Characteristics of virulence, resistance and genetic diversity of strains of Salmonella Infantis isolated from broiler chicken in Brazil / Características de virulência, resistência e diversidade genética de estirpes de Salmonella Infantis isoladas de frangos de corte no Brasil

Salmonella Infantis is frequently associated with human infections worldwide and is transmitted by consumption of contaminated foods, particularly those of animal origin, especially the chicken meat. We aimed to evaluate virulence characteristics, antimicrobial resistance and the genetic similarity of 51 strains of S. Infantis isolated from samples of poultry origin. The strains were isolated from 2009 to 2010 in a company with full cycle of broiler's production in the state of São Paulo, Brazil. The antimicrobial susceptibility test was performed and, by PCR, we evaluated the presence of the genes lpfA (hem-adhesion), agfA (hem-biofilm) and sefA (hem-adhesion) and resistance genes to beta-lactams (blaTEM, blaSHV, bla CTX-M and blaAmpC ). The phylogenetic relationship was determined by RAPD-PCR method. Among the drugs tested, the highest percentages of resistance were to amoxicillin (35.3%) and to sulfonamide (15.7%). Eleven antimicrobial resistance patterns were identified (A1 to A11), none of them presented a multiresistance profile (> 3 antimicrobials classes). There was 100% of positivity for the agfA gene, 92.2% for the lpfA gene, and no strain presented the sefA gene. Most of the isolates showed similarities in virulence potential, since they were simultaneously positive for two studied genes, agfA and lpfA (92.2%, 47/51). Of the 18 (35.3%) strains resistant to antimicrobials of the β-lactam class, 10 (55.5%) were positive to blaAmpC gene, five (27.8%) for blaCTX-M , two (11.1%) to blaSHV and no strain presented the blaTEM gene. The phylogenetic evaluation has shown the presence of five clusters (A, B, C, D and E) with similarity greatSalmonella Infantis is frequently associated with human infections worldwide and is transmitted by consumption of contaminated foods, particularly those of animal origin, especially the chicken meat. We aimed to evaluate virulence characteristics, antimicrobial resistance and the genetic similarity of 51 strains of S. Infantis isolated from samples of poultry origin. The strains were isolated from 2009 to 2010 in a company with full cycle of broiler's production in the state of São Paulo, Brazil. The antimicrobial susceptibility test was performed and, by PCR, we evaluated the presence of the genes lpfA (hem-adhesion), agfA (hem-biofilm) and sefA (hem-adhesion) and resistance genes to beta-lactams (blaTEM, blaSHV, bla CTX-M and blaAmpC ). The phylogenetic relationship was determined by RAPD-PCR method. Among the drugs tested, the highest percentages of resistance were to amoxicillin (35.3%) and to sulfonamide (15.7%). Eleven antimicrobial resistance patterns were identified (A1 to A11), none of them presented a multiresistance profile (> 3 antimicrobials classes). There was 100% of positivity for the agfA gene, 92.2% for the lpfA gene, and no strain presented the sefA gene. Most of the isolates showed similarities in virulence potential, since they were simultaneously positive for two studied genes, agfA and lpfA (92.2%, 47/51). Of the 18 (35.3%) strains resistant to antimicrobials of the ß-lactam class, 10 (55.5%) were positive to blaAmpC gene, five (27.8%) for blaCTX-M , two (11.1%) to blaSHV and no strain presented the blaTEM gene. The phylogenetic evaluation has shown the presence of five clusters (A, B, C, D and E) with similarity greater than 80%, and three distinct strains which were not grouped in any cluster. Cluster B grouped 33 strains, all positive for lpfA and agfA genes, from both, the broiler farming facility and the slaughterhouse, persistent throughout all the study period. This cluster also grouped 18 strains clones with genetic similarity greater than 99%, all isolated in the slaughterhouse. The presence of virulence genes associated with persistent strains clones for a long period, warns to the possibility of S. Infantis to form biofilm, and should be constantly monitored in broilers' production chain, in order to know the profile of the strains that may contaminate the final product and evaluate the hazards that represents to public health.er than 80%, and three distinct strains which were not grouped in any cluster. Cluster B grouped 33 strains, all positive for lpfA and agfA genes, from both, the broiler farming facility and the slaughterhouse, persistent throughout all the study period. This cluster also grouped 18 strains clones with genetic similarity greater than 99%, all isolated in the slaughterhouse. The presence of virulence genes associated with persistent strains clones for a long period, warns to the possibility of S. Infantis to form biofilm, and should be constantly monitored in broilers' production chain, in order to know the profile of the strains that may contaminate the final product and evaluate the hazards that represents to public health.(AU)

