Phenotypic analyses of Salmonella enterica serovar Enteritidis strains isolated in the pre- and post-epidemic period in Brazil.

Salmonella Enteritidis has caused, since the 1980s, a sustained epidemic of human infections in many countries. This study analyzed S. Enteritidis strains isolated before and after the epidemic period in Brazil regarding their capacities to survive to acid, oxidative, and high-temperature stresses, and capacity to grow in egg albumen. Moreover, the ability to invade human epithelial cells (Caco-2) and to survive inside human (U937) and chicken (HD11) macrophages was checked. Post-epidemic strains showed a better ability to survive after 10 min under acid stress at 37 °C (P ≤ 0.05). However, both groups of strains showed similar ability to survive after 1 h under acid stress at 37 °C and at 42 °C independently of the time of exposure. Similar ability was verified in both groups of strains regarding oxidative stress, growth in egg albumen, high-temperature stress, invasion to Caco-2 cells, and invasion and survival in macrophages. In conclusion, post-epidemic S. Enteritidis strains showed a better ability to survive under the acid stress found in the stomach, which might be an advantage to reach the intestine and colonize chickens and humans. However, both groups of strains did not differ significantly in the majority of the phenotypic tests analyzed in this study.

Changing of the Genomic Pattern of Salmonella Enteritidis Strains Isolated in Brazil Over a 48 year-period revealed by Whole Genome SNP Analyses.

Salmonella Enteritidis became the main serovar isolated from gastroenteritis cases in Brazil after the 90's. In this study we used whole genome sequence analysis to determine the phylogenetic relationships among a collection of strains isolated in Brazil to identify possible genomic differences between the strains isolated in the pre and post-epidemic period. Also, we compared our data from strains isolated in Brazil to strains available in the public domain from other South American countries. Illumina technology was used to sequence the genome of 256 Salmonella Enteritidis strains isolated over a 48 year-period in Brazil, comprising the pre- and post-epidemic period. Phylogenetic analyses revealed distinct lineages for strains isolated before and after 1994. Moreover, the phage region SE20 that may be related to the emergence of Salmonella Enteritidis worldwide was present only in strains of the post-epidemic cluster. In conclusion, our results showed that the genomic profile of Salmonella Enteritidis strains isolated in Brazil shifted after 1994, replaced by a global epidemic group of strains. It may be hypothesized that the presence of the prophage SE20 might have conferred to these strains a better ability to colonize chicken and consequently to infect and cause disease in humans, which might better explain the increase in the number of S. Enteritidis cases in Brazil and other South American countries. However, to verify this hypothesis further studies are needed.

Draft Genome Sequences of 256 Salmonella enterica subsp. enterica Serovar Enteritidis Strains Isolated from Humans, Food, Chickens, and Farm Environments in Brazil.

Salmonella enterica subsp. enterica serovar Enteritidis emerged in the late 1980s as the most isolated Salmonella serovar worldwide. Here, we report the draft genomes of 256 S Enteritidis strains isolated from humans, food, chickens, and farm environments in Brazil. These draft genomes will help enhance our understanding of this serovar in Brazil.

Prevalence of gyrA Mutations in Nalidixic Acid-Resistant Strains of Salmonella Enteritidis Isolated from Humans, Food, Chickens, and the Farm Environment in Brazil.

Salmonella Enteritidis strains that are resistant to nalidixic acid and exhibit reduced susceptibility to fluoroquinolones have been increasing worldwide. In Brazil, few studies have been conducted to elucidate the quinolone resistance mechanisms of S. Enteritidis strains. This study analyzed the profile of gyrA, gyrB, parC, and parE mutations and plasmid-mediated quinolone resistance (PMQR) mechanisms in S. Enteritidis NalR strains isolated in Brazil. Moreover, the minimum inhibitory concentrations (MICs) of ciprofloxacin were evaluated in 84 NalR strains and compared with 20 NalS strains. The mutation profiles of the gyrA gene were accessed by high-resolution melting analysis and gyrB, parC, and parE by quinolone resistance-determining region sequencing. The MICs of ciprofloxacin were accessed with Etest®. The strains were divided into five gyrA melting profiles. The NalR strains exhibited the following amino acid substitutions: Ser97→Pro, Ser83→Phe, Asp87→Asn, or Asp87→Tyr. The average MICs of ciprofloxacin was 0.006 µg/ml in the NalS and 0.09 µg/ml in the NalR strains. No points of mutation were observed in the genes gyrB, parC, and parE. The qnrB gene was found in two strains. In conclusion, the reduced susceptibility to ciprofloxacin observed in NalR strains may cause treatment failures once this drug is commonly used to treat Salmonella infections. Moreover, this reduced susceptibility in these Brazilian strains was provided by target alteration of gene gyrA and not by mobile elements, such as resistance plasmids.

