•Quorum sensing (QS) modulates virulent factor on enterotoxigenic E. coli (ETEC).•Conditioned media (CM) increase growth, motility, F4 and LT expression on ETEC.•CM and epinephrine on ETEC culture ...increase growth, motility, F4 and LT expression.•Auto-inducer and host molecules might play important role in the pathogenesis of the ETEC.
Quorum sensing (QS) is a signaling system among bacteria mediated by auto-inducer substances (AI). Whenever the concentration of these molecules reaches a threshold corresponding to a high cell density or quorum, the whole population starts a coordinated expression of specific genes. Studies have shown that epinephrine is also responsible for activating specific bacterial genes. This work aimed to investigate the role of conditioned medium (containing AI), epinephrine and their association on growth, motility, F4 fimbriae and heat-labile toxin (LT) expression on enterotoxigenic Escherichia coli (ETEC, E68). A significant increase in motility, F4 and LT expression, was observed in the ETEC culture supplemented with conditioned medium and epinephrine. These findings suggest that ETEC uses some components of conditioned medium (e.g., AI molecules), host molecules (epinephrine), and their association to modulate the expression of important virulence genes.
Vibrio parahaemolyticus is an important pathogen for both fish industry and consumers. It forms biofilm which makes it difficult to eliminate this microorganism using sanitizers. This study aimed to ...assess biofilm formation on different surfaces and effect of biofilm on resistance to sanitizers. Eight isolates of biofilm-forming V. parahaemolyticus were tested for the ability to form biofilms on a number of surfaces including high density polyethylene, stainless steel, glass, exoskeleton of Farfantepenaeus paulensis (Pink Shrimp), and operculum of Micropogonias furnieri (Whitemouth Croaker). Efficiency of sanitizer sodium hypochlorite against the bacteria was evaluated in the biofilms formed on the surface of the materials used; out the eight strains analyzed four formed biofilm on different surfaces. The present study shows that there are variations between surfaces in terms of biofilm formation, with more than one bacterial strain being able to form biofilm on the surface of the operculum of M. furnieri and on high density polyethylene as well. One isolate formed biofilm on glass, and one isolate formed biofilm on stainless steel. Sanitizers reduced biofilm formation on all surfaces. Based on our findings, we concluded that V. parahaemolyticus isolates have different ability to form biofilm on different surfaces. No isolates formed biofilm on shrimp shells. Results of this study also showed that sodium hypochlorite eat a concentration of 20 parts per million (20ppm) of Cl2, albeit not able to eliminate bacteria reported in biofilms, is still capable of reducing bacterial populations.
RESUMO: Vibrio parahaemolyticus é uma bactéria patogênica importante tanto para a indústria como para os consumidores de pescados, uma vez que pode formar biofilme, dificultando a sua eliminação por sanitizantes. Este estudo teve como objetivo verificar a formação de biofilme em diferentes superfícies e o efeito do biofilme sobre a resistência a sanitizante. Oito isolados de V. parahaemolyticus formadores de biofilme foram testados quanto à capacidade de formar biofilme em superfícies de polietileno de alta densidade, aço inoxidável, vidro, exoesqueleto de Farfantepenaeus paulensis (Camarão-rosa) e opérculo de Micropogonias furnieri (Corvina). A eficiência do sanitizante hipoclorito de sódio foi avaliada frente às bactérias nos biofilmes formados sobre a superfície dos materiais utilizados. Das oito cepas analisadas, quatro foram consideradas formadoras de biofilme em diferentes superfícies. Os resultados mostraram variação entre as superfícies, sendo que mais de uma cepa formou biofilme na superfície do opérculo de M. furnieri e do polietileno de alta densidade. Um isolado formou biofilme em vidro e um em aço inoxidável. Nenhum isolado formou biofilme na carapaça de camarão. O sanitizante reduziu a formação do biofilme em todas as superfícies. Conclui-se que os isolados de V. parahaemolyticus apresentam distinta capacidade de formar biofilme em diferentes superfícies e que o hipoclorito de sódio na concentração de 20 partes por milhão (20ppm) de Cl2, embora não elimine as bactérias que se encontram em biofilme, reduz a sua população.
Essential oils have been studied to replace synthetic chemical preservatives, among these oils, the thyme essential oil stands out. However, in addition to promoting microbial control, synthetic ...chemical preservatives impart a characteristic color to inlaid products, a parameter not attained by the application of essential oils. This study aimed to evaluate the effect of the joint addition of thyme essential oil and powdered beet juice to meat sausage, for a partial or total reduction of the addition of nitrates and nitrites. The thyme essential oil showed a high inhibitory action against Staphylococcus aureus and Escherichia coli in the concentration of 9.17 mg/mL, and bactericidal effect in the concentrations of 9.17 mg/mL and 36.68 mg/mL respectively, in all tests applied in vitro. When applied to meat sausages, it showed an inhibitory effect against coagulase-positive Staphylococcus, reducing the microorganism count, and partial inhibition of aerobic mesophilic bacteria. No interference was observed in the multiplication of thermotolerant coliforms in the meat product. When evaluated sensorially, the sausage presented good general acceptability, with acceptance levels above 75%. The combined uses of thyme essential oil and powdered beet juice showed potential viability for use in substitution for nitrates and nitrites in meat sausages.
