Comparative genomics reveals high genetic similarity among strains of Salmonella enterica serovar Infantis isolated from multiple sources in Brazil.

Background: Salmonella enterica serovar Infantis (Salmonella Infantis) is a zoonotic, ubiquitous and foodborne pathogen of worldwide distribution. Despite Brazil's relevance as a major meat exporter, few studies were conducted to characterize strains of this serovar by genomic analyses in this country. Therefore, this study aimed to assess the diversity of 80 Salmonella Infantis strains isolated from veterinary, food and human sources in Brazil between 2013 and 2018 by comparative genomic analyses. Additional genomes of non-Brazilian countries (n = 18) were included for comparison purposes in some analyses. Methods: Analyses of whole-genome multi-locus sequence typing (wgMLST), using PGAdb-builder, and of fragmented genomes, using Gegenees, were conducted to compare the 80 Brazilian strains to the 18 non-Brazilian genomes. Pangenome analyses and calculations were performed for all Salmonella Infantis genomes analyzed. The presence of prophages was determined using PHASTER for the 80 Brazilian strains. The genome plasticity using BLAST Ring Image Generator (BRIG) and gene synteny using Mauve were evaluated for 20 selected Salmonella Infantis genomes from Brazil and ten from non-Brazilian countries. Unique orthologous protein clusters were searched in ten selected Salmonella Infantis genomes from Brazil and ten from non-Brazilian countries. Results: wgMLST and Gegenees showed a high genomic similarity among some Brazilian Salmonella Infantis genomes, and also the correlation of some clusters with non-Brazilian genomes. Gegenees also showed an overall similarity >91% among all Salmonella Infantis genomes. Pangenome calculations revealed an open pangenome for all Salmonella Infantis subsets analyzed and a high gene content in the core genomes. Fifteen types of prophages were detected among 97.5% of the Brazilian strains. BRIG and Mauve demonstrated a high structural similarity among the Brazilian and non-Brazilian isolates. Unique orthologous protein clusters related to biological processes, molecular functions, and cellular components were detected among Brazilian and non-Brazilian genomes. Conclusion: The results presented using different genomic approaches emphasized the significant genomic similarity among Brazilian Salmonella Infantis genomes analyzed, suggesting wide distribution of closely related genotypes among diverse sources in Brazil. The data generated contributed to novel information regarding the genomic diversity of Brazilian and non-Brazilian Salmonella Infantis in comparison. The different genetically related subtypes of Salmonella Infantis from Brazil can either occur exclusively within the country, or also in other countries, suggesting that some exportation of the Brazilian genotypes may have already occurred.

Hierarchical Virtual Screening of Potential New Antibiotics from Polyoxygenated Dibenzofurans against Staphylococcus aureus Strains.

Staphylococcus aureus is a microorganism with high morbidity and mortality due to antibiotic-resistant strains, making the search for new therapeutic options urgent. In this context, computational drug design can facilitate the drug discovery process, optimizing time and resources. In this work, computational methods involving ligand- and structure-based virtual screening were employed to identify potential antibacterial agents against the S. aureus MRSA and VRSA strains. To achieve this goal, tetrahydroxybenzofuran, a promising antibacterial agent according to in vitro tests described in the literature, was adopted as the pivotal molecule and derivative molecules were considered to generate a pharmacophore model, which was used to perform virtual screening on the Pharmit platform. Through this result, twenty-four molecules were selected from the MolPort® database. Using the Tanimoto Index on the BindingDB web server, it was possible to select eighteen molecules with greater structural similarity in relation to commercial antibiotics (methicillin and oxacillin). Predictions of toxicological and pharmacokinetic properties (ADME/Tox) using the eighteen most similar molecules, showed that only three exhibited desired properties (LB255, LB320 and LB415). In the molecular docking study, the promising molecules LB255, LB320 and LB415 showed significant values in both molecular targets. LB320 presented better binding affinity to MRSA (-8.18 kcal/mol) and VRSA (-8.01 kcal/mol) targets. Through PASS web server, the three molecules, specially LB320, showed potential for antibacterial activity. Synthetic accessibility (SA) analysis performed on AMBIT and SwissADME web servers showed that LB255 and LB415 can be considered difficult to synthesize and LB320 is considered easy. In conclusion, the results suggest that these ligands, particularly LB320, may bind strongly to the studied targets and may have appropriate ADME/Tox properties in experimental studies.

Salmonella enterica in soybean production chain: Occurrence, characterization, and survival during soybean storage.

