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

Análisis del perfil oxidativo de diferentes muestras biológicas de pacientes con lesión de ligamento cruzado anterior

O joelho é uma das articulações mais importantes para locomoção. No entanto, devido a sua complexidade, torna-se suscetível a diversos tipos de lesões, como a ruptura do ligamento cruzado anterior (LCA). Essa complicação desencadeia um processo inflamatório, que pode culminar em formação de radicais livres e, consequentemente, em estresse oxidativo (EO). O objetivo do estudo foi comparar o perfil oxidativo de pacientes com lesão do LCA, analisando duas amostras biológicas: líquido sinovial e soro. Foram analisados 11 indivíduos do gênero masculino, com ruptura total do LCA, com idade superior a 18 anos. Coletou-se amostras de sangue e líquido sinovial 15 minutos antes da artroplastia e se analisou biomarcadores de EO, catalase, flavonoides e peroxidação lipídica, isto é, substâncias reativas ao ácido tiobarbitúrico (TBARS). Os resultados apontam menor concentração de flavonoides, combinada a aumento de TBARS e de atividade de catalase no soro quando comparado com o líquido sinovial. A análise dos resultados indica que a lesão de LCA induz a quadro de EO, caracterizado por consumo de antioxidantes e elevação de dano lipídico no líquido sinovial quando comparado com o soro, indicando que análises séricas podem não ser adequadas para medir EO em partes como a articulação do joelho.The knee is one of the most important joints for locomotion. However, due to its complexity, it becomes susceptible to several types of injuries, such as the anterior cruciate ligament (ACL) rupture. This complication triggers an inflammatory process, which can lead to the formation of free radicals and, consequently, oxidative stress (OS). The objective of this study was to compare the oxidative profiles of patients with ACL injury, analyzing two biological samples: synovial fluid and serum. Eleven male subjects with total ACL rupture, older than 18 were analyzed. Blood samples and synovial fluid were collected fifteen minutes before arthroplasty. OS catalase biomarkers, flavonoids and lipid peroxidation (TBARS) were analyzed. The results indicate a lower flavonoid concentration, combined with an increase in TBARS and serum catalase activity when compared with synovial fluid. Analysis of the results indicates that the ACL injury induces OS, characterized by antioxidant consumption and elevated lipid damage in the synovial fluid, when compared with the serum, which indicates that serumal analyses may not be adequate to measure OS in compartments such as the knee joint.La rodilla es una de las articulaciones más importantes para locomoción. Sin embargo, debido a su complejidad, se torna susceptible a diversos tipos de lesiones, como la ruptura del ligamento cruzado anterior (LCA). Esa complicación desencadena un proceso inflamatório, que puede culminar en formación de radicales libres y, en consecuencia, en estrés oxidativo (EO). El objectivo del estudio fue comparar el perfil oxidativo de pacientes con lesión del LCA, analizando dos muestras biológicas: fluido sinovial y suero. Fueron analizados 11 individuos del género masculino, con ruptura total del LCA, con edad superior a 18 años. Se recogió muestras de sangre y fluido sinovial 15 minutos antes de la artroplastia y se analizó biomarcadores de EO, catalasa, flavonoides y peroxidación de las grasas, o sea, substancias reactivas al ácido tiobarbitúrico (TBARS). Los resultados apuntan menor concentración de flavonoides, combinada a aumento de TBARS e de actividad de catalasa en el suero cuando comparado con el fluido sinovial. El análisis de los resultados indica que la lesión de LCA induce a cuadro de EO, caracterizado por consumo de antioxidantes y elevación de daño de las grasas en el fluido sinovial cuando comparado con el suero, indicando que análisis séricas pueden no ser adecuadas para medir EO en partes como la articulación de la rodill

Salmonella Enteritidis forma biofilme sob baixas temperaturas em diferentes superfícies da indústria de alimentos

