Recent Evolution and Genomic Profile of Salmonella enterica Serovar Heidelberg Isolates from Poultry Flocks in Brazil.

Salmonella enterica serovar Heidelberg is isolated from poultry-producing regions around the world. In Brazil, S. Heidelberg has been frequently detected in poultry flocks, slaughterhouses, and chicken meat. The goal of the present study was to assess the population structure, recent temporal evolution, and some important genetic characteristics of S. Heidelberg isolated from Brazilian poultry farms. Phylogenetic analysis of 68 S. Heidelberg genomes sequenced here and additional whole-genome data from NCBI demonstrated that all isolates from the Brazilian poultry production chain clustered into a monophyletic group, here called S. Heidelberg Brazilian poultry lineage (SH-BPL). Bayesian analysis defined the time of the most recent common ancestor (tMRCA) as 2004, and the overall population size (Ne) was constant until 2008, when an ∼10-fold Ne increase was observed until circa 2013. SH-BPL presented at least two plasmids with replicons ColpVC (n = 68; 100%), IncX1 (n = 66; 97%), IncA/C2 (n = 65; 95.5%), ColRNAI (n = 43; 63.2%), IncI1 (n = 32; 47%), ColMG828, Col156, IncHI2A, IncHI2, IncQ1, IncX4, IncY, and TrfA (each with n < 4; <4% each). Antibiotic resistance genes were found, with high frequencies of fosA7 (n = 68; 100%), mdf(A) (n = 68; 100%), tet(34) (n = 68; 100%), sul2 (n = 64; 94.1%), and blaCMY-2 (n = 56; 82.3%), along with an overall multidrug resistance (MDR) profile. Ten Salmonella pathogenicity islands (SPI1 to SPI5, SPI9, and SPI11 to SPI14) and 139 virulence genes were also detected. The SH-BPL profile was like those of other previous S. Heidelberg isolates from poultry around the world in the 1990s. In conclusion, the present study demonstrates the recent introduction (2004) and high level of dissemination of an MDR S. Heidelberg lineage in Brazilian poultry operations. IMPORTANCES. Heidelberg is the most frequent serovar in several broiler farms from the main Brazilian poultry-producing regions. Therefore, avian-source foods (mainly chicken carcasses) commercialized in the country and exported to other continents are contaminated with this foodborne pathogen, generating several national and international economic losses. In addition, isolates of this serovar are usually resistant to antibiotics and can cause human invasive and septicemic infection, representing a public health concern. This study demonstrates the use of whole-genome sequencing (WGS) to obtain epidemiological information for one S. Heidelberg lineage highly spread among Brazilian poultry farms. This information will help to define biosecurity measures to control this important Salmonella serovar in Brazilian and worldwide poultry operations.

Retrospective whole-genome comparison of Salmonella enterica serovar Enteritidis from foodborne outbreaks in Southern Brazil.

Salmonella enterica serovar Enteritidis is frequently isolated from animal-source foods associated with human salmonellosis outbreaks. This serovar was spread to animal (mainly poultry) farms worldwide in the 1980s, and it is still detected in foods produced in many countries, including Brazil. The present study reports a retrospective genome-wide comparison of S. Enteritidis from foodborne outbreaks in Southern Brazil in the last two decades. Fifty-two S. Enteritidis isolates were obtained from foodborne outbreaks occurring in different cities of the Brazilian southernmost State, Rio Grande do Sul (RS), from 2003 to 2015. Whole-genome sequences (WGS) from these isolates were obtained and comparatively analyzed with 65 additional genomes from NCBI. Phylogenetic and Bayesian analyses were performed to study temporal evolution. Genes related to antibiotic resistance and virulence were also evaluated. The results demonstrated that all S. Enteritidis isolates from Southern Brazil clustered in the global epidemic clade disseminated worldwide originally in the 1980s. Temporal analysis demonstrated that all Brazilian isolates had a tMRCA (time to most recent common ancestor) in 1986 with an effective population size (Ne) increase soon after until 1992, then becoming constant up to now. In Southern Brazil, there was a significant decrease in the spreading of S. Enteritidis in the last decade. In addition, three antibiotic resistance genes were detected in all isolates: aac(6')-Iaa, mdfA, and tet(34). These results demonstrate the high frequency of one only specific S. Enteritidis lineage (global epidemic clade) in foodborne outbreaks from Southern Brazil in the last two decades.