This study aimed to determine Salmonella enterica occurrence along the soybean meal production chain (raw material, in-processing samples, final products, and in the environment of five processing plants), characterize the isolates, and assess the survival of Salmonella Senftenberg 775W in soybeans stored under different temperature conditions. Among 713 samples analyzed, 12.9% (n = 92) were positive for Salmonella enterica. Dust collected inside and outside processing plants (n = 148) comprised the samples with the highest positivity for Salmonella enterica, 47.3%. The occurrence of Salmonella enterica varied among the different processing plants. Twenty-nine (n = 29) Salmonella serotypes were isolated, with S. Mbandaka as the most frequent serotype, whereas S. Typhimurium was mainly linked to final product samples (soybean meal). S. Senftenberg 775W did not survive for a long time in soybean stored at 20-37 °C, but at 20 °C, cells were viable for more than 60 days. This study suggests that soybean meal may harbor Salmonella serotypes related to foodborne disease outbreaks in humans and can be responsible for Salmonella introduction into livestock and, consequently, in foods of animal origin. This study provides crucial data on contamination pathways of Salmonella in the soybean production chain, contributing to the understanding of Salmonella epidemiology which is strategic for the development of preventive and control measures to reduce the burden of salmonellosis linked to products of animal origin.

Partial Correlation Between Phenotypic and Genotypic Antimicrobial Resistance of Salmonella enterica Serovar Enteritidis Strains from Brazil.

Whole-genome sequencing analyses have provided important data and information on the repertoire of resistance genes in several bacterial species. This study examined to what extent the antimicrobial resistance genes found in a set of whole-genome-sequenced Salmonella Enteritidis strains from Brazil correlated with the phenotypic antimicrobial resistance possibly related to these genes. The genotypic resistance data from the strains studied were compared with publicly available data from strains isolated in other countries. The genotypic resistance profiles were accessed on the NCBI Pathogen Detection website, and the phenotypic resistance profiles were determined by the disk diffusion technique according to the Clinical and Laboratory Standards Institute guidelines. Fourteen of the 256 sequenced strains presented antimicrobial resistance genes, with the highest prevalence of resistance genes to aminoglycosides-with 16 genes detected in seven strains-not only in Brazilian strains but also in the strains from other parts of the world. The strongest correlation between phenotypic and genotypic resistance was found for tetracycline (75%). The genotypic and phenotypic profiles of the S. Enteritidis strains studied only partially matched, and they diverged in some antimicrobial classes more strongly than in other classes. The advances on whole-genome sequencing analyses associated with a better understanding of the correlation between phenotypic and genotypic resistance data may improve this powerful tool for antimicrobial resistance prediction.

Survey of Salmonella spp. in beef meat for export at slaughterhouses in Brazil / Pesquisa de Salmonella spp. em carcaças bovinas durante o processamento em abatedouros-frigoríficos exportadores

The aim of the present study was to investigate the presence of Salmonella spp. in samples collected from beef meat at three points of the slaughter line (after skinning, washing and cooling) at three slaughterhouses in Brazil that export meat. Detection was based on ISO 6579:2002 and confirmed by PCR and qPCR. The isolates were typified using slide agglutination tests and PFGE. The antibiotic sensitivity profile was determined using the disk diffusion method. Contamination was detected in only one slaughterhouse. The overall frequency of contamination by Salmonella spp. was 6.7% of carcasses (6/90) and 2.6% of carcass surface samples (7/270). All isolates were confirmed by PCR and qPCR. The serological analysis and the PFGE showed a single profile: Typhimurium. The strains demonstrated 100% susceptibility to ampicillin, cefotaxime, ciprofloxacin, chloramphenicol, gentamicin and tetracycline. Positive carcasses after cooling pose a direct risk to consumers, since the meat is considered ready to be marketed after this process.(AU)

O objetivo deste trabalho foi investigar a presença de Salmonella spp. em amostras coletadas de carcaças de bovinos, em três pontos da linha de abate (após a esfola, lavagem e refrigeração) de três frigoríficos exportadores no Brasil. A detecção foi realizada pela ISO 6579:2002, e confirmada por PCR e qPCR. Os isolados foram tipificados por testes de soroaglutinação e PFGE e avaliado o perfil de sensibilidade aos antibióticos pelo método de difusão em disco. A contaminação foi detectada em apenas um abatedouro-frigorífico. As contaminações das carcaças (n=90) e amostras de carne (n=270) por Salmonella spp. foram 6 (6,7%) e 7 (2,6%), respectivamente. Todos os isolados foram confirmados por PCR e qPCR. A análise sorológica e o PFGE mostraram um único perfil: Typhimurium. As cepas apresentaram 100% de suscetibilidade à ampicilina, cefotaxima, ciprofloxacina, cloranfenicol, gentamicina e tetraciclina. As carcaças positivas após a refrigeração apresentam um risco direto para o consumidor, uma vez que, após este processo, a carne está pronta para ser comercializada.(AU)

MALDI-TOF MS as a tool for the identification of Vibrio alginolyticus from Perna perna mussels (Linnaeus, 1758) / MALDI-TOF MS como ferramenta na identificação de Vibrio alginolyticus isolados de mexilhões Perna perna (Linnaeus, 1758)