•Thyme essential oil has antimicrobial activity in vitro against Staphylococcus aureus and Escherichia coli.•Thyme essential oil and powdered beet juice promoted the control of microorganisms in meat sausages.•Meat sausages with thyme essential oil or powdered beet juice have good sensory acceptance.
Intense manipulation during beef jerky production increases the possibility of contamination with pathogenic microorganisms. This study evaluated the contamination by thermotolerant coliforms, ...Escherichia coli and Salmonella spp., on processing surfaces and raw materials during beef jerky production, as well as in the final product. Thermotolerant coliforms were found on all surfaces tested and in the raw material. Escherichia coli was identified in 6.7% of the surface samples, while Salmonella spp. was found in 3.3% of the surface samples and 8.6% of raw material samples. Virulence genes were detected in Salmonella spp. isolates. One Salmonella spp. isolate was resistant to sulfonamide, while one E. coli isolate was multiresistant, including the presence of resistance genes sul2, strA, strB, tetA and tetB. The presence of coliforms demonstrates failings in hygienic-sanitary procedures. The presence of pathogenic microorganisms causing foodborne diseases in the production line indicates persistent contamination in the production plant. Although the drying process applied to beef jerky should guarantee the safety of the final product, the presence of multiresistant pathogenic microorganisms, presenting virulence genes, should be a matter of concern. Because beef jerky is a ready-to-eat product, a failure in the production process may cause such microorganisms to pose a public health risk.
Genus Salmonella bacteria are among the major pathogenic microorganisms in food. This bacterium pathogenicity is related to a number of virulence factors, among which its flagella. Flagellum ...expression is one of the virulence factors modulated by Quorum Sensing. Epinephrine produced by mammals uses the same signaling pathway of the 3 bacteria autoinducer. This study evaluated the effect of molecules inducer (epinephrine) and autoinducers (autoinducer 2 and autoinducer 3) and their association with the motility, growth and expression genes flhC, fliA, fliY, motA, motB e fliC of Salmonella Typhimurium (ST). Initially, ST was inoculated in BHI. Then, motility assays, growth curves and gene expression were performed by testing different concentrations of epinephrine (50, 125, 250, 500µM), conditioned medium (10 and 50%) and a combination of these. ST was exposed to different concentrations of epinephrine, conditioned medium and an association of both. Following, motility assays, bacterial growth and gene expression were performed. The results obtained showed that the combination of 500uM epinephrine with 50% conditioned medium increased ST bacterial motility by increasing the expression of genes involved in flagellum assembly.
Salmonella está entre os principais micro-organismos patogênicos veiculados por alimentos. A patogenicidade dessa bactéria está relacionada a uma série de fatores de virulência e, dentre estes, podemos citar os flagelos. A expressão do flagelo está entre os fatores de virulência modulados por Quorum Sensing. A adrenalina produzida pelos mamíferos utiliza a mesma via de sinalização do autoindutor 3 das bactérias. Nesse sentido, este trabalho teve como objetivo avaliar o efeito de moléculas indutora (adrenalina) e autoindutoras (auto-indutor 2 e auto-indutor 3) e a associação destas na motilidade, no crescimento celular e na expressão dos genes flhC, fliA, fliY, motA, motB e fliC de Salmonella Typhimurium (ST). Inicialmente, ST foi semeada em caldo BHI. Após, ensaios de motilidade, curvas de crescimento e expressão gênica foram feitos, testando diferentes concentrações de adrenalina (50, 125, 250, 500µM), meio condicionado (10 e 50%) e a associação destes. A partir dos resultados obtidos, observou-se que o tratamento que utilizou 50% de meio condicionado + 500µM de adrenalina aumentou a motilidade de ST, em decorrência do aumento de genes envolvidos com montagem do flagelo.
Abstract This study aimed to evaluate the application of good practices in hotel restaurants in Southern Brazil, relate these practices to the microbiological quality of the food offered, in addition ...to identifying contamination sources. The food and beverage sector of four hotels was evaluated by means of a checklist based on the Resolution 216/2014 of Agência Nacional de Vigilância Sanitária and microbiological analyzes. Surface, utensil, equipment and handlers’ hand samples were collected for coagulase positive Staphylococcus and thermotolerant coliform count. Isolates obtained from these culture counts were compared by band profiles obtained by rep-PCR. According to non-conformities observed, only one hotel was rated as excellent in good practices avaliation. Some microbiological analysis exceeded the allowed limit for the analyzed microorganism count, revealing failure in the hygiene-sanitary process. Genetic similarities between some food samples and handlers’ hands and utensils were identified. These results showed inadequacies in the process of food care and handling which may compromise the quality of food offered to custumers.
