Dinâmica de formação de biofilmes multiespécies de Salmonella enteritidis, Campylobacter jejuni, Listeria monocytogenes e Escherichia coli e efeitos de procedimentos de higienização

A carne de frango pode servir de veículo para inúmeros microrganismos patogênicos como Salmonella spp., Campylobacter spp., Escherichia coli, Listeria monocytogenes, entre outros, potencialmente capazes de desencadear doenças transmitidas por alimentos. Estes microrganismos podem se aderir em superfícies bióticas e abióticas, formando biofilmes. Microrganismos, na forma de biofilme, são mais resistentes a adversidades ambientais, como falta de nutrientes e variações de temperatura, além de serem mais resistentes à sanitizantes químicos e físicos. Na indústria avícola, procedimentos de higienização, incluindo limpeza e desinfecção, são fundamentais para reduzir o número de microrganismos a um nível seguro. A sanitização, pelo uso de água quente, detergentes e sanitizantes, é a última etapa do procedimento de higienização com a finalidade de garantir um produto de boa qualidade higiênicosanitária. Porém, falhas na etapa de higienização levam a permanência de resíduos de alimentos nos equipamentos e sob determinadas condições, os microrganismos se aderem, interagem com as superfícies de processamento e iniciam a formação de biofilmes. Na primeira etapa deste estudo, foi realizada uma triagem entre as amostras de Salmonella Enteritidis, Escherichia coli, Listeria monocytogenes e Campylobacter jejuni quanto à capacidade de formação de biofilme monoespécie em placa de poliestireno, pela coloração de cristal violeta. As amostras foram classificadas como: não aderentes, fracamente, moderadamente e fortemente formadoras de biofilmes nas temperaturas de 3°C, 9°C, 25°C, 36°C e 42°C e foram selecionadas 4 cepas formadoras de biofilme de cada espécie. Na segunda etapa, as 4 cepas selecionadas foram testadas individualmente quanto a sua resistência aos sanitizantes peróxido de hidrogênio a 0,3% e hipoclorito de sódio a 2%, para avaliar a eficácia destes sanitizantes em células na fase planctônica. A maioria das cepas foram resistentes ao peróxido de hidrogênio a 0,3%, com exceção de C. jejuni. Quanto ao hipoclorito de sódio a 2%, apenas a amostra de Listeria monocytogenes foi resistente. Na terceira etapa, 4 cepas selecionadas foram testadas quanto a formação de biofilme multiespécie, nas superfícies de aço inoxidável, poliuretano e polietileno, coletadas de matadouro-frigorífico avícola, nas temperaturas de 42±1ºC, 36±1ºC, 25±1ºC, 9±1ºC e 3±1ºC, e nos intervalos de tempo 0, 4, 12 e 24 horas, utilizando microbiologia convencional. Além disso, perante a formação de biofilme, foi avaliada a eficácia dos procedimentos de higienização mimetizados nas diferentes condições ambientais, verificando a sensibilidade destes biofilmes frente aos tratamentos com água quente a 85ºC, hipoclorito de sódio a 2% e peróxido de hidrogênio a 0,3%. As cepas de S. Enteritidis, E. coli e L. monocytogenes foram capazes de formar biofilme multiespécie, com maior prevalência de S. Enteritidis, considerando todas as temperaturas testadas. As superfícies de polietileno e poliuretano proporcionaram formação de biofilme estatisticamente semelhantes, onde o polietileno foi a superfície mais difícil de higienizar. O aço inoxidável proporcionou menos adesão interespécie e foi mais facilmente higienizado. Os sanitizantes hipoclorito de sódio a 2% e a água quente a 85°C possuíram eficácia semelhante nas superfícies testadas. O peróxido de hidrogênio não demonstrou eficácia na higienização das superfícies. Estes resultados