Vibrio species are ubiquitous in aquatic environments, including coastal and marine habitats. Vibrio alginolyticus is an opportunistic pathogen for fish, crustaceans and mussels and their identification by biochemical tests may be impaired due their nutritional requirements. The study used Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) to identify 49 Vibrio spp. isolates associated with mussels (Perna perna) from different locations along the Rio de Janeiro coast. The rpoA gene was used as a genus-specific marker of Vibrio spp. and was positive in all 209 isolates. MALDI-TOF MS confirmed 87.8% of V. alginolyticus when compared to the results of the biochemical tests. Four isolates were identified as Shewanella putrefaciens (8.16%) and one was identified as V. parahaemolyticus (2.0%). Just one isolate was not identified by this technique (2.0%). The pyrH sequencing confirmed 75% of the proteomic technique results. MALDI-TOF MS is an excellent option for characterization of bacterial species, as it is efficient, fast and easy to apply. In addition, our study confirms its high specificity and sensitivity in these marine bacteria identification.(AU)

Espécies de Vibrio são ubiquitárias em ambientes aquáticos, incluindo habitats costeiros e marinhos. A espécie Vibrio alginolyticus é oportunista para peixes, crustáceos e moluscos e a sua identificação através de testes bioquímicos pode ter a qualidade prejudicada devido às suas exigências nutricionais. O presente estudo utilizou Espectrometria de Massa por Tempo de Vôo de Ionização/Desorção por Laser Assistida por Matriz (MALDI-TOF MS) para identificar diferentes espécies de Vibrio provenientes de mexilhões (Perna perna) coletados em diferentes locais ao longo da costa do Rio de Janeiro. O gene rpoA foi utilizado como um marcador gênero-específico de Vibrio spp. sendo positivo em todos os 209 isolados. MALDI-TOF MS confirmou 87,75% de V. alginolyticus quando comparados com os resultados dos testes bioquímicos. Quatro isolados foram identificados como Shewanella putrefaciens (8,16%), um como V. parahaemolyticus (2,0%) e apenas um (2,0%) não foi identificado pela técnica proteômica. E o sequenciamento do pyrH confirmou 75% dos resultados da técnica proteomica. MALDI-TOF MS tem sido considerada uma excelente opção para a caracterização bacteriana, por ser eficiente, rápida, de fácil aplicação e este estudo confirmou a sua elevada especificidade e sensibilidade na identificação de bactérias marinhas.(AU)

Prevalência e susceptibilidade antimicrobiana de sorotipos de Salmonella spp. isolados de frangos vivos e carcaças no estado do Rio de Janeiro / Prevalence and antimicrobial susceptibility of Salmonella spp. serotypes in broiler chickens and carcasses in the State of Rio de Janeiro, Brazil

A presença de Salmonella spp. em produtos de origem avícola e seus subprodutos se mostra um grande desafio para a produção comercial. Dados de prevalência, dos sorotipos circulantes e do perfil de susceptibilidade antimicrobiana de cepas de Salmonella spp. no Estado do Rio de Janeiro são escassos. Portanto, objetivou-se detectar a presença Salmonella spp. em frangos vivos e carcaças em matadouros do Estados do Rio de Janeiro, identificar os sorotipos e avaliar a susceptibilidade antimicrobiana dessas cepas para fluoroquinolonas e betalactâmicos. Foram coletadas 60 amostras cloacais de frangos vivos e 60 amostras de carcaça de seis matadouros sob Inspeção Estadual (SIE). Os isolados foram sorotipificados e testados frente a oito antimicrobianos: enrofloxacina, ciprofloxacina, norfloxacina, cefalotina, ceftiofur, cefotaxima, amoxicilina/ácido clavulânico e ampicilina pelo método de difusão em disco. Os resultados mostraram uma prevalência de Salmonella spp. de 1,66% (1/60) em amostras de suabe de cloaca e de 26,66% (16/60) em carcaças. Em amostras de suabe de cloaca, somente o sorotipo Senftenberg (1,66%) foi isolado. No total, foram isolados sete sorotipos diferentes nas carcaças: Senftenberg (15%) o mais frequente, seguido por Mbandaka (8,3%), Schwarzengrund (3,3%), Cerro (3,3%), Ohio (3,3%), Minnesota (1,66%) e Tennessee (1,66%). Em relação à susceptibilidade antimicrobiana, 29 (87,87%) isolados foram sensíveis a todos os antimicrobianos testados e 4 (12,12%) isolados foram resistentes a pelo menos três antimicrobianos betalactâmicos ou mais. Não foi observada resistência às fluoroquinolonas. Os resultados encontrados demonstram uma prevalência de Salmonella spp. acima da esperada em matadouros do Estado do Rio de Janeiro, além da presença de vários sorotipos de Salmonella spp. A resistência encontrada para betalactâmicos alerta para a disseminação dessas cepas pela cadeia alimentar.(AU)