Assessing the growth of Staphylococcus aureus and Escherichia coli on fruits and vegetables.

INTRODUCTION: The number of registered foodborne diseases involving fresh produce is a preoccupation in many countries. For this reason, the aim of this study was to better understand the growth of Staphylococcus aureus and Escherichia coli, two indicators of hygienic and sanitary conditions, on fruits and vegetables that were exposed at different temperatures. METHODOLOGY: The main salads served at the buffets of commercial restaurants were artificially contaminated with separate pools of both pathogens and subsequently exposed at 10, 20 and 30 °C and at different time intervals. Then, the growth potential of S. aureus and E. coli on each fruit and vegetable was determined. RESULTS: There was no significant S. aureus and E. coli growth on all evaluated foods exposed at 10 °C until 6 hours. When comparing both microorganisms, E. coli demonstrated higher growth potential than S. aureus on all analysed salads. Peculiarly, E. coli had the highest growth rate for the tomato (α = 6.43 at 30 °C), a fruit with low pH. CONCLUSION: We suggest that fruits and vegetables should be distributed at temperatures equal to or lower than 10 °C and should not be kept for more than 2 hours at room temperature.

Biosurfactant Produced by Salmonella Enteritidis SE86 Can Increase Adherence and Resistance to Sanitizers on Lettuce Leaves (Lactuca sativa L., cichoraceae).

Salmonella Enteritidis SE86 is an important foodborne pathogen in Southern Brazil and it is able to produce a biosurfactant. However, the importance of this compound for the microorganism is still unknown. This study aimed to investigate the influence of the biosurfactant produced by S. Enteritidis SE86 on adherence to slices of lettuce leaves and on resistance to sanitizers. First, lettuce leaves were inoculated with S. Enteritidis SE86 in order to determine the amount of biosurfactant produced. Subsequently, lettuce leaves were inoculated with S. Enteritidis SE86 with and without the biosurfactant, and the adherence and bacterial resistance to different sanitization methods were evaluated. S. Enteritidis SE86 produced biosurfactant after 16 h (emulsification index of 11 to 52.15 percent, P < 0.05) and showed greater adherence capability and resistance to sanitization methods when the compound was present. The scanning electron microscopy demonstrated that S. Enteritidis was able to adhere, form lumps, and invade the lettuce leaves' stomata in the presence of the biosurfactant. Results indicated that the biosurfactant produced by S. Enteritidis SE86 contributed to adherence and increased resistance to sanitizers when the microorganism was present on lettuce leaves.

Inspection Score and Grading System for Food Services in Brazil: The Results of a Food Safety Strategy to Reduce the Risk of Foodborne Diseases during the 2014 FIFA World Cup.

In 2014, Brazil hosted one of the most popular sport competitions in the world, the FIFA World Cup. Concerned about the intense migration of tourists, the Brazilian government decided to deploy a food safety strategy based on inspection scores and a grading system applied to food services. The present study aimed to evaluate the results of the food safety strategy deployed during the 2014 FIFA World Cup in Brazil. To assess food safety, an evaluation instrument was applied twice in 1927 food service establishments from 26 cities before the start of the competition. This instrument generated a food safety score for each establishment that ranged from 0.0 (no flaws observed) to 2565.95, with four possible grades: A (0.0-13.2); B (13.3-502.6); C (502.7-1152.2); and pending (more than 1152.3). Each food service received a stamp with the grade of the second evaluation. After the end of the World Cup, a study was conducted with different groups of the public to evaluate the acceptance of the strategy. To this end, 221 consumers, 998 food service owners or managers, 150 health surveillance auditors, and 27 health surveillance coordinators were enrolled. These participants completed a survey with positive and negative responses about the inspection score system through a 5-point Likert scale. A reduction in violation scores from 393.1 to 224.4 (p < 0.001) was observed between the first and second evaluation cycles. Of the food services evaluated, 38.7% received the A stamp, 41.4% the B stamp, and 13.9% the C stamp. All positive responses on "system reliability" presented a mean of 4.0 or more, indicating that the public believed this strategy is reliable for communicating risks and promoting food safety. The strategy showed positive results regarding food safety and public acceptance. The deployed strategy promoted improvements in the food safety of food services. The implementation of a permanent policy may be well accepted by the public and may greatly contribute to a reduction in foodborne diseases (FBDs).