Salmonella Infantis é frequentemente associada a infecções humanas no mundo todo sendo transmitida pelo consumo de alimentos contaminados, principalmente aqueles de origem animal, com destaque para a carne de frango. Objetivou-se avaliar características de virulência, resistência antimicrobiana e a similaridade genética de 51 estirpes de S. Infantis isoladas em amostras de origem avícola. As estirpes foram isoladas no período de 2009 a 2010 em uma empresa com ciclo completo de produção de frango de corte, localizada no estado de São Paulo, Brasil. Foi realizado o teste de susceptibilidade antimicrobiana e pela técnica de PCR, foi avaliada a presença dos genes lpfA (fímbria-adesão), agfA (fímbria-biofilme) e sefA (fímbria-adesão) e os genes de resistência aos beta-lactâmicos (bla TEM, blaSHV, blaCTX-M e blaAmpC ). A relação filogenética foi determinada pelo método de RAPD-PCR. Dentre as drogas testadas, os maiores percentuais de resistência foram para amoxacilina com 35,3% e sulfonamida com 15,7%. Onze perfis de resistência aos antimicrobianos foram identificados (A1 a A11), sendo que nenhum deles apresentou perfil de multirresistência (>3 classes de antimicrobianos). Houve 100% de positividade para o gene agfA, 92,2% para o gene lpfA e nenhuma estirpe apresentou o gene sefA. A maioria dos isolados apresentaram semelhanças no potencial de virulência, pois foram positivos simultaneamente para dois genes estudados, agfA e lpfA (92,2% - 47/51). Das 18 (35,3%) estirpes resistentes aos antimicrobianos da classe dos ß-lactâmicos, 10 (55,5%) foram positivas para o gene blaAmpC , cinco (27,8%) para blaCTX-M , duas (11,1%) para blaSHV e nenhuma estirpe apresentou o gene bla TEM . A avaliação filogenética demonstrou a presença de cinco clusters (A, B, C, D e E) com similaridade superior a 80%, e três estirpes distintas que não foram agrupadas em nenhum dos clusters. O cluster B agrupou 33 estirpes, todas positivas para os genes lpfA e agfA, provenientes tanto do aviário quanto do matadouro frigorífico, persistentes durante todo o período do estudo. Este cluster ainda agrupou 18 estirpes clones com similaridade genética superior a 99%, todas isoladas no matadouro frigorífico. A presença dos genes de virulência, associada à persistência das estirpes clones durante um longo período do estudo, alertam para a possibilidade de S. Infantis em formar biofilme, devendo ser constantemente monitorada na cadeia de produção avícola, especialmente no ambiente de abate, de forma a conhecer o perfil das estirpes que podem contaminar o produto final e assim avaliar os perigos que representam para a saúde pública.(AU)

Strain variability of foodborne pathogens in microbiological risk assessment - a review / 生物工程学报

Strain variability is one of the most important factors to influence the accuracy of foodborne pathogens risk assessment, such as Listeria monocytogenes, Salmonella spp. Strain-to-strain variation is defined as the inherent differences among identically treated strains of the same microbial species. The differences cannot be eliminated by changing test methods or improving test protocols. This review addresses presently related studies of strain variability. Based on the effect of strain variability on the outcome of risk assessment, we summarize sources of variabilities in food chain, strain phenotypic variabilities and the methods to integrate strain variability in growth and inactivation into predictive modelling, and indicate the inadequacies in the study of strain variability. We suggest further study the mechanism of strain variability, expand the comparison of variability among different sources, and integrate the variability of gene expression, protein and cell metabolism into the predictive modelling.

Características de virulência de Salmonella Heidelberg isoladas na cadeia avícola brasileira / Virulence characteristics of Salmonella Heidelberg isolated in the Brazilian poultry chain

S. Heidelberg é frequentemente isolada em produtos avícolas e associada a infecções alimentares humanas. Objetivou-se determinar a presença de genes de virulência em 62 cepas de S. Heidelberg isoladas na cadeia de produção avícola brasileira e inferir o seu potencial em causar infecções em humanos. Foi avaliado, por PCR, a presença dos genes avrA, invA, lpfA, agfA, sefA, luxS e sodC. Houve 100% de positividade para os genes avrA e invA, 98,4% para agfA, 96,8% para sodC, 87,1% para lpfA, 77,4% para luxS e ausência d o gene sefA. Os genes de virulência encontrados nas amostras alertam para a possível gravidade das infecções causadas por este sorovar. Estes resultados são importantes para maior compreensão dos perfis de patogenicidade de cepas circulantes de S. Heidelberg nos sistemas de produção avícola brasileiros.

Multidrogorresistencia de Salmonella infantis en Perú: un estudio mediante secuenciamiento de nueva generación / Multidrug resistance of Salmonella infantis in Peru: a study through next generation sequencing

RESUMEN Objetivos. Describir los patrones fenotípicos y genotípicos de la resistencia antimicrobiana de Salmonella Infantis en Perú. Materiales y Métodos. Se analizaron 297 cepas de Salmonella sp. remitidas al Instituto Nacional de Salud (INS) en el periodo 2014-2016. Las cepas fueron caracterizadas fenotípicamente mediante pruebas microbiológicas, serológicas y de susceptibilidad antimicrobiana. En base a los patrones de resistencia antimicrobiana se seleccionaron 46 cepas que fueron caracterizadas genéticamente mediante secuenciamiento de nueva generación. Resultados. Se identificaron 193/297 (65,0%) cepas de Salmonella Infantis, de la cuales 143 (74,1%) fueron multidrogorresistentes productoras de betalactamasas de espectro extendido (BLEE). Con el secuenciamiento genómico se evidenció un nuevo perfil para Salmonella Infantis, además, se identificó la presencia de 15 diferentes determinantes genéticos de resistencia a los antimicrobianos codificados en cromosoma bacteriano y cinco codificados en un megaplásmido. Los patrones de resistencia fenotípicos y genotípicos coincidieron, a excepción de la ceftazidima. Asimismo, las 46 cepas presentaron resistencia y/o sensibilidad disminuida a las quinolonas. Conclusiones. Salmonella Infantis se ha convertido en una de las serovariedades más frecuentemente referidas al INS, la cual incluye cepas multidrogoresistentes productoras de BLEE con resistencia a las quinolonas. Finalmente, se reafirma la relevancia del secuenciamiento de nueva generación en la caracterización de nuevas variantes de patógenos de importancia para la salud pública y su uso potencial en los sistemas de vigilancia de resistencia antimicrobiana.