são de grande relevância para estimular novas estratégias de higienização diante de biofilmes em matadouro-frigoríficos avícolas e para instigar outros estudos de formação de biofilme multiespécie.The poultry meat can serve as a vehicle for numerous pathogens such as Salmonella, Campylobacter spp, Escherichia coli, Listeria monocytogenes, among others, potentially capable of triggering foodborne illness. These microorganisms can adhere to biotic and abiotic surfaces forming biofilms. Microorganisms in biofilms are more resistant to environmental adversities, such as starvation of nutrients and temperature variations as well as being more resistant to physical and chemical sanitizers. In the poultry industry, cleaning procedures, including cleaning and disinfection are essential to reduce microorganisms to a safe level. The sanitization, by the use of hot water, detergents and chemical and physical sanitizers is the last step of the cleaning procedure in order to ensure product of good sanitary conditions. However, failures in sanitizing step lead to permanence of food residues in equipment and under certain conditions, microorganisms adhere interact with the surfaces of processing and trigger the biofilms formation. In the first step of this study, screening was performed for the strains of Salmonella Enteritidis, Escherichia coli, Listeria monocytogenes, and Campylobacter jejuni as monospecies biofilm formation capacity in the polystyrene plate by crystal violet staining. Strains were classified in: nonadherent weakly, moderately and strongly forming biofilms in temperature of 3°C, 9°C, 25°C, 36°C and 42°C and selected 4 strains forming biofilms by species. In the second step, the 4 selected strains were tested individually as their resistance to the sanitizers of hydrogen peroxide at 0.3% and sodium hypochlorite at 2% for the effectiveness of these sanitizers with cells in planktonic phase. Most strains were resistant to hydrogen peroxide at 0.3%, with the exception of C. jejuni, which was sensitive to action this sanitizer. As for the sodium hypochlorite at 2%, only L. monocytogenes was resistance and the rest of the strains were sensitive to the action of sanitizing. In the third stage, the 4 selected strains were tested for the formation of multispecies biofilms on the surfaces of: stainless steel, polyurethane and polyethylene collected from poultry slaughterhouse, incubated at temperatures of 42±1ºC, 36±1ºC, 25±1ºC, 9±1ºC e 3±1ºC, and ±1°C, and the time slots 0, 4, 12 and 24 hours, using conventional microbiology. Moreover, the effectiveness of simulated hygiene procedures in the different environmental conditions was evaluated by checking the sensitivity of the biofilms to the treatments with hot water at 85°C, sodium hypochlorite at 2% and hydrogen peroxide at 0.3%. The strains of S. enteritidis, E. coli and L. monocytogenes were able to form multi-species biofilm, with higher prevalence of S. Enteritidis, considering all temperatures tested. The surfaces of polyethylene and polyurethane provided statistically similar biofilm formation, where the polyethylene was the hardest surface to sanitize. Stainless steel interspecies provided less adhesion and is more easily sanitized. Sanitizers sodium hypochlorite at 2% and hot water at 85 ° C possessed similar efficacy at the tested surfaces. Hydrogen peroxide has not demonstrated efficiency in the cleaning of surfaces. These results are of great importance to promote new strategies for cleaning before biofilms in poultry slaughterhouses and to instigate other multispecies biofilm formation studies

