4, 25, 30 e 35°C por quatro dias e a -20°C por 48 dias, e os resultados estatísticos indicam tendência à estabilidade. Conclui-se que o material apresentou todos os requisitos necessários de um MR de qualidade e poderia ser transportado aos laboratórios participantes de um EP à temperatura máxima de 35°C por até quatro dias, uma vez que os resultados indicaram a manutenção da concentração celular nesse período. Esse foi o primeiro trabalho a descrever uma metodologia de produção de MR contendo L. monocytogenes em matriz queijo.

The standard most used in testing laboratories, ISO/IEC 17025:2005, describes the participation of laboratories in periodic proficiency testing (PT), as a criteria for quality assurance of analytical results. The analyses used in PT are reference materials (MR) from the same lot, and must have characteristics of homogeneity and stability. This study aimed to produce a qualitative RM for detection of Listeria monocytogenes assays in cheese matrix. For the production of RM, Minas Frescal cheese (MFQ) was used as matrix. The matrix was distributed in flasks, contaminated with the target bacteria and submitted to freeze-drying. Sucrose was used as cryoprotector. The RM produced was considered homogeneous and stable at -70°C during the entire period of study (10 months). The material showed stability at 4, 25, 30 and 35°C for 4 days and at -20°C the RM showed stability for 48 days, and the statistical results indicate a tendency to maintain stability. It was concluded that the material showed all the requirements of an RM quality and could be transported to the laboratory participants of a PT at 35°C up to 4 days, since the results indicate the maintenance of cell concentration during this period. This is the first study to describe a methodology for producing MR containing L. monocytogenes in cheese matrix.

O uso de materiais de referência (MR) é uma das principais ferramentas utilizadas para garantia e controle da qualidade de laboratórios de microbiologia de alimentos. No Brasil, a RDC n.º 12/01 da Anvisa prevê como um dos parâmetros para a avaliação da qualidade de queijos a enumeração de estafilococos coagulase positiva (ECP). O grande desafio na produção de MR destinados a ensaios microbiológicos é a instabilidade natural dos micro-organismos, o que dificulta o desenvolvimento e a manutenção desses MR. O objetivo deste estudo foi produzir um MR quantitativo destinado ao ensaio de enumeração de ECP em matriz queijo. Uma amostra de queijo ultrafiltrado com contagem de ECP <10 UFC/g e número de aeróbios viáveis de 1,22 × 10³ UFC/g foi utilizada como matriz para produção do MR. A matriz foi distribuída em frascos, contaminada com a bactéria alvo em concentração específica e submetida à liofilização. Como crioprotetor, foi utilizada sacarose. O MR produzido foi considerado homogêneo e estável à temperatura < -70 ºC durante todo o período estudado (dez meses). O material apresentou estabilidade a 4, 25, 30 e 35 ºC durante quatro dias; contudo, os resultados indicam que, a 35 ºC, ocorre um decréscimo na concentração celular. A -20 ºC, o MR apresentou-se estável durante 48 dias. Conclui-se que o material apresentou todos os requisitos necessários de um MR de qualidade e poderia ser transportado aos laboratórios participantes de um ensaio de proficiência a temperaturas de até 35 ºC por até quatro dias, uma vez que os resultados indicaram a manutenção da concentração celular neste período. Esse foi o primeiro trabalho a descrever uma metodologia de produção de MR contendo ECP em matriz queijo.

The use of reference materials (RM) is one of the principal tools used for assurance and quality control in food microbiology laboratories. In Brazil, Anvisa RDC nº 12/01 specifies the enumeration of coagulase-positive staphylococcus (CPS) as one of the parameters for evaluating cheese quality. The main challenge in the production of RM destined for microbiological assays is the natural instability of the microorganisms, which makes it difficult to develop and maintain them. This study aimed to produce a quantitative RM for use in enumeration assays of CPS in cheese matrixes. A sample of an ultra-filtered cheese with a CPS count of <10 CFU/g and a total nº of viable aerobes of 1.2 × 10³ CFU/g was used as the matrix to produce the RM. The matrix was distributed in flasks, contaminated with specific concentrations of the target bacteria and submitted to freeze-drying. Sucrose was used as the cryo-protector. The RM produced was considered homogeneous and stable at < -70ºC during the entire study period (10 months). The material was shown to be stable at 4, 25, 30 and 35 ºC for 4 days, although the results indicated a decrease in cell concentration at 35 ºC. At -20 ºC the RM was stable for 48 days. It was concluded that the material showed all the necessary requirements for a quality RM and could be transported to the laboratories taking part in a proficiency test at up to 35 ºC for up to 4 days, since the results indicated maintenance of the cell concentrations during this period. This is the first study to describe a methodology for producing MR containing CPS in a cheese matrix.