We evaluated the influence of temperature on the ability of Salmonella Enteritidis (SE) to form biofilms on stainless steel, polyethylene, and polyurethane surfaces under different hygiene procedures. These materials were placed on SE culture and incubated at 42±1 ºC, 36±1 ºC, 25±1 ºC, 9±1 ºC, and 3±1 ºC for 4, 8, 12, and 24 h. Hot water at 45 ºC and 85 ºC, 0.5% peracetic acid solution, and 1% quaternary ammonia were used for hygienization. Biofilm formation occurred at all temperatures evaluated, highlighting at 3 ºC which has not been reported as an ideal temperature for the adhesion of SE to these materials. The SE adhered more often to polyethylene surfaces than to polyurethane and stainless steel surfaces (P<0.05). Peracetic acid and water at 85 ºC had similar hygienization efficiency (P<0.05) followed by quaternary ammonia whereas water at 45 ºC was not effective. SE adhered to these materials under low temperatures which to date have been deemed safe for food preservation.Avaliou-se o efeito da temperatura na capacidade de Salmonella Enteritidis (SE) formar biofilme em superfícies de aço inoxidável, polietileno e poliuretano e diferentes processos de higienização. Corpos de prova destes materiais foram postos frente a culturas de SE e incubados a 42±1 ºC, 36±1 ºC, 25±1 ºC, 9±1 ºC e 3±1 ºC por 4, 8, 12 e 24 horas. Para a higienização foram testados água aquecida a 45ºC e 85 ºC e soluções de ácido peracético 0,5% e amônia quaternária 1%. Verificou-se a formação de biofilmes em todas as temperaturas avaliadas, ressaltando-se a 3 ºC, ainda não citada como propícia para adesão de SE. Houve maior adesão ao polietileno do que ao poliuretano e ao aço inoxidável (P<0.05). Para higienização, o ácido peracético e a água a 85 ºC tiveram ação semelhante (P<0.05), seguidos por amônia quaternária, enquanto que a água a 45 ºC não foi eficaz. Todos os materiais avaliados propiciaram a aderência de SE, mesmo sob temperaturas baixas, consideradas até então seguras para a conservação dos alimentos

Detection of virulence genes in Salmonella Heidelberg isolated from chicken carcasses

During the last years, Brazilian government control programs have detected an increase of Salmonella Heidelberg in poultry slaughterhouses a condition that poses a threat to human health However, the reasons remain unclear. Differences in genetic virulence profiles may be a possible justification. In addition, effective control of Salmonella is related to an efficient epidemiological surveillance system through genotyping techniques. In this context, the aim of this study was the detection of 24 virulence-associated genes in 126 S. Heidelberg isolates. We classified the isolates into 56 different genetic profiles. None of the isolates presented all the virulence genes. The prevalence of these genes was high in all tested samples as the lowest number of genes detected in one isolate was 10/24. The lpfA and csgA (fimbriae), invA and sivH (TTSS), and msgA and tolC (intracellular survival) genes were present in 100% of the isolates analyzed. Genes encoding effector proteins were detected in the majority of SH isolates. No single isolate had the sefA gene. The pefA gene was found in only four isolates. We have also performed a screening of genes associated with iron metabolism: 88.9% of isolates had the iroN gene and 79.4% the sitC gene. Although all the isolates belong to the same serotype, several genotypic profiles were observed. These findings suggest that there is a diversity of S. Heidelberg isolates in poultry products. The fact that a single predominant profile was not found in this study indicates the presence of variable sources of contamination caused by SH. The detection of genetic profiles of Salmonella strains can be used to determine the virulence patterns of SH isolates

Detection of virulence genes in Salmonella Heidelberg isolated from chicken carcasses

During the last years, Brazilian government control programs have detected an increase of Salmonella Heidelberg in poultry slaughterhouses a condition that poses a threat to human health However, the reasons remain unclear. Differences in genetic virulence profiles may be a possible justification. In addition, effective control of Salmonella is related to an efficient epidemiological surveillance system through genotyping techniques. In this context, the aim of this study was the detection of 24 virulence-associated genes in 126 S. Heidelberg isolates. We classified the isolates into 56 different genetic profiles. None of the isolates presented all the virulence genes. The prevalence of these genes was high in all tested samples as the lowest number of genes detected in one isolate was 10/24. The lpfA and csgA (fimbriae), invA and sivH (TTSS), and msgA and tolC (intracellular survival) genes were present in 100% of the isolates analyzed. Genes encoding effector proteins were detected in the majority of SH isolates. No single isolate had the sefA gene. The pefA gene was found in only four isolates. We have also performed a screening of genes associated with iron metabolism: 88.9% of isolates had the iroN geneand 79.4% the sitC gene. Although all the isolates belong to the same serotype, several genotypic profiles were observed. These findings suggest that there is a diversity of S. Heidelberg isolates in poultry products. The fact that a single predominant profile was not found in this study indicates the presence of variable sources of contamination caused by SH. The detection of genetic profiles of Salmonella strains can be used to determine the virulence patterns of SH isolates