Biofilm formation by Salmonella enteritidis at different incubation temperatures

Background: The genus Salmonella, associated with poultry products, is considered the leading cause of foodborne outbreaks in humans in many countries. In Brazil, Salmonella Enteritidis (SE) is the serovar remains as one most frequently isolated from humans, and it is also a major serovar found in animals, food, animal feed, and environmental samples, despite all the efforts to control this pathogen. Also, the bacterium is able to form biofilms on different surfaces, protecting cells from both cleaning and sanitizing procedures in the food industries. This study aimed to verify the ability of Salmonella Enteritidis isolates to form biofilm on polystyrene at different incubation temperatures. Materials, Methods & Results: A total of 171 SE samples were isolated from foodborne outbreaks (foods and stool cultures) and poultry products between 2003 and 2010. The biofilm-forming ability of samples was measured at four different temperatures (3°C, 9ºC, 25ºC, and 36ºC), for 24 h, simulating temperatures usually found in poultry slaughterhouses. Later, 200 μL of each bacterial suspension was inoculated, in triplicate, onto 96-well, flat-bottomed sterile polystyrene microtiter plates, washed, after that, the biofilm was fixed with methanol. The plates were dried at ambient temperature, stained with 2% Hucker’s crystal violet. Afterwards, absorbance was read using an ELISA plate reader and the optical density (OD) of each isolate was obtained by the arithmetic mean of the absorbance of three wells and this value was compared with the mean absorbance of negative controls (ODnc). The following classification was used for the determination of biofilm formation: no biofilm production, weak biofilm production, moderate biofilm production and strong biofilm production. Results demonstrated all isolates from stool cultures and foods involved in foodborne outbreaks, at least one of the four temperatures tested, were able to form biofilm, even at 3°C, undescribed as possible for the growth of SE. SE strains from poultry products also formed biofilm at least at one of the temperatures. Discussion: The prevention of biofilms formation is very important, once they can be difficult to remove from utensils and food equipment surfaces, becoming a chronic source of microbial contamination of foods, possible dissemination of diseases, and increase of resistance to cleaning and sanitization procedures. A high ability for biofilm formation on plastic surfaces was observed. We may consider that Salmonella has the capacity to bind to surfaces, with relevant impacts on public health. Although biofilm formation could be affected by temperature, most of the SE isolates analyzed in our study were strong biofilm producers at all temperatures, including at 3°C, a temperature used for food preservation and until then not acknowledged as worrisome regarding the development of Salmonella spp. There is a common sense that maintenance of food at low temperatures, particularly below 5°C, is safer to consumers as low temperatures reduce microbial multiplication. However, our results show that the growth of SE in its sessile form is possible under refrigeration. These findings lead to the assumption that the ability of SE to form biofilms, especially at low temperatures, is related to its endurance in inhospitable environments, eventually infecting humans, and that may be one of the factors associated with the high prevalence of this serovar in outbreaks of foodborne diseases. To our knowledge, this is the first publication about biofilm formation by Salmonella Enteritidis at 3ºC

Biofilm former salmonella enteritidis are multiresistant to antibiotics

Background: The Salmonella Enteritidis is one of the most isolated pathogens in outbreaks of foodborne illness, which can occur due to various factors such as cooking temperature, inadequate storage and cross-contamination. The choice of the appropriate disinfectant in food industries is essential to prevent the spread of contamination and control of biofilms on surfaces. It is also extremely important the concern with resistance to antimicrobials used both as growth promoters and in human and animal treatments, which may generate a selective pressure favoring the emergence of resistant bacteria. Materials, Methods & Results: Twenty samples of Salmonella Enteritidis were tested, 10 from outbreaks of foodborne diseases and 10 of poultry origin, as for the formation of biofilms, antibiotic resistance and sanitizers. The samples were stored frozen in BHI with 20% glycerol. For reactivation were incubated in BHI broth, plated on XLD agar and subsequently performed biochemical tests to check purity. Firstly were evaluated for biofilm formation on polystyrene at temperature of 36 ± 1ºC. We tested the sanitizing resistance to biguanide concentrations 0.6%, 1.0% and 1.5%, peracetic acid at concentrations 0.1%, 0.5% and 1.0%, and quaternary ammonia at concentrations of 0.3%, 1.0% and 2.0%. For tests of antimicrobial resistance the cultures were evaluated front 10 μg ampicillin, 30 μg cephalexin, 30 μg chloramphenicol, 5 μg enrofloxacin, 15 μg erythromycin, 30 μg neomycin, 25 μg sulphazotrim, 300 μg sulfonamides. According to the results, 25% of samples were strongly biofilm formers, 35% moderately formers, 35% weakly formers and 10% not biofilm formers. In sanitizers, quaternary ammonia and peracetic acid were effective at all concentrations and at all times, but tests with biguanide resulted in resistance in the time of 1 min at concentrations 0.6%, 1.0% and 1,5%, at time 5 min at concentrations of 1.0% and 1.5% and at time 10 min at concentrations of 0.6% and 1.0%. As for antimicrobial susceptibility testing, 10 samples of S. Enteritidis presented pattern of multidrug resistance to the antibiotics tested. In relation to the active principles, 25% of S. Enteritidis were resistant to ampicillin, 5% to cephalexin, 55% to enrofloxacin, 90% to erythromycin, 80% to neomycin, 5% to sulphazotrim, 70% to sulfonamides. There was 100% sensitivity to chloramphenicol. Discussion: All S. Enteritidis from outbreaks of foodborne diseases and 80% of S. Enteritidis from poultry products produced biofilm. Regarding S. Enteritidis outbreaks of foodborne illness, 30% were strongly biofilm formers, 50% moderately former and 20% poorly formers. Those isolated from poultry products were 10% strongly formers, 10% moderately formers and 60% poorly formers. Besides the formation of biofilms, 50% of S. Enteritidis were multiresistant to antimicrobials been tested, and of these, 35% corresponded to S. Enteritidis isolates from outbreaks of foodborne illness and only 15% were of poultry origin. Still, 50% of Salmonella Enteritidis were also resistant to biguanide, of which 30% were S. Enteritidis isolates from outbreaks of foodborne illness and 20% isolated from poultry products. These results denotes great relevance due to the possibility of permanence of these microorganisms in food manipulation environments in the form of biofilms and, in the case of transmission to humans, present more difficulty in treatment due to the multidrug resistance