ABSTRACT Objectives. To describe the phenotypic and genotypic patterns of the antimicrobial resistance of Salmonella Infantis in Peru. Materials and Methods. Two hundred and ninety-seven strains of Salmonella sp. submitted to the National Institute of Health (INS, in Spanish) during 2014-2016 were analyzed. The strains were phenotypically characterized by microbiological, serological, and antimicrobial susceptibility tests. Based on antimicrobial resistance patterns, 46 strains were selected and genetically characterized by next generation sequencing. Results. 193/297 (65%) strains of Salmonella Infantis were identified, of which 143 (74.1%) were multidrug-resistant producers of extended spectrum beta-lactamases (ESBL). The genomic sequencing evidenced a new profile for Salmonella Infantis; additionally, it identified the presence of 15 different genetic determinants of antimicrobial resistance coded in bacterial chromosome and five coded in a megaplasmid. The phenotypic and genotypic resistance patterns matched, with the exception of ceftazidime. Moreover, the 46 strains presented resistance and/or decreased sensitivity to quinolones. Conclusions. Salmonella Infantis has become one of the sero-varieties most frequently referred to the INS, which includes ESBLproducing multidrug-resistant strains with resistance to quinolones. Finally, the relevance of next generation sequencing is reasserted in the characterization of new variants of pathogens that are important for public health, and their potential use in antimicrobial resistance surveillance systems.

Caracterización de cepas clínicas y ambientales de Salmonella enterica subsp. enterica serovar Heidelberg aisladas en Chile / Characterization of Salmonella Heidelberg strains isolated in Chile

Background: Salmonella Heidelberg (S. Heidelberg) causes gastroenteritis and sometimes bacteremia and endocarditis. In other countries, this serovar has multidrug resistance including extended-spectrum β-lactamases (ESBLs) and AmpC (β-lactamases (AmpC), associated with the blaCMY-2 gene. In Chile, an outbreak by S. Heidelberg occurred in 2011, the phenotypic and genetic characteristics of Chilean strains are unknown. Aim: To determine the antimicrobial susceptibility, presence of plasmids and virulence factor genes in S. Heidelberg strains isolated in Chile over the period 2006-2011. Material and Methods: In sixty-one S. Heidelberg clinical and environmental strains collected by the Public Health Institute in Chile during 2006-2011, antimicrobial susceptibility, plasmids and virulence factor genes (invA, sifA, pefA, agfA, lpfA and, stkD) were studied. Results: S. Heidelberg had a high susceptibility to sulfamethoxazole-trimethoprim, gentamicin, ceftriaxone, ceftiofur, chloramphenicol, amoxicillin-clavulanic acid and ampicillin. However, 52% had decreased susceptibility to ciprofloxacin and 33% resistance to tetracycline. ESBLs were detected in three strains isolated from blood cultures, environment and human feces. The latter strain was positive for AmpC and blaCMY-2 gene. Fifty three of 61 strains showed one to seven plasmids of 0.8 to approximately 30 kb. Most plasmids were small with sizes between 0.8 and 2 kb. All isolates were positive for all genes except pefA. Conclusions: S. Heidelberg isolated from Chilean samples was susceptible to first-line antimicrobials, except tetracycline and ciprofloxacin. The emergence of strains with ESBLs and AmpC should be a warning. The strains were homogeneous for virulence genes, but heterogeneous in their plasmids.

Survival of Salmonella spp. in minced meat packaged under vacuum and modified atmosphere

Abstract The effect of different modified atmosphere packaging regimes on the behavior of Salmonella spp. on minced meat was studied. Minced meat was experimentally contaminated with a Salmonella spp. cocktail (S. Enteritidis, S. Typhimurium, S. Infantis and S. Arizonae), packaged under vacuum or modified atmosphere with initial headspaces containing 20%O2/50%CO2/30%N2 and 20%O2/30%CO2/50%N2) and stored at 3 ± 1 °C for 12 days. Samples were analyzed for Salmonella spp., viable and lactic acid bacteria count every third day. Salmonella spp. counts decreased during storage in all packaging types, with reductions of about 1.5 log CFU/g. A significant difference (p < 0.01) was noted between Salmonella spp. counts in meat packaged in vacuum and modified atmospheres, although there was no significant difference in Salmonella spp. count between meat packaged in 50%CO2, and meat packaged in 30%CO2. At the end of the study, there were significant differences (p < 0.01; p < 0.05) in total viable and lactic acid bacterial counts between meat packaged in vacuum and modified atmosphere, and the lowest counts were noted in meat packaged in modified atmosphere with 50%CO2.