Background: The buffalo milk mozzarella cheese is a new product in the market, with high consumer acceptance and excellent prospects for trade. The cheese is rich in nutrients, which favors the ...proliferation of microorganisms that can cause food-borne diseases in the consumer. Staphylococcus aureus can cause gastro-enteritis in humans by the production of enterotoxins in food. One problem that may hinder the elimination of undesirable microorganisms in the food industry is the formation of biofilms. The objective of this study was to determine the effect of biofilm formation by Staphylococcus aureus isolated from buffalo mozzarella cheese on sensitivity to sanitizers.Materials, Methods & Results: Fifty samples of buffalo mozzarella cheese were analyzed to investigate the presence of S. aureus. The isolates were obtained through microbiological analysis and identified by PCR. The similarity of the strains was compared through rep-PCR. The distinct strains were tested for biofilm formation in microtiter plates. Soy Tripticase Broth (TSB) was placed in each well of the microtiter plate and overnight cultures of each strain was added. Wells without bacterial culture were used as controls. A villous cap was then placed on the plate and incubated for 48 h at 37°C. During incubation, the biofilms formed on the surface of the villi of the caps. For quantification of biofilm formation, material that remained attached to the cap was stained with crystal violet, the stained biofilm was extracted and the OD570 of each well was measured. Each strain was classified as non-biofilm forming, weak forming, moderately formed or formative strong. Strong forming and non-biofilm forming strains were tested on high density polyethylene, stainless steel and glass surfaces. Plates of 4 cm² of the different materials were placed in TSB where the culture of each isolate was inoculated separately. At each 48 h incubation the plates were washed to remove unbound cells and re-inserted into TSB without the inoculum. After five replicates of the procedure, sterile swabs were passed over the entire surface of each plate for counting in Baird-Parker agar. They were also tested for sensitivity to sodium hypochlorite and iodine after biofilm formation. The biofilm plates were immersed in flasks containing sanitizers, where they remained for 10 min. At the established contact time, the plates were immersed in neutralizing solution for 30 s. After washing with PBS, a sterile swab was passed on the surface of each plate and counts on Baird-Parker agar were performed. The bands profiles obtained on rep-PCR were identical when compared to isolates from the same sample, indicating that each sample was contaminated with only one S. aureus strain. From the twenty S. aureus strain identified, two isolates were classified as strong biofilm formers, seven as moderate formers, ten weak formers and one as non-biofilm builder. The two strong forming strains produced biofilm on the three surfaces tested. The application of sodium hypochlorite and iodine sanitizers promoted a reduction of approximately 2 log bacterial populations on all surfaces of both the biofilm and non-forming strains.Discussion: Most strains of S. aureus isolated from buffalo milk mozzarella cheese have the ability to form biofilm on the surfaces of equipment and utensils that have stainless steel, glass or high density polyethylene components. Although biofilm forming strains are no longer resistant to sanitizers sodium hypochlorite and iodine than non-forming sanitizers, they reach higher concentrations in the biofilm, resulting in larger bacterial populations remaining after application of the sanitizers. These results support the recommendation that the good hygienic practices adopted by industries processing buffalo milk mozzarella cheese should include specific measures to control the Staphylococcus aureus contamination.
The immunomagnetic separation (IMS) is a technique that has been used to increase sensitivity and specificity and to decrease the time required for detection of Salmonella in foods through different ...methodologies. In this work we report on the development of a method for detection of Salmonella in chicken cuts using in house antibody-sensitized microspheres associated to conventional plating in selective agar (IMS-plating). First, protein A-coated microspheres were sensitized with polyclonal antibodies against lipopolysacharide and flagella from salmonellae and used to standardize a procedure for capturing Salmonella Enteritidis from pure cultures and detection in selective agar. Subsequently, samples of chicken meat experimentally contaminated with S. Enteritidis were analyzed immediately after contamination and after 24h of refrigeration using three enrichment protocols. The detection limit of the IMS-plating procedure after standardization with pure culture was about 2x10 CFU/mL. The protocol using non-selective enrichment for 6-8h, selective enrichment for 16-18h and a post-enrichment for 4h gave the best results of S. Enteritidis detection by IMS-plating in experimentally contaminated meat. IMS-plating using this protocol was compared to the standard culture method for salmonellae detection in naturally contaminated chicken cuts and yielded 100% sensitivity and 94% specificity. The method developed using in house prepared magnetic microespheres for IMS and plating in selective agar was able to diminish by at least one day the time required for detection of Salmonella in chicken products by the conventional culture method.
In order to evaluate the survival of
Salmonella typhimurium,
Listeria monocytogenes,
Escherichia coli O157:H7 and
Staphylococcus aureus in
dulce de leche, aliquots of this sweet were experimentally ...contaminated with these pathogenic microorganisms at 10
3 (C1) and 10
1 (C2) bacterial cells per gram, and later analysed to evaluate microorganism viability after storage for 0,
1,
2,
3,
5,
10,
20 and 30
days.
Salmonella and
L. monocytogenes were recovered from all aliquots.
E. coli O157:H7 and
S. aureus were recovered from all aliquots at C1 concentration, and up to the 5th and 10th day, respectively, at C2 concentration. Relevant public-health danger pathogenic microorganisms were found to survive up to 30
days in
dulce de leche.
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