The presence of Salmonella spp. in poultry products and their by-products is a major challenge for commercial production. Data about the prevalence, the circulating serotypes and the antimicrobial susceptibility profile of Salmonella spp. strains in the State of Rio de Janeiro are scarce. Therefore, the aim of this study was to detect the presence of Salmonella spp. in live chickens and carcasses in slaughterhouses of the State of Rio de Janeiro, to identify the serotypes and to evaluate the antimicrobial susceptibility of these strains for fluoroquinolones and beta-lactams. Sixty cloacal swabs samples from broiler chickens and sixty samples of carcasses from six slaughterhouses under State Inspection were collected. The isolates were serotyped and resistance was tested to eight antimicrobials: enrofloxacin, ciprofloxacin, norfloxacin, cephalothin, ceftiofur, cefotaxime, amoxicillin/clavulanic acid and ampicillin by disc diffusion method. The results showed a prevalence of Salmonella spp. of 1.66% (1/60) in cloacal swabs samples and 26.66% (16/60) in carcasses. In cloacal swabs sample only Senftenberg (1.66%) serotype was isolated. In total, seven different serotypes were obtained from carcasses: Senftenberg (15%), followed by Mbandaka (8.3%), Schwarzengrund (3.3%), Cerro (3.3%), Ohio (3.3%), Minnesota (1.66%) and Tennessee (1.66%). Regarding antimicrobial susceptibility, 29 (87.87%) isolates were sensitive to all antimicrobials tested and 4 (12.12%) isolates were resistant to three or more beta-lactams antimicrobials. No susceptibility to fluoroquinolones was observed. These results showed a prevalence of Salmonella spp. higher than expected in slaughterhouses in the State of Rio de Janeiro, besides the presence of several serotypes of Salmonella spp. The resistance found for beta-lactams alerts to the spread of these strains through the food chain.(AU)

Genetically distinct lineages of Salmonella Typhimurium ST313 and ST19 are present in Brazil.

In sub-Saharan Africa, two genetically distinct lineages of multi-drug resistant non-typhoidal Salmonella (NTS) serovar Typhimurium sequence type 313 (ST313) are known to cause invasive disease among people. S. Typhimurium ST313 has evolved to become more human-adapted and is commonly isolated from systemic sites (eg., blood) from febrile patients in sub-Saharan Africa. Epidemiological studies indicate that S. Typhimurium is frequently isolated from systemic sites from human patients in Brazil, however, it is currently unknown if this pathogen has also evolved to become more invasive and human-adapted in this country. Here we determined genotypic and phenotypic divergence among clinical S. Typhimurium strains isolated from systemic and non-systemic sites from human patients in Brazil. We report that a subset (8/38, 20%) of epidemiologically diverse human clinical strains of S. Typhimurium recovered from systemic sites in Brazil show significantly higher intra-macrophage survival, indicating that this subset is likely more invasive. Using the whole genome sequencing and phylogenetic approaches, we identified S. Typhimurium ST313-lineage in Brazil that is genetically and phenotypically distinct from the known African ST313-lineages. We also report the identification of S. Typhimurium ST19-lineage in Brazil that is evolving similar to ST313 lineages from Africa but is genetically and phenotypically distinct from ST19-lineage commonly associated with the gastrointestinal disease worldwide. The identification of new S. Typhimurium ST313 and ST19 lineages responsible for human illnesses in Brazil warrants further epidemiological investigations to determine the incidence and spread of a genetically divergent population of this important human pathogen.

Draft Genome Sequences of 11 Salmonella enterica Serovar Typhimurium Strains Isolated from Human Systemic and Nonsystemic Sites in Brazil.

Salmonella enterica serovar Typhimurium strains isolated from systemic sites outside sub-Saharan Africa have been rarely sequenced. Here, we report the draft genome sequences of S Typhimurium sequence type 19 (ST19) (n = 9), ST1649 (n = 1), and ST313 (n = 1) strains isolated from human systemic (e.g., blood) and nonsystemic (e.g., stool and wounds) sites in Brazil.

Draft Genome Sequences of Salmonella enterica subsp. enterica Serovar Typhimurium Strains Isolated from Chicken and Swine Carcasses in Two Distinct Geographical Regions from Rio de Janeiro State, Brazil.

Salmonella enterica subsp. enterica serovar Typhimurium is a surveyed worldwide serotype with well-characterized genomes for several different strains. In Brazil, very few studies have submitted whole-genome sequences to GenBank. This genome may be useful to analyze the genetic mechanisms comparable to those of other related studies conducted in Brazil and globally.