Rapid detection of food-borne Salmonella contamination using IMBs-qPCR method based on pagC gene

Abstract Detection of Salmonella is very important to minimize the food safety risk. In this study, the recombinant PagC protein and PagC antibody were prepared and coupled with immunomagnetic beads (IMBs) to capture Salmonella cells from pork and milk samples. And then the SYBR Green qualitative PCR was developed to detect the pathogenic Salmonella. The results showed that the PagC polyclonal antiserum is of good specificity and the capture rate of 0.1 mg IMBs for Salmonella tended to be stable at the range of 70-74% corresponding to the concentrations between 101 and 104 CFU/mL. The method developed demonstrated high specificity for the positive Salmonella samples when compared to non-specific DNA samples, such as Escherichia coli, Staphylococcus aureus, Yersinia enterocolitica, and Yersinia pseudotuberculosis. The limit of detection of this assay was 18 CFU/mL. Detection and quantitative enumeration of Salmonella in samples of pork or milk shows good recoveries of 54.34% and 52.07%. In conclusion, the polyclonal antibody of recombinant PagC protein is effective to capture Salmonella from detected samples. The developed pagC antibody IMBs-qPCR method showed efficiency, sensitivity and specificity for 30 Salmonella detection, enabling detection within 10 h, which is a promising rapid method to detect Salmonella in emergency.

Survey on pathogenic Escherichia coli and Salmonella spp. in captive cockatiels (Nymphicus hollandicus)

Abstract We surveyed healthy captive cockatiels (Nymphicus hollandicus) for Escherichia coli and Salmonella spp. Cloacal swabs were collected from 94 cockatiels kept in commercial breeders, private residencies and pet shops in the cities of São Paulo/SP and Niterói/RJ (Brazil). Three strains of E. coli from each individual were tested for the presence of ExPEC-, APEC- and DEC-related genes. We evaluated the blaTEM, blaSHV, blaOXA, blaCMY, blaCTX-M, tetA, tetB, aadA, aphA, strAB, sul1, sul2, sul3, qnrA, qnrD, qnrB, qnrS, oqxAB, aac (6)′-Ib-cr, qepA resistance genes and markers for plasmid incompatibility groups. Salmonella spp. was not detected. E. coli was isolated in 10% of the animals (9/94). Four APEC genes (ironN, ompT, iss and hlyF) were detected in two strains (2/27-7%), and iss (1/27-4%) in one isolate. The highest resistance rates were observed with amoxicillin (22/27-82%), ampicillin (21/27-79%), streptomycin (18/27-67%), tetracycline (11/27-41%). Multiresistance was verified in 59% (16/27) of the isolates. We detected strAB, bla TEM, tetA, tetB, aadA, aphaA, sul1, sul2, sul3 resistance genes and plasmid Inc groups in 20 (74%) of the strains. E. coli isolated from these cockatiels are of epidemiological importance, since these pets could transmit pathogenic and multiresistant microorganisms to humans and other animals.

A comparative survey between non-systemic Salmonella spp. (paratyphoid group) and systemic Salmonella Pullorum and S. Gallinarum with a focus on virulence genes / Uma investigação comparativa entre Salmonella spp. não-sistêmicas (grupo paratifoide) e sistêmicas Salmonella Pullorum e S. Gallinarum com enfoque nos genes de virulência

A comparative survey between non-systemic (paratyphoid Salmonellae) and systemic (S. Pullorum and S. Gallinarum) Salmonella strains was performed to produce a virulence gene profile for differentiation among the groups. The following virulence genes were evaluated: invA, spvC, sefC, pefA, fimY, sopB, sopE1, stn and avrA. There are substantial differences among paratyphoid Salmonellae, S. Pullorum, and S. Gallinarum regarding the genes sefC, spvC, sopE1 and avrA. A higher frequency of sefC, spvC, sopE1 and avrA genes were detected in S. Gallinarum and S. Pullorum when compared with strains from the paratyphoid group of Salmonella. These results may be useful for differentiating among different groups and serotypes.(AU)

Uma investigação comparativa entre amostras de Salmonella não-sistêmicas (grupo paratifoide) e sistêmicas (S. Pullorum and S. Gallinarum) foi desenvolvida para produzir um perfil de genes de virulência para diferenciação entre os grupos. Os seguintes genes de virulência foram avaliados invA, spvC, sefC, pefA, fimY, sopB, sopE1, stn e avrA. Detectou-se uma diferença substancial entre Salmonella do grupo paratifoide, S. Pullorum e S. Gallinarum considerando os genes sefC, spvC, sopE1 e avrA. Os genes sefC, spvC, sopE1 e avrA foram detectados, em maior número, em S. Gallinarum e S. Pullorum quando comparados com as amostras de Salmonella do grupo paratifoide. Estes resultados podem ser úteis para a diferenciação entre os diferentes grupos e sorotipos de Salmonella.(AU)

[Pattern of serotyping and antibiotic resistance of Salmonella in children with diarrhea]

Gastroenteritis due to Salmonella is common in human and considered as a global dilemma of public health. This study was done to determine the Pattern of serotyping and antibiotic resistance of Salmonella in children with diarrhea in Iran. In this laboratory study, 306 stool samples were collected from children with diarrhea in public health centers in Robat-karim, Tehran province, Iran. The specimens were enriched in Selenite F medium and then cultured on Hekton agar. The identification of Salmonella was carried out by conventional method and antimicrobial susceptibility testing was performed according to CLSI procedures. Out of 306 stool samples, 7.2 % were identified as Salmonella species, as follow: 7 Salmonella typhi, 6 Salmonella paratyphi B, 3 Salmonella paratyphi C, 2 Salmonella paratyphi A and 4 samples were not identifiable. There was a significant relation between presence of WBC in fecal and salmonellosis [P<0.05]. In drug sensitivity trends, 92.3% of Salmonella species were sensitive to chloramphenicol, ceftizoxime, Nalidixic acid and Amikacin. This study showed that Salmonella was the cause of children diarrhea in 7.2% in this region