Salmonella spp. Isolated by Miniaturized Most Probable Number and Conventional Microbiology in Poultry Slaughterhouses

Background: Salmonella spp. are frequently isolated from fowls, and their detection in poultry products varies according to the breeding system and the slaughtering process, bringing risks to the consumer and compromising the marketability. The control of Salmonella in poultry slaughterhouses is based on the detection of bacteria, but the quantification of the agent would be important in assessing risk, as well as in obtaining data to determine the capacity of each step of the process to decrease or increase bacterial contamination. The aims of this study were to propose a method for the quantification of Salmonella in poultry slaughterhouses, frequency of isolation and serovars identified.Materials, Methods & Results: Twenty-one broiler flocks from seven federally inspected slaughterhouses in southern Brazil, totaling 1,071 samples, were assessed by miniaturized most probable number (mMPN) and conventional microbiology. The samples were collected in triplicate at 17 points, which included cloacae, transportation cages before and after sanitization, water (scald tank, supply, pre-chiller and chiller), and carcasses (before and after scalding, defeathering, rinsing, evisceration, final rinsing, chilling at 4ºC, and freezing at -12°C for 24 h, 30 and 60 days). Typical Salmonella colonies were submitted to TSI, LIA, SIM, urea, and polyvalent anti-O antiserum tests, and to final identification by Microarray by Check&Trace. Nine of the 1,071 (0.83%) samples analyzed by mMPN and by conventional microbiology were positive for Salmonella and the following serovars were identified: Anatum, Brandenburg, Agona, Tennessee, Bredeney, Schwarzengrund and Infantis.Discussion: This positive rate was lower than that described by other authors, whose rates ranged from 3% and 39% for the isolation of Salmonella spp. from different sources, such as slaughterhouses and retail sales in samples collected in Brazil. The low frequency of isolation of Salmonella in this study can be attributed to the efficiency of control systems used from the field to the slaughterhouse, such as Good Manufacturing Practices (GMP) and Sanitation Standard Operating Procedures (SSOP), which are HACCP requirements. Also, when slaughtering technology actions are properly managed, such as water replacement and temperatures lower than 4ºC in the chiller, the initial contamination by Salmonella spp. can be reduced, with a decline in contamination from 70% to 20%, and with a reduction in the contamination of broiler carcasses after chilling from 15.8% to 3.3%. On the other hand the contamination of carcasses by Salmonella before pre-chilling and in post-chilling might be due to the automated system, inadequate temperatures during chilling, and inappropriate water chlorination in the assessed meat-packing plant. Of the 17 points evaluated, seven were positive for Salmonella, especially the cages after sanitization and frozen carcasses. The contamination by Salmonella spp. in transportation cages after sanitization indicates inefficiency of the automated system as well as possible bacterial resistance to the sanitizers used in SSOP while the isolation in carcasses frozen for 24 h and 60 days demonstrates the thermal resistance of the bacterium to a conservation method widely used in the food industry. In this work, just one of the nine positive samples for Salmonella was identified by conventional methods (CM) and mMPN. The discrepancy between methods can be explained by the heterogeneous distribution of Salmonella and other bacteria in naturally contaminated samples. Samples that were positive in the qualitative test but negative in the mMPN protocol could have had a number of Salmonella below the detection amount

Genomics and phenotypical characterization of two new lytic bacteriophages for biocontrol of Salmonella enterica