Caracterização molecular de Listeria monocytogenes oriundas de cortes cárneos bovinos e de abatedouros frigoríficos de bovinos localizados no Distrito Federal, Brasil / Molecular characterization of Listeria monocytogenes from beef samples and cattle slaughterhouses located in the Federal District, Brazil

Este trabalho teve como objetivo realizar a detecção de cepas de Listeria monocytogenes de cortes cárneos bovinos bem como no ambiente de abatedouros frigoríficos localizados no Distrito Federal, promover a sorotipificação pela reação em cadeia da polimerase (PCR), realizar antibiograma e submeter às cepas à eletroforese de campo pulsado (Pulsed-field gel electrophoresis - PFGE). Foram analisados um total de 125 cortes cárneos bovinos, 45 amostras de swabs de carcaças e 43 amostras de swabs em que foram detectados 13 cepas de Listeria monocytogenes, sendo 11 em cortes cárneos bovinos e 2 swabs de ambiente em um abatedouro frigorifico. Não foram isoladas cepas de swabs de carcaça. Dentre as 13 cepas de Listeria monocytogenes foram encontradas seis cepas do sorotipo 4b, cinco do sorotipo 1/2c e duas cepas do sorotipo 1/2a. Dentre as 11 cepas de L. monocytogenes encontradas em cortes cárneos bovino, uma (9,1%) cepa apresentou resistência a eritromicina, outra (9,1%) cepa a gentamicina e outra a ciprofloxacina (9,1%) e todas as cepas (100%) apresentaram resistência ao Ác. Nalidíxico. Das duas (2) cepas oriundas de ralos de abatedouro frigorífico, todas (100%) apresentaram resistência ao Ác. Nalidíxico e a sulfonamidas. A análise por eletroforese de campo pulsante (PFGE) demonstrou 13 diferentes pulsotipos, em que foram agrupados em 3 diferentes grupos clonais, que coincidentemente se correlacionavam com os 3 diferentes sorotipos encontrados sugerindo uma ampla disseminação desses perfis no Distrito Federal.(AU)

The aim of the study was the analysis of Listeria monocytogenes strains in beef samples as well as slaughterhouse environment, located in the Federal District, promote serotyping by polymerase chain reaction (PCR), perform antibiotic susceptibility and submit the strains to Pulsed-field gel electrophoresis (PFGE). A total of 125 beef samples were analyzed, 45 samples of carcasses swabs and 43 swab samples. It detected 13 strains of Listeria monocytogenes, 11 in beef samples. and 2 in slaughterhouse environment. No carcass swabs strains were isolated. Among the 13 strains of L. monocytogenes six strains of serotype 4b were found, five serotype 1/2c and two strains of serotype 1/2a. Among the 11 strains of L. monocytogenes found in beef, one (9.1%) strain showed resistance to erythromycin, one (9.1%) strain to gentamicin, one to ciprofloxacin (9.1%) and all strains (100%) were resistant to nalidixic acid. The two strains coming from the slaughterhouse drains, all (100%) were resistant to nalidixic acid and Sulfonamides. The analysis by pulsed field gel electrophoresis (PFGE) showed 13 different pulsotypes; they were grouped into three different clonal groups, coincidentally correlated with the three different serotypes found, what suggests a widespread dissemination of these profiles in the Federal District, Brazil.(AU)

Lethal dose and clinical signs of Aeromonas hydrophila in Arapaima gigas (Arapaimidae), the giant fish from Amazon.

Aeromonas hydrophila is causing substantial economic losses in world aquaculture. This study determined the tolerance limit (LD50-96h) of A. hydrophila in Arapaima gigas, and also investigated the clinical signs after intradermal inoculation. Arapaima gigas fingerlings were inoculated intraperitoneally with 0 (control), 1.0×10(5), 1.0×10(6), 1.0×10(7), 1.0×10(9) and 1.0×10(10)CFU/mL of A. hydrophila for the determination of LD50-96h, which was 1.8×10(8)CFU/mL. In another trial with intradermal inoculation of 1.8×10(8)CFU/mL A. hydrophila, there was a 91.6% of mortality between 8 and 23h, and several clinical signs were found. As follows: depigmentation in the tegument, lesions in the tail and fins, loss of balance, reduction of respiratory movements, hemorrhagic foci, necrotic hemorrhages in the kidney, liver and swim bladder, splenomegaly, ascites in the abdominal cavity and hyperemia, enlargement of the gall bladder, among other clinical signs observed. The results showed that A. gigas has a relative tolerance to A. hydrophila when compared to other Neotropical fish species.

A temporal study of Salmonella enterica serotypes from broiler farms in Brazil.