Loop-mediated isothermal amplification assays for screening of bacterial integrons

BACKGROUND: The occurrence and prevalence of integrons in clinical microorganisms and their role played in antimicrobial resistance have been well studied recently. As screening and detection of integrons are concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for integrons detection. RESULTS: In this study, three loop-mediated isothermal amplification (LAMP) assays targeting on class 1, 2 and 3 integrons were implemented and evaluated. Optimization of these detection assays were performed, including studing on the reaction temperature, volume, time, sensitivity and specificity (both primers and targets). Application of the established LAMP assays were further verified on a total of 1082 isolates (previously identified to be 397 integron-positive and 685 integron-negative strains). According to the results, the indispensability of each primer had been confirmed and the optimal reaction temperature, volume and time were found to be 65°C, 45 min and 25 µL, respectively. As application was concerned, 361, 28 and 8 isolates carrying intI1, intI2 and intI3 yielded positive amplicons, respectively. Other 685 integron-negative bacteria were negative for the integron-screening LAMP assays, totaling the detection rate and specificity to be 100%. CONCLUSIONS: The intI1-, intI2- and intI3-LAMP assays established in this study were demonstrated to be the valid and rapid detection methodologies for the screening of bacterial integrons.

Molecular characterization of Salmonella strains in individuals with acute diarrhea syndrome in the State of Sucre, Venezuela / Caracterização molecular de cepas de Salmonella em indivíduos com síndrome da diarreia aguda no Estado de Sucre, Venezuela

INTRODUCTION:In Venezuela, acute diarrheic syndrome (ADS) is a primary cause of morbi-mortality, often involving the Salmonella genus. Salmonella infections are associated with acute gastroenteritis, one of the most common alimentary intoxications, and caused by the consumption of contaminated water and food, especially meat. METHODS: Conventional and molecular methods were used to detect Salmonella strains from 330 fecal samples from individuals of different ages and both sexes with ADS. Polymerase chain reaction (PCR) was used for the molecular characterization of Salmonella, using invA, sefA, and fliC genes for the identification of this genus and the serotypes Enteritidis and Typhimurium, respectively. RESULTS: The highest frequency of individuals with ADS was found in children 0-2 years old (39.4%), and the overall frequency of positive coprocultures was 76.9%. A total of 14 (4.2%) strains were biochemically and immunologically identified as Salmonella enterica subsp. enterica, of which 7 were classified as belonging to the Enteritidis serotype, 4 to the Typhimurium serotype, and 3 to other serotypes. The S. enterica strains were distributed more frequently in the age groups 3-4 and 9-10 years old. CONCLUSIONS: The molecular characterization method used proved to be highly specific for the typing of S. enterica strains using DNA extracted from both the isolated colonies and selective enrichment broths directly inoculated with fecal samples, thus representing a complementary tool for the detection and identification of ADS-causing bacteria.

INTRODUÇÃO: Na Venezuela, síndrome da diarreia aguda (SDA) é a principal causa de mórbi-mortalidade, muitas vezes envolvem o gênero Salmonella. Infecções por Salmonella são associadas com gastroenterite aguda, uma das mais comuns intoxicações alimentares causada pelo consumo de água e alimentos contaminados, principalmente carne. MÉTODOS: Métodos convencionais e moleculares foram usados para detectar cepas de Salmonella em 330 amostras de fezes de indivíduos com SDA de diferentes idades e ambos os sexos. A reação em cadeia da polimerase (PCR) foi utilizada para a caracterização molecular de genes Salmonella invA, sefA e fliC para identificar o gênero e os sorotipos Enteritidis e Typhimurium, respectivamente. RESULTADOS: A maior frequência de indivíduos com SDA foi encontrada em crianças de 0-2 (39,4%) anos, e a frequência total de culturas de fezes positiva foi de 76,9%. Um total de 14 (4,2%) cepas foram bioquímica e imunologicamente identificados como Salmonella enterica subsp. enterica, dos quais 7 foram classificados como pertencentes ao sorotipo Enteritidis, Typhimurium sorotipo 4 e 3 para outros sorotipos. Cepas S. enterica foram distribuídas mais frequentemente em grupos de 3-4 e 9-10 anos de idade. CONCLUSÕES: O método de caracterização molecular usada provou ser altamente específico para tipificar as estirpes dos S. enterica usando tanto DNA extraído de colônias isoladas e direta e caldos de enriquecimento seletivo inoculados com amostras fecais, o que representa uma ferramenta complementar para a detecção e identificação de bactérias que causam a SDA.

Dos aislamientos de Salmonella Infantis multirresistentes se comportan como hipoinvasivos pero con elevada proliferación intracelular

En este trabajo se investigó la presencia de determinantes característicos de plásmidos de virulencia en dos aislamientos clínicos de Salmonella Infantis portadores de plásmidos de multirresistencia. Además, se estudió la capacidad de invasión y proliferación en células eucariotas no fagocíticas. Ninguno de los aislamientos de S. Infantis mostró los determinantes genéticos que caracterizan a los plásmidos de virulencia para este género (operón spv). Los ensayos de invasión sobre líneas celulares eucariotas mostraron que los aislamientos de S. Infantis presentan una capacidad de invasión disminuida pero persisten y proliferan en el citoplasma, independientemente de utilizar una línea celular permisiva (HeLa) o no permisiva (NRK) para tal fin. Finalmente, no se observaron indicios microscópicos que podrían hacer sospechar un efecto bactericida de estas líneas celulares sobre los aislamientos estudiados.

