RESUMO
Previous studies have demonstrated the antagonistic potential of lactic acid bacteria (LAB) present in raw milk microbiota over Staphylococcus aureus, albeit the molecular mechanisms underlying this inhibitory effect are not fully understood. In this study, we compared the behavior of S. aureus ATCC 29213 alone and in the presence of a cheese-isolated LAB strain, Enterococcus faecalis 41FL1 in skimmed milk at 30⯰C for 24â¯h using phenotypical and molecular approaches. Phenotypic analysis showed the absence of classical staphylococcal enterotoxins in co-culture with a 1.2-log decrease in S. aureus final population compared to single culture. Transcriptional activity of several exotoxins and global regulators, including agr, was negatively impacted in co-culture, contrasting with the accumulation of transcripts coding for surface proteins. After 24â¯h, the number of transcripts coding for several metabolite responsive elements, as well as enzymes involved in glycolysis and acetoin metabolism was increased in co-culture. The present study discusses the complexity of the transcriptomic mechanisms possibly leading to S. aureus attenuated virulence in the presence of E. faecalis and provides insights into this interspecies interaction in a simulated food context.
Assuntos
Proteínas de Bactérias/genética , Enterococcus faecalis/crescimento & desenvolvimento , Leite/microbiologia , Staphylococcus aureus/genética , Animais , Proteínas de Bactérias/metabolismo , Bovinos , Queijo/microbiologia , Técnicas de Cocultura , Microbiologia de Alimentos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/metabolismo , Transcrição GênicaRESUMO
Hospital bloodstream infection (BSI) caused by methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality and is frequently related to invasive procedures and medically complex patients. An important feature of MRSA is the clonal structure of its population. Specific MRSA clones may differ in their pathogenic, epidemiological, and antimicrobial resistance profiles. Whole-genome sequencing is currently the most robust and discriminatory technique for tracking hypervirulent/well-adapted MRSA clones. However, it remains an expensive and time-consuming technique that requires specialized personnel. In this work, we describe a pangenome protocol, based on binary matrix (1,0) of open reading frames (ORFs), that can be used to quickly find diagnostic, apomorphic sequence mutations that can serve as biomarkers. We use this technique to create a diagnostic screen for MRSA isolates circulating in the Rio de Janeiro metropolitan area, the RdJ clone, which is prevalent in BSI. The method described here has 100% specificity and sensitivity, eliminating the need to use genomic sequencing for clonal identification. The protocol used is relatively simple and all the steps, formulas and commands used are described in this work, such that this strategy can also be used to identify other MRSA clones and even clones from other bacterial species.
RESUMO
Staphylococcus spp. are microorganisms that are naturally present in milk and dairy products and are often associated with food-borne diseases outbreaks due to the ability of some strains to produce thermostable enterotoxins. This ability is usually associated with coagulase and thermonuclease production, characteristics that are considered in the microbiological analyses for the control of such microorganisms. The objective of this study was to evaluate the culture media and the methodologies used for the enumeration of coagulase and thermonuclease-positive Staphylococcus spp. in raw milk and fresh soft cheese. Samples of artificially contaminated milk (with coagulase-positive Staphylococcus reference strains) and samples of naturally contaminated raw milk and cheese were submitted for enumeration in Baird-Parker agar (BP), Rabbit Plasma Fibrinogen agar (RPFA) and in the Petrifilm Staph Express count system (STX). No significant differences (P > 0.05) were observed between the mean counts obtained in all of the evaluated culture media. RPFA and STX had good correlation indices between the total and typical colony counts as well as with coagulase and the thermonuclease-positive colony counts. Thus, there is a better association between coagulase and thermonuclease production to typical colony morphology developed on these culture media, leading to more accurate and reliable results than with BP, which demonstrated lower correlation indices between these counts.
