Alzira Maria Morato Bergamini

Quantification of Listeria monocytogenes in minimally processed leafy vegetables using a combined method based on enrichment and 16S rRNA real-time PCR

Modern lifestyle markedly changed eating habits worldwide, with an increasing demand for ready-to-eat foods, such as minimally processed fruits and leafy greens. Packaging and storage conditions of those products may favor the growth of psychrotrophic bacteria, including the pathogen Listeria monocytogenes. In this work, minimally processed leafy vegetables samples (n = 162) from retail market from Ribeirão Preto, São Paulo, Brazil, were tested for the presence or absence of Listeria spp. by the immunoassay Listeria Rapid Test, Oxoid. Two L. monocytogenes positive and six artificially contaminated samples of minimally processed leafy vegetables were evaluated by the Most Probable Number (MPN) with detection by classical culture method and also culture method combined with real-time PCR (RTi-PCR) for 16S rRNA genes of L. monocytogenes. Positive MPN enrichment tubes were analyzed by RTi-PCR with primers specific for L. monocytogenes using the commercial preparation ABSOLUTE QPCR SYBR Green Mix (ABgene, UK). Real-time PCR assay presented good exclusivity and inclusivity results and no statistical significant difference was found in comparison with the conventional culture method (p < 0.05). Moreover, RTi-PCR was fast and easy to perform, with MPN results obtained in ca. 48 h for RTi-PCR in comparison to 7 days for conventional method.

Fiscalização de verduras comercializadas no município de Ribeirão Preto, SP

Resumo O consumo de verduras cruas constitui importante meio de transmissao de varias doencasinfecciosas. Este estudo tem como objetivo a avaliacao microbiologica e parasitologica de verdurascomercializadas no municipio de Ribeirao Preto, SP, abrangendo todos os pontos de venda ao consumidor.Do total de 172 estabelecimentos fixos ou ambulantes analisados, 115 (67%) apresentaram hortalicas comirregularidades: elevada concentracao de coliformes fecais em 63%, presenca de Salmonella em 9% e deenteroparasitas em 33%. Os pontos de venda com maior frequencia de hortalicas com resultados inadequadosforam: mercearias (92%), CEAGESP (75%), quitandas (71%), vendedores ambulantes (71%), feiras-livres(69%), supermercados (52%) e hortas (18%). O tipo de contaminacao apresentou distribuicao uniforme emrelacao aos locais de venda e a variedade da hortalica. A maioria (61%) das verduras contaminadas eraprocedente de hortas localizadas no municipio de Ribeirao Preto. Considerando a elevada frequencia decontaminacao fecal e o potencial risco de doencas veiculadas pelas hortalicas, sugerimos uma vigilânciasanitaria mais atuante na fiscalizacao de alimentos oferecidos a populacao.Palavras-chaves: Verduras. Coliformes fecais. Salmonella. Cisticercose.Abstract The ingestion of raw vegetables represents an important means of transmission of several infectiousdiseases. The objective of the present study was to perform a microbiological and parasitological evaluationof the vegetables commercially sold in the municipality of Ribeirao Preto, SP, Brazil. Of a total of 172 commercialconcerns analyzed, 115 (67%) presented irregularities in the vegetables they sold, such as elevatedconcentration of fecal coliforms in 63%, presence of Salmonella in 9%, and presence of enteroparasites in33%. The commercial concerns with the highest frequencies of vegetables showing inadequate results were:grocery stores (92%), CEAGESP (75%), fruit and vegetables stores (71%), traveling vendors (71%), fairs(69%), supermarkets (52%), and vegetable gardens (18%). The type of contamination was uniformly distributedamong these commercial concerns. Most of the contaminated vegetables (61%) were from gardens located inthe municipality of Ribeirao Preto. Considering the high frequency of fecal contamination and the potential riskof disease transmitted by vegetables, we suggest greater enforcement in the sanitary surveillance of the foodoffered to the population.Key-words: Lettuce. Vegetable. Fecal coliforms. Salmonella. Cysticercosis.The ingestion of raw vegetables represents an important means of transmission of several infectious diseases. The objective of the present study was to perform a microbiological and parasitological evaluation of the vegetables commercially sold in the municipality of Ribeirão Preto, SP, Brazil. Of a total of 172 commercial concerns analyzed, 115 (67%) presented irregularities in the vegetables they sold, such as elevated concentration of fecal coliforms in 63%, presence of Salmonella in 9%, and presence of enteroparasites in 33%. The commercial concerns with the highest frequencies of vegetables showing inadequate results were: grocery stores (92%), CEAGESP (75%), fruit and vegetables stores (71%), traveling vendors (71%), fairs (69%), supermarkets (52%), and vegetable gardens (18%). The type of contamination was uniformly distributed among these commercial concerns. Most of the contaminated vegetables (61%) were from gardens located in the municipality of Ribeirão Preto. Considering the high frequency of fecal contamination and the potential risk of disease transmitted by vegetables, we suggest greater enforcement in the sanitary surveillance of the food offered to the population.