Aims: To perform the isolation, characterization and sequencing of the bacteriophages. To demonstrate that the bacteriophages can be used for biocontrol of different Salmonella enterica serovars. Study Design: This study was an experimental study. Place and Duration of Study: Bacteriology and Mycology Laboratory in the Veterinary Hospital at the Faculty of Agronomy and Veterinary Medicine of the University of Passo Fundo (FAMV/UPF), Biotechnology Center (CBiotec) of the Federal University of Paraíba (UFPB), Center for Microscopy and Microanalysis at the Faculty of Veterinary of the Federal University of Rio Grande do Sul (UFRGS), between January – September 2016. Methodology: Twelve Salmonella enterica serovars (S. Anatum, S. Agona, S. Brandenburg, S. Bredeney, S. Infantis, S. Lexington, S. Panama, S. Rissen, S. Schwarzengrund, S. Tennessee, S. Enteritidis ATCC 13076 and S. Typhimurium ATCC 14028) were selected to be the hosts. We isolate, purify, produce and determine the bacteriophage titers to verify the potential for lysis of these phages against the hosts. Having determined the action of the phages against the hosts, we performed the sequencing of the bacteriophages on the Illumina Mi-Seq platform and the morphology was performed by transmission electron microscopy (TEM). Results: We isolated, characterized and sequenced the genome of two new bacteriophages, Salmonella phage UPF_BP1, belonging to the family Podoviridae and Salmonella phage UPF_BP2, family Myoviridae. UPF_BP1 has lytic action against seven tested Salmonella enterica serovars, while UPF_BP2 has action against the twelve tested serovars. Conclusion: The two new bacteriophages have a lytic action against different Salmonella enterica serovars, feeding our expectations for the development of alternatives for the use of antimicrobials, being possible candidates for use as a biocontrol of Salmonella enterica in food, animals and the environment

Staphylococcus aureus and Mesophilic Aerobic Bacteria Quantification in Hygienization Process of Milking Equipment

Background:Milk’s composition is an excellent substrate for microorganism’s multiplication. Presence of Staphylococcus aureus and aerobic mesophilic bacteria are one of the most common problems in dairy farms. On dairy industry’s and milk farms Clean in Place (CIP) system higyenization are commonly used, then the cleaning occurs as a closed process, for better results sanitizans are applied, in order to obtain a safety food. This project aim to evaluate Staphylococcus aureus and aerobic mesophilic bacteria reduction after two milking higyenization process. Materials, Methods &amp; Results:This research was done on a Rio Grande do Sul North Milk farm, with mechanized milking and Clean in Place system for cleaning. For liners and CIP tubes higyenization commercial products as Sodium Hipoclorite 3% and phosphoric acid 11.3% are used for detergency, and peracetic acid 5% for sanitization. Milk bunk tank are higyenized with sodium hypoclorite 3.8% alcalin detergent. After higyenization steps liners, CIP’s water process, bulk milk tank and milk set were collected. At process 1, liners and CIP water were collected after milking, detergency and sanitization that occurred immediately at the detergency’s finish, while process 2 the sanitization was realized 8 h after detergency, before following milking. Cooling milk bulk tank was collected before and after detergency, and milk set after milkings Convencional microbiology were used to count and results in log10 UFC.cm-2. In CIP water’s after process 1 was 3.81 log10 reduction to aerobic mesophilic bacteria (P &gt; 0.05) and reducing 4.51 log10 (P = 0.03). Meanwhile there was no significant reduction for mesophilic aerobic bacteria and S. aureus on the others samples (liners, bulk milk tank and milk set).Discussion: This results show the maintenance of milking machine contamination, and that even bacterial load reduced among hygienization steps this was not significant, suggesting that deteriorate and pathogenic microorganisms can remain on milk produced. Highlights are teat taps of milking machine as the major cause of contamination among cows. The results are worrying because Staphylococcus aureus contamination, once this bacteria causes alimentar diseases, even after higyenization process, which can damage public health that can reflect milk chain economically. Since amount of this microorganism found in milk is already sufficient to synthesize enterotoxins. In addition, resistance to disinfectants is another concern, as it may result in resistance to antimicrobial agents. So reduction of bacteria level among cleaning steps there was no significance, once the products and equipments on dairy and farms act as a constant elimination point of deteriorate and pathogenic microorganisms for the final product, milk. With this studies aim to aprimorate hygienization process on milk chain, in order to obtain a hygienic sanitary good product

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