Prevalence and antimicrobial susceptibility profile of Enterococcus spp isolated from frozen chicken carcasses

Prevalence and antimicrobial susceptibility of Enterococcus spp. were evaluated in 360 frozen unse asoned chicken carcasses samples collected from September 2004 to June 2006 from the retail stores in São Paulo State, Brazil. Enterococcus spp. was isolated from all analyzed samples, and 1,332 strains were identified from them. Among the ten identified species, the predominance of E. faecalis, E. gallinarum, E.casseliflavus and E. faecium was occurred. All of the Enterococci strains showed some degree of resistanceto the nine antimicrobials utilized in the study. The percentages of antimicrobial resistance were: 89.2% for tetracycline, 91.4% for quinupristin-dalfopristin, 83.5% for erythromycin, 65% for ciprofloxacin, 55.4% for chloramphenicol, 6.5% for linezolid, 2.3% for vancomycin, 2.3% for teicoplanin and 0.2% for ampicillin. The occurrence of the high level resistance to amyno glicosides (HLR-A) was detected in 57.4% of the isolates. E. faecalis and E. faecium species, which are considered as important agents in nosocomial infections, showed resistance to eight and seven antibiotics, respectively.

Queijo minas frescal: perfil higiênico-sanitário e avaliação da rotulagem / Minas frescal cheese: hygienic-sanitary characteristic and assessment of label information

O queijo Minas Frescal, em função do alto teor de umidade, torna-se susceptível a contaminações por micro-organismos. Vários estudos têm mostrado a ocorrência variável de patógenos em queijo Minas Frescal, sendo comuns altas contagens de coliformes termotolerantes. Neste estudo, foram analisadas as condições higiênico-sanitárias, a presença de micro-organismos potencialmente patogênicos e as informações no rótulo em 30 amostras (22 industrializadas e oito caseiras) de queijos Minas Frescal, comercializadas na região de Ribeirão Preto-SP. A qualidade microbiológica foi avaliada por enumeração de coliformes termotolerantes e de estafilococos coagulase-positiva e pela pesquisa de Salmonella spp.e Listeria monocytogenes, seguindo-se a Resolução – RDC nº 12, de 2 de janeiro de 2001, da ANVISA. Foram também realizadas a enumeração de coliformes totais e a pesquisa de Shigella spp. A análise do rótulo foi efetuada seguindo-se as legislações da ANVISA/MS, do MAPA e do INMETRO. Do total de amostras, 63,4 apresentaram coliformes termotolerantes acima de 1,1 x 103 NMP/g, sendo 23,4 e 40,0 correspondentes, respectivamente, às amostras de queijos dos tipos caseiro e industrializado. Não foram isoladas Salmonella spp., Listeria monocytogenes e Shigella spp. Das 22 amostras industrializadas, 71 apresentaram rótulos em não conformidade. As amostras caseiras e as industrializadas mostraram baixa qualidade microbiológica.

Avaliação do perfil microbiológico e microscópico do caldo de cana in natura comercializado por ambulantes / Study on microbiological and microscopic profiles of fresf sugar-cane juice marketed by street vendors

A cana-de-açúcar (Saccharum spp.) é a matéria prima constituída basicamente de fibras e caldo. O caldo, conhecido como garapa, é uma bebida de baixo custo, refrescante, energética e muito popular no Brasil. O presente trabalho avaliou a qualidade microbiológica, microscópica e as condições higiênicas durante os procedimentos de manipulação do caldo de cana in natura, comercializado por ambulantes em Ribeirão Preto/SP. Foram avaliadas 90 amostras de caldo de cana in natura, adquiridas no período de maio de 2007 a janeiro de 2008, quanto ao isolamento de Salmonella sp, contagem de coliformes a 35ºC e a 45ºC, detecção de bolores e leveduras e pesquisa de matérias estranhas. Do total de amostras avaliadas, 31,0% apresentaram coliformes a 45ºC acima de 2 (logNMP/mL), porém Salmonella spp. não foi isolada em nenhuma das amostras. Quanto aos parâmetros microscópicos, 32,2% estavam em desacordo com a legislação em vigor principalmente em virtude da presença de fragmentos de insetos e de insetos inteiros pertencentes às ordens Hymenoptera (abelhas e formigas), Díptera (moscas domésticas, varejeiras e drosófilas) e Arachinida (aranha). Estes resultados evidenciam as deficiências higiênico-sanitárias durante o procedimento de obtenção do caldo de cana, o qual sugere a necessidade de implantação de programas para capacitação dos manipuladores desse produto.