Assuntos
Queijo/microbiologia , Contagem de Colônia Microbiana/métodos , Contaminação de Alimentos/análise , Leite/microbiologia , Staphylococcus/isolamento & purificação , Ágar , Animais , Técnicas Bacteriológicas , Coagulase/metabolismo , Meios de Cultura/química , Microbiologia de Alimentos , Humanos , Nuclease do Micrococo/metabolismo , Staphylococcus/enzimologia , Staphylococcus/metabolismoRESUMO
This study aimed to characterize the microbiological quality and safety of raw milk and soft cheese, verifying possible associations between microbial populations, and the detection of lactic acid bacteria (LAB) with antagonistic activity against foodborne pathogens. Raw milk (n = 36) and soft cheese (n = 18) samples were collected and submitted for the analysis of mesophilic aerobes, total coliforms, Escherichia coli, LAB, coagulase-positive Staphylococcus (CPS), Listeria monocytogenes, and Salmonella spp. In all, 389 LAB isolates were randomly selected and submitted for antagonistic tests against L. monocytogenes, St. aureus, Salmonella Typhimurium, and Lactobacillus sakei. The samples presented high counts of mesophilic aerobes, total coliforms, and LAB, and also high and significant correlation indices between these populations. Low levels of CPS and E. coli were observed, as well as an absence of Salmonella spp. and L. monocytogenes. A substantial portion of the analyzed samples presented LAB cultures with antagonistic activity, but not against Salmonella Typhimurium. The obtained results indicate the antimicrobial potential of the autochthonous microbiota of raw milk and soft cheese. Despite the spoilage potential, the LAB present in the studied food products can be isolated and properly characterized as antagonistic cultures, to be used in bioconservation studies for pathogen control in foods.
Assuntos
Antibiose , Queijo/microbiologia , Lactobacillaceae/fisiologia , Leite/microbiologia , Animais , Brasil , Queijo/normas , Contagem de Colônia Microbiana , Qualidade de Produtos para o Consumidor , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Conservação de Alimentos/métodos , Humanos , Lactobacillaceae/isolamento & purificação , Listeria monocytogenes/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Leite/normas , Salmonella/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
The consumption of raw milk soft cheeses (RMSC), which are typically manufactured in small dairy farms under unsatisfactory hygiene conditions, is common in Brazil. Due to these production characteristics, this type of cheese is a potential carrier of pathogenic microorganisms, such as Listeria monocytogenes, Salmonella, and enterotoxin-producing Staphylococcus spp. Considering these characteristics, in this work, we aimed to detect the presence of these pathogenic microorganisms in RMC and to evaluate their microbiological quality. Fifty-five samples of this product were collected from different noninspected commercial establishments and submitted to the enumeration of mesophilic aerobes (MA), total coliforms (TC), Escherichia coli, and coagulase-positive staphylococci (CPS), and detection of L. monocytogenes and Salmonella spp. All analyzed samples were negative for Salmonella spp. and L. monocytogenes. All samples presented counts of MA higher than 10(6) colony forming units/g (CFU/g; range, 3.0x10(6) to 4.0x10(9)). TC were present at levels between 1.0x10(3) and 1.8x10(8) CFU/g, and E. coli between 1.0x10(2) and 3.5x10(6) CFU/g. CPS were detected in 17 (30.9%) samples at levels higher than 10(4) CFU/g. These results confirm the poor microbiological quality of raw milk used in the manufacturing of RMC samples, and also the inadequate production conditions. Therefore, the evaluation of microbiological safety and quality of these products must be constantly reported to alert the official agencies about the significance of proper inspection.