Fiscalização de hortas produtoras de verduras do município de Ribeirão Preto, SP

The ingesting of raw vegetables plays an important role in the transmission of several infectious diseases due to the high frequency of irrigation with wastewater. The objective of this study was to evaluate the sanitary conditions of all lettuce producing crops in Ribeirao Preto through microbiological and parasitological analysis of both irrigation water and lettuce together with the implantation of an effective crop monitoring. Laboratory analysis of 129 crops showed irregularities in 26 (20.1%) of these: high concentration of fecal coliforms in 17% of the lettuce, presence of Salmonella in 3.1% and several enteroparasites (Ascaris sp, Ancylostomidae, Strongyloides sp, Hymenolepis nana, and Giardia sp ) in 13.1%. Persistent irregularities determined the definitive closing down of one producer; all of the remaining 128 crops were eventually approved, thus demonstrating the efficacy of lettuce crop monitoring. Crops approved in the laboratory analysis were awarded a Sanitary Inspection Certificate - an unprecedented procedure in our Country - that resulted in a better acceptance of the monitoring.The ingesting of raw vegetables plays an important role in the transmission of several infectious diseases due to the high frequency of irrigation with wastewater. The objective of this study was to evaluate the sanitary conditions of all lettuce producing crops in Ribeirão Preto through microbiological and parasitological analysis of both irrigation water and lettuce together with the implantation of an effective crop monitoring. Laboratory analysis of 129 crops showed irregularities in 26 (20.1%) of these: high concentration of fecal coliforms in 17% of the lettuce, presence of Salmonella in 3.1% and several enteroparasites (Ascaris sp, Ancylostomidae, Strongyloides sp, Hymenolepis nana, and Giardia sp ) in 13.1%. Persistent irregularities determined the definitive closing down of one producer; all of the remaining 128 crops were eventually approved, thus demonstrating the efficacy of lettuce crop monitoring. Crops approved in the laboratory analysis were awarded a Sanitary Inspection Certificate - an unprecedented procedure in our Country - that resulted in a better acceptance of the monitoring.

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 Sao 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 Ribeirao Preto (114 samples) and Campinas (136 samples), Sao 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%) collected in Ribeirao 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.

Avaliação da contaminação de hortas produtoras de verduras após a implantação do sistema de fiscalização em Ribeirão Preto, SP

This study evaluated the microbiological and parasitological contamination of 88 market gardens producing green vegetables, of which 47 had been investigated previously. The contamination rate was 38.3% in the market gardens previously evaluated and 43.9% in the new market gardens. Food safety requires a continuous and efficient sanitary surveillance system for market gardens.

Análise da cadeia de produção de verduras em Ribeirão Preto, SP

With the aim of assessing the cumulative risk of lettuce contamination, 45 production chains were investigated. The presence of thermotolerant coliforms, Salmonella and/or parasites was detected in 69% of these, in all steps of the production chain. Quality control in all steps of the lettuce production process should be intensified.

Monitoring of lettuce crops of Ribeirão Preto, SP, Brazil

The ingesting of raw vegetables plays an important role in the transmission of several infectious diseases due to the high frequency of irrigation with wastewater. The objective of this study was to evaluate the sanitary conditions of all lettuce producing crops in Ribeirao Preto through microbiological and parasitological analysis of both irrigation water and lettuce together with the implantation of an effective crop monitoring. Laboratory analysis of 129 crops showed irregularities in 26 (20.1%) of these: high concentration of fecal coliforms in 17% of the lettuce, presence of Salmonella in 3.1% and several enteroparasites (Ascaris sp, Ancylostomidae, Strongyloides sp, Hymenolepis nana, and Giardia sp ) in 13.1%. Persistent irregularities determined the definitive closing down of one producer; all of the remaining 128 crops were eventually approved, thus demonstrating the efficacy of lettuce crop monitoring. Crops approved in the laboratory analysis were awarded a Sanitary Inspection Certificate - an unprecedented procedure in our Country - that resulted in a better acceptance of the monitoring.The ingesting of raw vegetables plays an important role in the transmission of several infectious diseases due to the high frequency of irrigation with wastewater. The objective of this study was to evaluate the sanitary conditions of all lettuce producing crops in Ribeirão Preto through microbiological and parasitological analysis of both irrigation water and lettuce together with the implantation of an effective crop monitoring. Laboratory analysis of 129 crops showed irregularities in 26 (20.1%) of these: high concentration of fecal coliforms in 17% of the lettuce, presence of Salmonella in 3.1% and several enteroparasites (Ascaris sp, Ancylostomidae, Strongyloides sp, Hymenolepis nana, and Giardia sp ) in 13.1%. Persistent irregularities determined the definitive closing down of one producer; all of the remaining 128 crops were eventually approved, thus demonstrating the efficacy of lettuce crop monitoring. Crops approved in the laboratory analysis were awarded a Sanitary Inspection Certificate - an unprecedented procedure in our Country - that resulted in a better acceptance of the monitoring.

Comparison of four molecular methods to type Salmonella Enteritidis strains

This study compared the pulsed‐field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus‐PCR (ERIC‐PCR), multilocus variable‐number of tanden‐repeat analysis (MLVA), and multilocus sequence typing (MLST) methods for typing 188 Salmonella Enteritidis strains from different sources isolated over a 24‐year period in Brazil. PFGE and ERIC‐PCR were more efficient than MLVA for subtyping the strains. However, MLVA provided additional epidemiological information for those strains. In addition, MLST showed the Brazilian strains as belonging to the main clonal complex of S. Enteritidis, CC11, and provided the first report of two new STs in the S. enterica database but could not properly subtype the strains. Our results showed that the use of PFGE or ERIC‐PCR together with MLVA is suitable to efficiently subtype S. Enteritidis strains and provide important epidemiological information.

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.

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

ABSTRACT 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.