Genotypic and antimicrobial characterization of pathogenic bacteria at different stages of cattle slaughtering in southern Brazil.

Meat can be contaminated in different stages of the slaughtering process and the identification of these stages is the starting point to implement adequate control measures. The objectives of this study were to assess the presence of pathogenic microorganisms in cattle carcasses, to identify the most important contamination points of the slaughtering process, and to evaluate the possible risk factors related to them in a cattle slaughterhouse. To this aim, 108 cattle carcasses were sampled at three stages of the slaughtering process: Point 1 (hides after bleeding); Point 2 (carcasses after hide removal); and Point 3 (carcasses immediately after division). Escherichia coli O157:H7, Listeria monocytogenes and Salmonella Livingstone were isolated from the carcasses. Phenotypic and genotypic characterization indicated that there was cross-contamination among animals, since bacteria with identical genotypic and phenotypic profiles were isolated from different animals at the same sampling day. Furthermore, this is the first report about the isolation of E. coli O157:H7 in a bovine slaughterhouse from southern Brazil.

Serological characterization and prevalence of spvR genes in Salmonella isolated from foods involved in outbreaks in Brazil.

Salmonella strains (n = 75) isolated from foods involved in foodborne outbreaks occurred in Rio Grande do Sul State, Brazil, during 1999 and 2000 were studied. Strains were serotyped and submitted to PCR analysis to verify the prevalence of Salmonella plasmid virulence (spvR) regulatory gene. Among the 75 isolates, 73 (97%) were classified as Salmonella enterica serovar Enteritidis. All of the Salmonella strains isolated in 1999 were classified as serotype Enteritidis, whereas in 2000 two isolates were serotyped as Salmonella Derby and Salmonella Typhimurium. Regarding the prevalence of spvR gene, 62 strains (82.7%) were PCR positive, and a positive correlation (P < 0.05) between the strains of Salmonella Enteritidis and the presence of spvR gene was demonstrated, which suggests that this gene is a characteristic of the Salmonella Enteritidis analyzed.

Biofilm formation capacity of Salmonella serotypes at different temperature conditions / Capacidade de produção de biofilme por cepas de diferentes sorovares de Salmonella em quatro temperaturas de incubação

Salmonella spp. are one of the most important agents of foodborne disease in several countries, including Brazil. Poultry-derived products are the most common food products, including meat and eggs, involved in outbreaks of human salmonellosis. Salmonella has the capacity to form biofilms on both biotic and abiotic surfaces. The biofilm formation process depends on an interaction among bacterial cells, the attachment surface and environmental conditions. These structures favor bacterial survival in hostile environments, such as slaughterhouses and food processing plants. Biofilms are also a major problem for public health because breakage of these structures can cause the release of pathogenic microorganisms and, consequently, product contamination. The aim of this study was to determine the biofilm production capacity of Salmonella serotypes at four different temperatures of incubation. Salmonella strains belonging to 11 different serotypes, isolated from poultry or from food involved in salmonellosis outbreaks, were selected for this study. Biofilm formation was investigated under different temperature conditions (37°, 28°, 12° and 3°C) using a microtiter plate assay. The tested temperatures are important for the Salmonella life cycle and to the poultry-products process. A total of 92.2% of the analyzed strains were able to produce biofilm on at least one of the tested temperatures. In the testing, 71.6% of the strains produced biofilm at 37°C, 63% at 28°C, 52.3% at 12°C and 39.5% at 3°C, regardless of the serotype. The results indicate that there is a strong influence of temperature on biofilm production, especially for some serotypes, such as S. Enteritidis, S. Hadar and S. Heidelberg. The production of these structures is partially associated with serotype. There were also significant differences within strains of the same serotype, indicating that biofilm production capacity may be strain-dependent.(AU)