Assuntos
Bactérias Aeróbias/isolamento & purificação , Queijo/microbiologia , Qualidade de Produtos para o Consumidor , Contaminação de Alimentos/análise , Higiene , Animais , Brasil , Queijo/normas , Contagem de Colônia Microbiana , Enterobacteriaceae/isolamento & purificação , Escherichia coli/isolamento & purificação , Inspeção de Alimentos , Microbiologia de Alimentos , Humanos , Leite/microbiologia , Controle de Qualidade , Salmonella/isolamento & purificação , Staphylococcus/isolamento & purificaçãoRESUMO
Genes encoding staphylococcal enterotoxins (SEs) are carried by mobile genetic elements, and enterotoxin gene clusters (egc) are pathogenicity island-borne structures comprising several SE genes, which are frequently found among clinical Staphylococcus aureus isolates. In the present study, we investigated the distribution and the genetic variability of egc loci in S. aureus strains isolated from raw milk and soft cheese in Minas Gerais, Brazil. Ninety-two isolates were submitted to PCR detection of individual egc-borne SE genes (seg, sei, sem, sen, seo, seu), and egc loci were typed using PCR-RFLP. PCR products of egc positive isolates were sequenced. Ninety-one isolates harbored at least one SE gene, which generated 14 different genotypes. The sei gene was the most widely distributed (97.8%), and was found in combination with seg in 49 isolates (53.3%). Altogether, a complete set of individual egc genes was detected in 37 isolates (40%). However, egc loci were detected by PCR-RFLP in only 4 isolates, and classified as egc1 (n=2), egc3 (n=1), and egc4 (n=1). This investigation demonstrated the low occurrence of the egc in S. aureus isolated from dairy products. However, the frequency of complete sets of individual egc-borne genes reflects either the presence of these SE genes outside egc or the existence of new egc types in these strains.
Assuntos
Queijo/microbiologia , Enterotoxinas/genética , Leite/microbiologia , Staphylococcus aureus/genética , Animais , Brasil , Enterotoxinas/isolamento & purificação , Variação Genética , Genótipo , Família Multigênica/genética , Reação em Cadeia da Polimerase , Staphylococcus aureus/isolamento & purificaçãoRESUMO
A qualidade do leite cru é influenciada diretamente pelas condições de higiene na ordenha earmazenamento, bem como sanidade dos animais. Isso determina que a adoção de Boas Práticas de Produção éfundamental para obtenção de produtos final com baixas contagens microbianas, característica indicativa de boa qualidade. Visando avaliar a eficácia de práticas higiênicas durante o procedimento de ordenha em uma propriedade rural, pontos específicos foram selecionados e amostrados em duas etapas: uma fase inicial, com as práticas do produtor, e uma faseseguinte, em que práticas higiênicas específicas foram adotadas (soluções de cloro para pré-dipping, a 750ppm, e higiene de utensílios, a 300 ppm, além de descarte de primeiros jatos de leite). As amostras foram coletadas em 3 repetições em cada etapa e submetidas a contagens de aeróbios mesófilos (Petrifilm AC, 35ºC por 48h), psicrotróficos (superfície de ágar padrão de contagem, 7ºC por 10 dias), coliformes totais e Escherichia coli (Petrifilm EC, 35ºC por 48h). Osresultados finais obtidos foram comparados em cada etapa. Na etapa inicial os grupos identificados como principais contaminantes da linha de ordenha foram os aeróbios mesófilos e psicrotróficos, presentes em altos níveis nos tetos e leite dos animais. As práticas adotadas geraram redução da contaminação dos microrganismos indicadores pesquisados em vários pontos da linha de ordenha, indicando a eficácia das soluções de cloro para higienização de tetos dos animais eutensílios de ordenha.
The quality of raw milk is directly influenced by hygiene conditions during milking and storage, and also by animal sanity. Considering these characteristics, Good Producing Practices (GPP) must be adoptedsystematically in milk production in order to obtain final products with low microbial counts, what is indicative of quality. A dairy farm was selected to evaluate the efficiency of some GPP during milking process, when milk and surface samples were collected from specific milking steps in two stages: a first one, when hygienic procedures from the farmers were conducted, and a following one, when specific hygienic procedures were adopted (chlorine solutions for pre-dipping, at 750ppm, and utensils cleaning, at 300ppm, and discard of the forestrip milk). The samples were collected in threerepetitions in each phase and submitted to microbiological analysis of mesophilic aerobes (Petrifilm AC, 35ºC for 48h),psychrotrophics (surface of plate count agar, 7ºC for 10 days), total coliforms e Escherichia coli (Petrifilm EC, 35ºC for48h). The final results were then compared in each phase. At the initial phase, mesophilic aerobes and psychrotrophics were identified as the main contaminants of milking procedure by their high levels in the teats and milk. The adopted procedures generated reduction of microbial contamination in several sampling points, indicating the efficacy of chlorine solutions for hygiene of teats and milking utensils.