FORMAÇÃO DE BIOFILME E RESISTÊNCIA A ANTIMICROBIANOS POR Salmonella spp. ISOLADAS DE SALAME E LINGÜIÇA

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El Adelanto : Diario político de Salamanca: Año XXXVIII Número 11776 - 1922 Octubre 20

Background: The genus Salmonella, associated with poultry products, is considered the leading cause of foodborne outbreaks in humans in many countries. In Brazil, Salmonella Enteritidis (SE) is the serovar remains as one most frequently isolated from humans, and it is also a major serovar found in animals, food, animal feed, and environmental samples, despite all the efforts to control this pathogen. Also, the bacterium is able to form biofilms on different surfaces, protecting cells from both cleaning and sanitizing procedures in the food industries. This study aimed to verify the ability of Salmonella Enteritidis isolates to form biofilm on polystyrene at different incubation temperatures. Materials, Methods & Results: A total of 171 SE samples were isolated from foodborne outbreaks (foods and stool cultures) and poultry products between 2003 and 2010. The biofilm-forming ability of samples was measured at four different temperatures (3°C, 9ºC, 25ºC, and 36ºC), for 24 h, simulating temperatures usually found in poultry slaughterhouses. Later, 200 μL of each bacterial suspension was inoculated, in triplicate, onto 96-well, flat-bottomed sterile polystyrene microtiter plates, washed, after that, the biofilm was fixed with methanol. The plates were dried at ambient temperature, stained with 2% Hucker’s crystal violet. Afterwards, absorbance was read using an ELISA plate reader and the optical density (OD) of each isolate was obtained by the arithmetic mean of the absorbance of three wells and this value was compared with the mean absorbance of negative controls (ODnc). The following classification was used for the determination of biofilm formation: no biofilm production, weak biofilm production, moderate biofilm production and strong biofilm production. Results demonstrated all isolates from stool cultures and foods involved in foodborne outbreaks, at least one of the four temperatures tested, were able to form biofilm, even at 3°C, undescribed as possible for the growth of SE. SE strains from poultry products also formed biofilm at least at one of the temperatures. Discussion: The prevention of biofilms formation is very important, once they can be difficult to remove from utensils and food equipment surfaces, becoming a chronic source of microbial contamination of foods, possible dissemination of diseases, and increase of resistance to cleaning and sanitization procedures. A high ability for biofilm formation on plastic surfaces was observed. We may consider that Salmonella has the capacity to bind to surfaces, with relevant impacts on public health. Although biofilm formation could be affected by temperature, most of the SE isolates analyzed in our study were strong biofilm producers at all temperatures, including at 3°C, a temperature used for food preservation and until then not acknowledged as worrisome regarding the development of Salmonella spp. There is a common sense that maintenance of food at low temperatures, particularly below 5°C, is safer to consumers as low temperatures reduce microbial multiplication. However, our results show that the growth of SE in its sessile form is possible under refrigeration. These findings lead to the assumption that the ability of SE to form biofilms, especially at low temperatures, is related to its endurance in inhospitable environments, eventually infecting humans, and that may be one of the factors associated with the high prevalence of this serovar in outbreaks of foodborne diseases. To our knowledge, this is the first publication about biofilm formation by Salmonella Enteritidis at 3ºC