Salmonella spp. são um dos mais importantes agentes causadores de doenças transmitidas por alimentos em vários países, inclusive no Brasil. Produtos avícolas e ovos são os principais alimentos envolvidos na transmissão dos sorovares de Salmonella que são responsáveis por surtos de salmonelose em humanos. Salmonella possui a capacidade de formar biofilmes em diversas superfícies. O processo de formação de biofilme depende da interação entre as células bacterianas, a superfície de adesão e as condições do ambiente onde a bactéria se encontra. Estas estruturas favorecem a sobrevivência bacteriana em ambientes hostis, como em matadouros-frigoríficos e em indústrias processadoras de alimentos. Biofilmes são um grande problema em saúde pública, pois a ruptura destas estruturas pode provocar a liberação de microrganismos patogênicos e, consequentemente, a contaminação dos produtos. O objetivo deste estudo foi avaliar a capacidade de produção de biofilme por diferentes sorovares de Salmonella submetidos a quatro temperaturas de incubação. Cepas de Salmonella de 11 sorovares foram selecionadas. A produção de biofilme foi avaliada através do método de incubação em microplacas de poliestireno incubadas a 37°, 28°, 12° e 3°C. Estas temperaturas são importantes durante o ciclo de vida de Salmonella e para o processamento de produtos avícolas. Do total de cepas avaliadas, 92,2% foram capazes de produzir biofilme em pelo menos uma das quatro temperaturas testadas. Neste estudo, 71,6% das cepas produziram biofilme a 37°C, 63% a 28°C, 52,3% a 12°C e 39,5% a 3°C, independentemente do sorovar. Os resultados indicam uma forte influência da temperatura na produção de biofilme, especialmente para os sorovares S. Enteritidis, S. Hadar e S. Heidelberg. A produção de biofilme está parcialmente associada com o sorovar da cepa. Também foi observado que existe variação quanto à produção destas estruturas dentro de um mesmo sorovar, indicando que possivelmente a produção de biofilme é cepa-dependente.(AU)

Análise de perigos e pontos críticos de controle em uma unidade de alimentaçäo e nutriçäo / Analyse hazard and critical control points in a food and nutrition unit

Objetivando a implantaçäo do sistema APPCC/HACCP em uma Unidade de Alimentaçäo e Nutriçäo, a presença de coliformes totais e fecais e Staphylococcus aureus foi verificada nas mäos de manipuladores de alimentos e na superfície de equipamentos ou utensílios. Também foram analisadas amostras de água, amostras de matérias-primas e amostras de produtos finais, os quais foram comparados aos padröes estipulados pela legislaçäo brasileira vigente. Análises de tempo e temperatura foram realizadas em diversas etapas das preparaçöes consideradas de maior risco (costela assada, maionese de batata e frango assado). As temperaturas das 4 câmaras frias também foram avaliadas. A partir dos resultados obtidos, constatou-se que 63 por cento dos manipuladores demonstraram condiçöes adequadas de higienizaçäo das mäos, 76 por cento dos equipamentos ou utensílios apresentaram resultados dentro do padräo interno especificado e 92 por cento das amostras de água näo apresentaram contaminaçäo por coliformes totais ou fecais. Em relaçäo às amostras de matéria-prima e produto final, 92 por cento e 87 por cento respectivamente, apresentaram resultados dentro do padräo estabelecido pela Portaria 451/97 - MS. Quanto à temperatura e tempo das etapas de preparaçäo dos alimentos avaliados, verificou-se a necessidade de procederem-se acompanhamentos sistemáticos, uma vez que foram ultrapassados os limites estipulados em algumas etapas. Com base nos resultados obtidos verificou-se a necessidade de algumas melhorias nas boas práticas de fabricaçäo e processamento de alimentos; mesmo assim, as análises demonstraram condiçöes básicas para a implementaçäo completa do sistema APPCC/HACCP.(AU)