The present study analyzes the characteristics of Salmonella spp. from broiler chicken farms in Brazil. In total, 82 Salmonella spp. strains were characterized by serotyping, determining susceptibility to antimicrobials, and using pulsed-field gel electrophoresis (PFGE). Fifteen Salmonella serotypes were identified, among which Minnesota (40.24%), Infantis (14.63%), Heidelberg (7.31%), Senftenberg (6.09%), and Mbandaka (6.09%) were the most frequent. Salmonella Minnesota occurred mostly in the state of Mato Grosso do Sul and in one of the broiler companies surveyed. Approximately 60% of the strains were resistant to at least one of the antimicrobials tested. From these isolates, 17.07% were resistant to only one antimicrobial (tetracycline or streptomycin), and 9.75% were resistant to 3 or more antimicrobial classes. Thirteen resistance profiles were characterized, the most frequent of which were the resistance to tetracycline (15.85%); to the combination of trimethroprim with sulfamethoxazole, and tetracycline (10.97%); and to the combination of streptomycin and tetracycline (9.75%). Multiple correspondence analysis revealed that susceptibility or resistance of the analyzed strains and also particular Salmonella serotypes were associated with broiler-producing companies where the samples were collected. Strains presented high intraserotype genetic variability, as shown by the 64 PFGE profiles, suggesting the existence of several contamination sources in the surveyed farms.

Antimicrobial susceptibility and serovars of salmonella circulating in commercial poultry carcasses and poultry products in Brazil.

Antimicrobial resistance was evaluated for 12,582 strains of Salmonella isolated by public and private laboratories from commercial poultry carcasses and poultry products from different regions in Brazil between 2007 and 2011. These isolates were submitted unsolicited to the National Reference Laboratory for Bacterial Enteroinfections, Oswaldo Cruz Institute, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, for conclusive antigenic characterization. The prevalent Salmonella enterica serogroups were O:4 (B) (28.9%), O:9 (D1) (22.3%), O:21 (L) (15.9%), and O:7 (C1) (14.3%); the most prevalent serovars were Enteritidis, Minnesota, Typhimurium, Schwarzengrund, and Mbandaka. The antimicrobial susceptibility profiles of 1,234 strains indicated that 54.5% were resistant to up to eight antimicrobial drugs. Multidrug resistance (three or more classes) was observed in 16.4% of the strains, with 190 distinct patterns. The results showed increased resistance to ampicillin (12.4 to 18.9%), tetracycline (≈ 15.2 to ≈ 18.9%), and gentamicin (f7.0 to ≈ 9.6%) during the study period. Decreased resistance to nitrofurans (61.9 to 9.2%), quinolones (44.4 to 15.5%), and folate inhibitors (11.7 to 7.2%) was observed. Resistance to third-generation cephalosporins was detected in 5.5% of the strains throughout the study period; resistance to fluoroquinolones has been observed in 0.3% of the strains since 2009. These findings highlight the importance of surveillance along the food chain for detection of altered patterns among foodborne zoonotic bacteria that are important to public health.

Effects of gamma irradiation on the viability and phenotypic characteristics of Salmonella Enteritidis inoculated into specific-pathogen-free eggs.

The goal of this study was to determine the effects of various levels of gamma irradiation on the phenotypic characteristics of 20 strains of Salmonella Enteritidis inoculated separately into specific-pathogen-free shell eggs. Bacterial strains were inoculated into egg yolks and exposed to (60)Co radiation at doses of 0.49 to 5.0 kGy. The eggs were maintained at 25°C and analyzed for the presence of Salmonella on days 1, 2, 4, and 7, and the recovered Salmonella isolates were characterized biochemically. All strains were resistant to doses of 0.49, 0.54, 0.59, 0.8, and 1 kGy; colony counts were ≥10(5) CFU/ml of egg yolk except for one strain, which was detected at 96 h and at 7 days after irradiation at 1 kGy, with a population reduction of 2 log CFU/ml. For the other evaluated doses, 12 strains (60.0%) were resistant at 1.5 kGy and 7 strains (35.0%) were resistant at 3.0 kGy. Among all analyzed strains, 5.0 kGy was more effective for reducing and/or eliminating the inoculated bacteria; only two (10%) strains were resistant to this level of irradiation. Salmonella colony counts were significantly reduced (P < 0.01) with increasing doses from the day 1 to 7 of observation, when microbial growth peaked. Loss of mobility, lactose fermentation, citrate utilization, and hydrogen sulfide production occurred in some strains after irradiation independent of dose and postirradiation storage time. Increases in antibiotic susceptibility also occurred: seven strains became sensitive to ß-lactams, two strains became sensitive to antifolates, and one strain each became sensitive to fluoroquinolone, phenicol, nitrofurans, tetracyclines, and aminoglycosides. The results indicate that up to 5.0 kGy of radiation applied to shell eggs inoculated with Salmonella Enteritidis at 4 log CFU per egg is not sufficient for complete elimination of this pathogen from this food matrix.