Two multidrug-resistant Salmonella Infantis isolates behave like hypo-invasive strains but have high intracellular proliferation. In this work, plasmid-encoded virulence factors in two Salmonella Infantis isolates carrying multiresistance plasmids were investigated. In addition, their invasion and proliferative ability in non-phagocytic cells was studied. None of them showed the typical determinants of virulence plasmids (spv operon). The invasion assays of S. Infantis isolates on eukaryotic cells showed a decreased ability to Invade but they remained and proliferated In the cytoplasm regardless of having used a permissive (HeLa) or non-permissive (NRK) cell line. Finally, there was no microscopic evidence suggesting a bactericidal effect of these eukaryotic cell lines on the Isolates tested.

Serotyping & molecular characterization for study of genetic diversity among seafood associated nontyphoidal Salmonella serovars.

Background & objectives: Infections due to seafood associated Salmonella serovars are great risk to public health. Different phenotypic characteristics have been used previously for epidemiological investigation of Salmonella. Beyond the phenotypic characterization, a reliable genetic level discriminatory method is required. Therefore, this study was attempted to use different phenotypic and molecular fingerprinting methods for investigation of genetic diversity among seafood associated nontyphoidal Salmonella serovars. Methods: Fifty eight seafood associated Salmonella isolates were included in this study. All isolates were serotyped and epidemiological investigation was carried out using molecular fingerprinting methods, random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus sequence based-PCR (ERIC-PCR) along with whole cell protein profiling using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in our study. Results: Among the 58 Salmonella isolates, S. Weltevreden was observed to be the most predominant serovar. Typing of Salmonella serovars using RAPD and ERIC-PCR suggested the existence of a genetic diversity. Though both PCR based techniques were found to have a good discriminatory index, a better discriminatory ability was observed when the results obtained by the two techniques were combined and taken for composite analysis. Protein profiling of whole cells using SDS-PAGE demonstrated the presence of several bands with two bands of sizes 38 kDa and 46 kDa common among all 58 isolates. Interpretation & conclusions: Our study shows that use of protein profiling in combination with established typing methods such as RAPD and ERIC-PCR may provide useful information in typing of non-typhoidal Salmonella isolates associated with seafood and to develop strategies to protect public from Salmonella infections.

Caracterização de beta-lactamases de espectro ampliado (ESBLs), genes de resistência aos antimicrobianos e conteúdo plasmidial em cepas de Escherichia coli e Salmonella spp. não tifóides isoladas do ambiente hospitalar e da comunidade / Characterization of extended spectrum Beta-lactamase (ESBLs), antimicrobial resistance genes, and plasmid content in Escherichia coli and Salmonella spp. isolates recovered from hospital and community

Enterobactérias produtoras de ESBLs são descritas tanto no ambiente hospitalar quanto na comunidade em todo o mundo. No Brasil, esses microrganismos também têm emergido como uma causa importante de infecções, sendo as enzimas CTX-M as prevalentes. O objetivo deste estudo foi analisar diferentes aspectos genotípicos relacionados à expressão da resistência aos antimicrobianos em cepas Escherichia coli e de Salmonella spp, tais como: a diversidade de ESBLs, os genes de resistência aos antimicrobianos e o conteúdo plasmidial. Os aspectos epidemiológicos das cepas produtoras de ESBLs também foram investigados. Foram estudadas 88 cepas de enterobactérias, sendo 43 E. coli e 45 cepas de Salmonella spp., de origem hospitalar e da comunidade (principalmente alimentos), isoladas na cidade do Rio de Janeiro. A expressão de ESBL foi observada em sete cepas de E. coli (7/43, 16,3%) e em uma cepa de Salmonella Typhimurium (1/45, 2,3%) e as enzimas foram identificadas como variantes de CTX-M e SHV-5, respectivamente. Entre as cepas de E. coli, a enzima CTX-M-2 foi a mais frequente (n = 4), sendo detectada em cepas isoladas de swab retal de pacientes hospitalizados, enquanto as enzimas CTX-M-59 (uma variante de CTX-M) (n = 1) e CTX-M-9 (n = 2) foram identificadas em cepas isoladas a partir de espécimes clínicos. Salmonella Typhimurium produtora de SHV-5 foi isolada do ambiente hospitalar (fórmula infantil). As cepas de E. coli produtoras das enzimas CTX-M pertenceram a grupos filogenéticos (A, B1, D) e STs (ST34, ST69, ST101) diferentes, sendo os genes blaCTX-M identificados em plasmídeos com tipo de replicon IncA/C de cerca de 150 kb (blaCTX-M-2, blaCTX-M-9, blaCTX-M-59) ou 80 kb (blaCTX-M-2)...