Análise de perigos e pontos críticos de controle em uma unidade de alimentaçäo e nutriçäo / Analyse hazard and critical control points in a food and nutrition unit

Objetivando a implantaçäo do sistema APPCC/HACCP em uma Unidade de Alimentaçäo e Nutriçäo, a presença de coliformes totais e fecais e Staphylococcus aureus foi verificada nas mäos de manipuladores de alimentos e na superfície de equipamentos ou utensílios. Também foram analisadas amostras de água, amostras de matérias-primas e amostras de produtos finais, os quais foram comparados aos padröes estipulados pela legislaçäo brasileira vigente. Análises de tempo e temperatura foram realizadas em diversas etapas das preparaçöes consideradas de maior risco (costela assada, maionese de batata e frango assado). As temperaturas das 4 câmaras frias também foram avaliadas. A partir dos resultados obtidos, constatou-se que 63 por cento dos manipuladores demonstraram condiçöes adequadas de higienizaçäo das mäos, 76 por cento dos equipamentos ou utensílios apresentaram resultados dentro do padräo interno especificado e 92 por cento das amostras de água näo apresentaram contaminaçäo por coliformes totais ou fecais. Em relaçäo às amostras de matéria-prima e produto final, 92 por cento e 87 por cento respectivamente, apresentaram resultados dentro do padräo estabelecido pela Portaria 451/97 - MS. Quanto à temperatura e tempo das etapas de preparaçäo dos alimentos avaliados, verificou-se a necessidade de procederem-se acompanhamentos sistemáticos, uma vez que foram ultrapassados os limites estipulados em algumas etapas. Com base nos resultados obtidos verificou-se a necessidade de algumas melhorias nas boas práticas de fabricaçäo e processamento de alimentos; mesmo assim, as análises demonstraram condiçöes básicas para a implementaçäo completa do sistema APPCC/HACCP.

Análise de perigos e pontos críticos de controle para garantir a segurança alimentar em restaurante da Universidade Federal do Rio Grande do Sul / Analyse hazard and critical control points to guarantee the alimentar safety in restaurant of Universidade Federal do Rio Grande do Sul

Muitos restaurantes universitários brasileiros produzem significativas quantidades de refeiçöes, mesmo näo havendo condiçöes ideais para tanto. As freqüentes reduçöes de orçamento e a falta de treinamento dos funcionários cooperam ainda mais no detrimento da qualidade dos alimentos ali processados. Objetivou-se analisar perigos e identificar pontos críticos de controle (PCC), os quais podem ser utilizados em um sistema HACCP/APPCC (Hazard Analysis Critical Control Point ou Análise de Perigos e Pontos Críticos de Controle) como fonte de resoluçäo dos problemas diários que afetam a qualidade das refeiçöes produzidas. A investigaçäo de perigos e pontos críticos de controle foi conduzida no restaurante universitário n§3 da Universidade Federal do Rio Grande do Sul através de elaboraçäo de fluxogramas, análises visuais, físicas e microbiológicas durante os meses de temperatura pico (janeiro e junho) no veräo e inverno dos anos de 1997 e 1998, na cidade de Porto Alegre. Os resultados obtidos foram comparados a fim de evidenciar as diferenças de contaminaçäo dentro de condiçöes climáticas diferentes. Treinamentos periódicos, destinaçäo de recursos para a manutençäo e aquisiçäo de equipamentos e utensílios, assim como a implantaçäo completa do sistema HACCP/APPCC reduziriam sensivelmente a possibilidade de toxinfecçöes e a instabilidade imposta pelas condiçöes näo ideais de trabalho desses restaurantes.(AU)