Kanagawa-negative, tdh- and trh-positive Vibrio parahaemolyticus isolated from fresh oysters marketed in Fortaleza, Brazil.

Between October 2008 and June 2009, 15 samples of 10 live oysters each (Crassostrea rhizophorae) measuring 8.31-10.71 cm were purchased from a restaurant on the seashore of Fortaleza, Brazil. The Vibrio count ranged from 75 (estimated) to 43,500 CFU/g. Fourteen species were identified among the 56 isolated Vibrio strains, with V. parahaemolyticus as the most prevalent. Two of the 17 V. parahaemolyticus strains were urease-positive and tdh- and trh-positive on multiplex PCR, but neither produced ß-hemolysis halos in Wagatsuma agar. Thus, fresh oysters served in natura in Fortaleza, Brazil, were found to contain Vibrio strains known to cause gastroenteritis in humans.

Antimicrobial susceptibility of Escherichia coli isolated from shrimp (Litopenaeus vannamei) and pond environment in northeastern Brazil.

This study aimed to test the susceptibility of Escherichia coli strains isolated from the water, bottom sediments and individuals cultivated in shrimp farm ponds, to antibiotics belonging to different families, namely B-Lactams: Imipenem (IPM; 10 micro g), Ampicillin (AMP; 10 micro g), Cephalothin (CEP; 30 micro g), Cefoxitin (FOX; 30 micro g), Ceftriaxone (CRO; 30 micro g); Tetracycline: Tetracycline (TCY; 30 micro g); Aminoglycosides: Gentamicin (GEN; 10 micro g), Amikacin (AMK; 30 micro g); Chloramphenicol: Chloramphenicol (CHO; 30 micro g); Fluoroquinolones: Ciprofloxacin (CIP; 5 micro g); Nitrofurans: Nitrofurantoin (NIT; 300 micro g); Sulfonamides: Trimethoprim-Sulfamethoxazole (SXT; 30 micro g); Quilononas: Nalidixic Acid (NAL; 30 micro g). In the laboratory, the method of dissemination (Test Kirby-Bauer) was performed in order to fulfill the antibiogram tests. The results showed high indices of resistance to Imipenem, Cephalothin and Ampicillin. Chloramphenicol, Nitrofurantoin, Cefoxitin, Ceftiaxone and Ciprofloxacin have displayed the highest index of sensitive strains. The antibiotic resistance index (ARI) and the multiple resistance index (MAR) varied within the ranges of 0.068-0.077 and 0.15-0.39, respectively. More than 90.5% of strains of Escherichia coli showed a variety of resistance profiles to the tested antibiotics. The high indices of resistance may be a consequence of indiscriminate use of antibiotics, but also the transfer of resistance through mobile genetic elements found in shrimp farms.

Enteropatógenos bacterianos em peixes criados em uma estação de reciclagem de nutrientes e no ecossistema relacionado / Bacterial enteropathogens from fishes of a nutrient recycle system and its ecosystem

Avaliou-se a presença de enteropatógenos bacterianos em 72 amostras obtidas a partir de peixes criados em sistema de reciclagem de nutrientes, em estação experimental, no município de Petrópolis, RJ. Paralelamente, foram obtidas amostras do lodo utilizado como adubo orgânico e da cama de aves localizada na área interna dos tanques criatórios. A metodologia empregada incluiu o pré-enriquecimento em Caldo Lactosado e Agua Peptonada Tamponada, seguido de enriquecimento em Agua Peptonada Alcalina (pH 8,4-8,6) e subseqüente semeadura em Agar GSP para o isolamento de Aeromonas spp. e Plesiomonas shigelloides. Para os demais microrganismos, alíquotas de 1ml foram inoculadas nos meios de enriquecimento Caldo Rappaport-Vassiliadis e Caldo Tetrationato de Kauffmann com posterior semeadura em Agar Entérico Hektoen e Agar Salmonella-Shigella. Com a finalidade de monitorar o índice de coliformes fecais, visando conhecer a qualidade da água para este sistema, paralelamente à coleta de peixes foram avaliadas amostras de água dos tanques criatórios e de macrófitas. No cômputo geral foram isoladas 116 cepas de enteropatógenos bacterianos, destacando-se Aeromonas spp (67,2 por cento) com 9 espécies (A. veronii biogrupo sobria, A. hydrophila, A. sobria, A. trota, A.eucrenophila, A. veronii biog. veronii, A. media, A. caviae e A jandaei) e Aeromonas spp., seguido de Edwardsiella tarda (16,4 por cento), Plesiomonas shigelloides (12,9 por cento) e Salmonella (3,4 por cento). A análise da qualidade da água empregada no sistema revelou, de um modo geral, índices mais elevados de coliformes fecais nos tanques dos peixes (>1800/100 ml).