ESBL-producing Enterobacteriaceae have been described in hospitals and in the community worldwide. In Brazil, ESBL-producing Enterobacteriaceae have also emerged as an important cause of infections, being CTX-M enzymes the most prevalent ESBLs. The objective of this study was to analyze different genotypic aspects related to expression of antimicrobial resistance in isolates of Escherichia coli and Salmonella spp., such as: diversity of ESBLs, antibiotic resistance genes and plasmid content. Epidemiological features of ESBL-producing isolates were also investigated. We studied 88 isolates of enterobacteria, 43 E. coli and 45 Salmonella serotypes of hospital and community (mainly food) origin, isolated in the city of Rio de Janeiro. ESBL expression was observed in seven E. coli isolates (7/43; 16,3%) and in one Salmonella Typhimurium (1/45; 2,3%) and the enzymes identified as CTX-M variants and SHV-5, respectively. Among the E. coli isolates, CTX-M-2 was the most frequent (n=4), being detected in isolates recovered from rectal swabs of hospitalized patients, whereas CTX-M-59 (a CTX-M-2-variant) (n=1) and CTX-M-9 (n=2) were identified in E. coli isolated from clinical specimens. SHV-5-producing S. Typhimurium was isolated from the hospital environment (infant formula). CTX-M-producing E. coli belonged to different phylogenetic groups (A, B1, D) and STs (ST34, ST69, ST101), being blaCTX-M genes were identified in IncA/C plasmids of approximately 150 kb (blaCTX-M-2, blaCTX-M-9, blaCTX-M-59) or 80 kb (blaCTX-M-2)...

Mecanismos moleculares utilizados por Salmonella para causar infecciones del tracto digestivo / Molecular mechanisms used by Salmonella to cause infections in the digestive tract

Salmonella enterica es uno de los principales causantes de gastroenteritis infecciosa en el mundo, debido al consumo de alimentos y aguas contaminadas con esta bacteria. Este grupo de bacterias Gram negativas se caracteriza por tener la capacidad de invadir células eucariontes y sobrevivir en el interior de ellas, evento fundamental para que la bacteria cause una enfermedad tanto localizada como sistémica. Las proteínas de virulencia que este agente utiliza para invadir y sobrevivir en células eucariontes son codificadas por genes presentes en islas de patogenicidad, que corresponden a grandes bloques de material genético integrados en el cromosoma bacteriano y que fueron posiblemente adquiridos a través de transferencia lateral de genes desde otros microorganismos. Estudios recientes han permitido identificar el rol que poseen estas proteínas de virulencia en el proceso infectivo de Salmonella y su impacto en el funcionamiento de la célula eucarionte. De este modo, ha sido posible entender de mejor manera los mecanismos moleculares utilizados para infectar a su hospedero y se han identificado posibles blancos terapéuticos para el tratamiento de los cuadros infecciosos causados por este patógeno.

Salmonella enterica is one of the main ethiological agents of infectious gastroenteritis in the world, due the consumption of food and water contaminated with these bacteria. This group of Gram negative bacterials characterized by its capacity to invade eukaryotic cells and survive inside them, an event that is fundamental for the bacteria to cause a localized as well as a systemic disease. The virulence proteins that this bacterium uses to invade and survive within eukaryotic cells are encoded by genes found in pathogenicity islands, big blocks of genetic material integrated in the bacterial chromosome, that were probably acquired through lateral gene transfer from other microorganisms. Recent studies have identified the role that these virulence proteins play in the infective process of Salmonella, and their impact in the function of the eukaryotic cell. This way, it has been possible to better understand the molecular mechanisms used by Salmonella to infect their hosts, and potential therapeutic targets have been identified to improve the treatment of the infection caused by this pathogen.

Detection, amplification & sequence homology of sodC in clinical isolates of Salmonella sp.

Background & objectives: Periplasmic copper and zinc superoxide dismutase (Cu,Zn-SOD or SodC) is an important component of the antioxidant shield which protects bacteria from the phagocytic oxidative burst. Cu,Zn-SODs protect Gram-negative bacteria against oxygen damage which have also been shown to contribute to the pathogenicity of these bacterial species. We report the presence of SodC in drug resistant Salmonella sp. isolated from patients suffering from enteric fever. Further sodC was amplified, cloned into Escherichia coli and the nucleotide sequence and amino acid sequence homology were compared with the standard strain sSalmonella Typhimurium 14028. Methods: Salmonella enterica serovar Typhi (S. Typhi) and Salmonella enterica serovar Paratyphi (S. Paratyphi) were isolated and identified from blood samples of the patients. The isolates were screened for the presence of Cu, Zn-SOD by PAGE using KCN as inhibitor of Cu,Zn-SOD. The gene (sodC) was amplified by PCR, cloned and sequenced. The nucleotide and amino acid sequences of sodC were compared using CLUSTAL X. Results: SodC was detected in 35 per cent of the Salmonella isolates. Amplification of the genomic DNA of S. Typhi and S. Paratyphi with sodC specific primers resulted in 519 and 515 bp amplicons respectively. Single mutational difference at position 489 was observed between the sodC of S. Typhi and S. Paratyphi while they differed at 6 positions with the sodC of S. Typhimurium 14028. The SodC amino acid sequences of the two isolates were homologous but 3 amino acid difference was observed with that of standard strain S. Typhimurium 14028. Interpretation & conclusions: The presence of SodC in pathogenic bacteria could be a novel candidate as phylogenetic marker.