Sugar cane (Saccharum spp.) is a raw plant, basically consisted of fibers and juice. The juice, also known as ‘garapa’ in Portuguese, is a cheap, refreshing and energetic beverage that is quite popular in Brazil. Thepresent study aimed at evaluating the microbiological and microscopic quality, and the hygienic conditions ofhandling procedures for the fresh sugar-cane juice sold by street vendors in Ribeirão Preto/Brazil. Ninety fresh sugar-cane samples were collected during the period between May 2007 and January 2008. Salmonella spp. isolation, coliform counts at 35ºC and 45ºC, and yeast and mold counts were performed, and the occurrence of extraneous foreign matters was investigated. Among the 90 analyzed samples, 31.0% showed coliforms at 45ºC above 2 (logMPN/mL), and no Salmonella spp. was isolated from any sample. On microscopy analyses, 32.2% of samples did not comply with the current legislation due to the presence of insect fragments and wholeinsects consisted of the following orders Hymenoptera (bees and ants), Diptera (house flies, blowflies anddrosophilas) and Arachnida (spiders). The hygienic-sanitary inadequacy in producing sugar-cane juice couldbe evidenced, thus, it might be inferred the need for establishing specific food-handlers training programs.

Avaliação microbiológica, parasitológica e da rotulagem de hortaliças minimamente processadas comercializadas no município de Ribeirão Preto, SP/Brasil / Microbiological and parasitic quality and labeling adequacy of minimally-processed vegetables, commercialized in Ribeirão Preto, SP/Brasil

O consumo de hortaliças minimamente processadas tem aumentado nos últimos anos no Brasil, principalmente em função da conveniência e da praticidade que o produto oferece. A contaminação microbiológica, a presença de parasitos e os dizeres da rotulagem foram avaliados em 70 amostras de hortaliças folhosas minimamente processadas, adquiridas em supermercados de Ribeirão Preto-SP, no período de janeiro a julho de 2006. Foram pesquisados os microrganismos aeróbios psicrotróficos, cloliformes a 35ºC e 45ºC, salmonella spp. e Escherichia coli. Para a avaliação da rotulagem foram utilizadas as legislações em vigor(Resoluções RDC nº259/2002, 359 e 360/2003, da ANVISA/MS e Lei nº10.674/2003). Das amostras analisadas, 20 estavam em descordo com a legislação em vigor nas análises microbiológicas, 18,6.

Prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) strains in ground beef in São Paulo, Brazil

This study aims to assess the prevalence of Shiga toxin-producing Escherichia coli (STEC) in ground beef collected in two cities located in the State of São Paulo, Brazil. A total of 250 samples of raw ground beef were collected in local grocery stores during the period of March to December 2002 in the cities of Ribeirão Preto (114 samples) and Campinas (136 samples), São Paulo State, Brazil. The samples were processed according to standard methods. The resulting 591 E.coli colonies were screened for STEC by hybridization assays using the specific DNA probes, stx1,stx2 and eae. Further characterization of STEC isolates included the search for the ehxA sequence, detection of enterohemolysin and expression of Shiga toxin using the Vero cell assay. STEC isolates belonging to serotypes O93:H19, ONT:HNT, ONT:H7, and O174:HNT we recovered from four samples (3.5 percent) collected in Ribeirão Preto. All samples from Campinas were negative for STEC. Three of the strains carried stx2 and ehxA sequences while one harbored stx1,stx2 and ehxA sequences. Considering that among foods of animal origin, ground beef is an important vehicle for STEC transmission, these data emphasize the need of a closer surveillance of these microorganisms. They can survive in unfavorable conditions specially when the products are refrigerated or frozen for long periods of time and can be the cause of outbreaks affecting a great number of consumers.

O objetivo deste estudo foi verificar a ocorrência de Escherichia coli produtora de toxina Shiga (STEC) em amostras de carne moída crua comercializadas em duas cidades do Estado de São Paulo, Brasil. Um total de 250 amostras de carne moída crua foi coletado de açougues locais durante o período de Março a Dezembro de 2002, nas cidades de Ribeirão Preto (114 amostras) e de Campinas (136 amostras), Estado de São Paulo, Brasil. As amostras foram processadas de acordo com os métodos de referência. Um total de 591 colônias de E.coli foi submetido à técnica de hibridização de colônias usando sondas específicas de DNA para a detecção das seqüências stx1,stx2 e eae. Caracterizações adicionais das cepas STEC incluíram a pesquisa da seqüência ehxA, a detecção da enterohemolisina e a pesquisa da expressão de toxina Shiga utilizando testes com células Vero. Em quatro amostras (3,5 por cento) coletadas em Ribeirão Preto, foram encontradas cepas STEC, mas todas aquelas da região de Campinas foram negativas. As cepas de STEC pertenciam aos sorotipos O93:H19, ONT:HNT, ONT:H7, e O174:HNT. Três cepas tinham o perfil stx2 ehxA e uma era portadora das seqüências stx1,stx2 e ehxA. Considerando que entre os alimentos de origem animal, a carne moída ainda representa um importante veículo de transmissão de STEC, estes dados alertam para a necessidade de uma vigilância da presença destes microrganismos capazes de sobreviver em condições desfavoráveis, especialmente quando os produtos são refrigerados ou congelados por longos períodos, podendo ser causas de importantes surtos afetando grande número de consumidores.