The presence of bacterial enteropathogens from fishes of a nutrient recycle system from a Experimental Station in Petropolis, RJ, was evaluated in 72 samples from april 2000 to july 2001 Simultaneously was collected the mud used as organic manure and poultry beds localized next to the tanks. The isolation procedures included preenrichment in Peptone Water followed by enrichment with alcaline Peptone Water (pH 8.4-8.6), and streaked onto GSP Agar for the isolation of Aeromonas spp. and Plesiomonas shigelloides. For enteropathogenic bacteria of the Enterobacteriaceae family, 1ml samples were transferred for enrichment in Rappaport- Vassiladis broth and Kauffmann Tetrathionate Broth followed by streak onto Hektoen Enteric Agar and Salmonella-Shigella Agar. Simultaneously at each visit samples of water from fish and macrophyte tanks were collected for monitoring faecal coliforms (MPN) using A1 medium. Among the 116 isolates, Aeromonas spp. were the most frequent (67.2 percent) with 9 species (A. veronii, biogroup sobria, A. hidrophila, A. sobria, A. trota, A. eucrenophila, A. veronii biog. veronii, A. media, A. cavia and A. jandaei), followed by Edwardsiella tarda (16.4 percent), Plesiomonas shigelloides (12.9 percent) and Salmonella spp. (3.4 percent). The NMP of fecal coliforms showed higher values in the fish tanks (>1800/100ml).

Vibrio spp. isolados de mamíferos marinhos capturados na região litorânea do Sudeste ao Sul do Brasil / Vibrio spp. isolated from marine mammals captured in coastal regions from southwestern to southern Brazil

Avaliou-se a incidência de Vibrio spp. a partir de lesões superficiais em mamíferos marinhos encalhados ou capturados em redes de pesca nas regiões litorâneas do Sudeste (Rio de Janeiro) e Sul (RS) do Brasil. Foram coletadas 198 amostras, pelas instituições de pesquisa DEENSP, GEMARS e Ceclimar, as quais foram enviadas ao Labent/IOC/FIOCruz, onde foram submetidas ao enriquecimento em Agua Peptonada Alcalina (APA) adicionada de 1 por cento e 3 por cento de NaCl e in-cubadas a 37°C por 18-24 horas. Em seqüência foram semeadas em meio Agar Tiossulfato Citrato Bile Sacarose (TCBS) e as colônias suspeitas submetidas à caracterização bioquímica. Foram isoladas 108 cepas bacterianas, destacando-se Vibrio alginolyticus, V. parahaemolyticus, V. vulnificus e V. fluvialis como os principais patógenos isolados. Os resultados obtidos apontam para a necessidade de implementar atividades de vigilância e monitorização bacteriológica, particularmente de espécies selvagens, e reforçar os programas de proteção ambiental em casos de mamíferos marinhos ameaçados de extinção.

In the present investigation was evaluated the incidence of Vibrio spp. from superficial lesions at marine mammals beached or captured by fishing net in the southwestern (RJ) and southern (RS) coastal regions of Brazil. One hundred and ninety eight swabs were collected by DEENSP, GEMARS and Ceclimar institutes and sent to Labent/IOC/FIOCruz where the samples were submitted to enrichment in Alkaline Peptone Water (APW) added with 1 percent and 3 percent of sodium chloride (NaCl) incubated at 37°C for 18-24 hours. After the samples were streaked onto Thiossulfate Citrate Bile Sucrose Agar (TCBS), the suspected colonies were submitted to biochemical characterization. The results showed 108 strains, and Vibrio alginolyticus, V. parahaemolyticus, V. vulnificus and V. fluvialis were the main pathogens isolated. These results appoint the importance of surveillance and microbiological monitoring accomplishment and reinforcement of environmental protective programs applied to marine mammals endangered with extinction.

Aeromonas spp. isolated from oysters (Crassostrea rhizophorea) from a natural oyster bed, Ceará, Brazil.

Between April and October 2002, thirty fortnightly collections of oysters (Crassostrea rhizophorea) from a natural oyster bed at the Cocó River estuary in the Sabiaguaba region (Fortaleza, Ceará, Brazil) were carried out, aiming to isolate Aeromonas spp. strains. Oyster samples were submitted to the direct plating (DP) and the presence/absence (P/A) methods. Aeromonas were identified in 15 (50%) samples analyzed by the DP method and in 13 (43%) analyzed by the P/A method. A. caviae, A. eucrenophila, A. media, A. sobria, A. trota, A. veronii bv. sobria, A. veronii bv. veronii and Aeromonas sp. were isolated. The predominant species was A. veronii (both biovars), which was identified in 13 (43%) samples, followed by A. media in 11 (37%) and A. caviae in seven (23%). From the 59 strains identified, 28 (48%) presented resistance to at least one of the eight antibiotics tested.