Plasmid-Encoded Multidrug Resistance: A Case study of Salmonella and Shigella from enteric diarrhea sources among humans

Salmonellosis and shigellosis are signifcant and persistent causes of diarrheal diseases among humans in developing countries. With that in mind, the current study investigates the occurrence of plasmid-encoded multidrug resistances in Salmonella and Shigella from diarrheal cases among humans. The isolates were characterized by serotyping, antimicrobial-susceptibility testing, transfer experiments and curing. The extended spectrum β-lactamase (ESBL) was detected by the double disc diffusion synergy test (DDST). A signifcant number of the plasmid-encoded multidrug resistant (PEMDR) Salmonella and Shigella isolates were found to harbour transferable plasmid genes resistant to antibiotics like ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, ceftriaxone, cefuroxime and to a lesser extent to ciprofoxacin and ofoxacin. The conjugative R-plasmids-encoded extended-spectrum β-lactamase also showed resistances to cephalosporins (ceftriaxone and cefuroxime) and ampicillin. Curing experiments showed chromosomal resistances to varied antibiotics. The fndings confrmed the presence of PEMDR in Salmonella and Shigella strains as a suitable adaptation to a changing antibiotic environment. The results therefore suggest the limited use of the commonly prescribed/or third generation cephalosporins as an empirical treatment of multidrug resistant Salmonella and Shigella because this may affect therapeutic outcomes.

Quantification and molecular characterization of Salmonella isolated from food samples involved in salmonellosis outbreaks in Rio Grande do Sul, Brazil / Quantificação e perfil molecular de Salmonella isolada de alimentos envolvidos em surtos de salmonelose no Rio Grande do Sul

Data concerning the prevalence and populations of Salmonella in foods implicated in outbreaks may be important to the development of quantitative microbial risk assessments of individual food products. In this sense, the objective of the present study was to assess the amount of Salmonella sp. in different foods implicated in foodborne outbreaks in Rio Grande do Sul occurred in 2005 and to characterize the isolated strains using phenotypic and genotypic methods. Nineteen food samples involved in ten foodborne outbreaks occurred in 2005, and positive on Salmonella isolation at the Central Laboratory of the Health Department of the State of Rio Grande do Sul, were included in this study. Food samples were submitted to estimation of Salmonella using the Most Probable Number (MPN) technique. Moreover, one confirmed Salmonella colony of each food sample was serotyped, characterized by its XbaI-macrorestriction profile, and submitted to antimicrobial resistance testing. Foods containing eggs, mayonnaise or chicken were contaminated with Salmonella in eight outbreaks. Higher counts (>10(7) MPN.g-1) of Salmonella were detected mostly in foods containing mayonnaise. The isolation of Salmonella from multiple food items in five outbreaks probably resulted from the cross-contamination, and the high Salmonella counts detected in almost all analyzed samples probably resulted from storing in inadequate temperature. All strains were identified as S. Enteritidis, and presented a unique macrorestriction profile, demonstrating the predominance of one clonal group in foods involved in the salmonellosis outbreaks. A low frequency of antimicrobial resistant S. Enteritidis strains was observed and nalidixic acid was the only resistance marker detected.

Dados sobre a prevalência e a população de Salmonella em alimentos implicados em surtos podem contribuir na condução de análises de risco. Dessa forma, o objetivo desse estudo foi determinar a quantidade de Salmonella sp. presente em alimentos implicados em surtos ocorridos no Rio Grande do Sul em 2005 e caracterizar os isolados por meio de técnicas fenotípicas e genotípicas. Dezenove amostras de alimentos obtidas em dez surtos ocorridos em 2005 e identificadas como positivas para Salmonella no Laboratório Central da Secretaria da Saúde do Estado do Rio Grande do Sul foram incluídas no estudo. A quantificação de Salmonella foi feita pela técnica do Número Mais Provável (NMP). Ao lado disto, uma colônia de Salmonella obtida de cada amostra de alimento foi submetida à sorotipificação, macro-restição com XbaI e determinação de resistência a antimicrobianos. Salmonella esteve presente em alimentos a base de ovos, maionese e frango em oito surtos. As contagens mais elevadas (>10(7) NMP.g-1) foram detectadas principalmente em alimentos contendo maionese. O isolamento de Salmonella de vários alimentos em cinco surtos resultou, provavelmente, da contaminação cruzada, enquanto as elevadas contagens encontradas, em quase todos os alimentos, podem ser explicadas por armazenamento em temperatura inadequada. Todos os isolados foram identificados como S. Enteritidis, e apenas um perfil foi encontrado na macro-restrição, demonstrando a predominância de um grupo clonal desse sorovar nos surtos de salmonelose. Uma baixa freqüência de isolados de S. Enteritidis resistentes a antimicrobianos foi encontrada, sendo a resistência ao ácido nalidíxico o único perfil encontrado.

Caracterización molecular de cepas colombianas de Salmonella spp. a través del RFLP-IS 200m / Molecular characterizacion of Colombian stumps of Salmonella spp. through the RFLP-IS200

Las técnicas moleculares empleadas para el análisis de ADN cromosomal como el RFLP IS200 han demostrado ser eficientes para resolver relaciones filogenéticas y epidemiológicas entre las cepas de Salmonella spp., aisladas de aves y humanos. El presente estudio tuvo como objetivo determinar si la IS200 estaba presente en las cepas de Salmonella aisladas de aves de corral y de humanos para establecer una posible cadena de transmisión entre ambos. El análisis filogenético de las cepas de Salmonella mostró 13 perfiles de IS200 que exhibieron de 1 a 11 copias de la secuencia de inserción, localizadas en un rango entre 13 y 1.71kb. El trabajo permitió concluir que IS200 es un marcador molecular eficiente, sensible y específico, útil para realizar estudios epidemiológicos ya que discriminó entre cepas de diferentes orígenes y comprobó que existe una relación clonal entre las cepas aviares y humanas. Los resultados obtenidos muestran la necesidad de implementar medidas sanitarias que conlleven a que en la producción se minimicen las condiciones que favorezcan la propagación de Salmonella entre las aves