Análise de rota de transmissão de Enterococcus spp. em infecções nosocomiais a partir de alimentos

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Evaluation of the relative expression of genes involved in biofilm formation by Acinetobacter baumannii

Acinetobacter baumannii é um patógeno oportunista, de grande importância no ambiente hospitalar devido à sua capacidade de adquirir mecanismos de resistência, de aderir e formar biofilmes sob diferentes superfícies. Devido a relevância da infecção causadas pelo A. baumannii torna-se fundamental o conhecimento dos mecanismos envolvidos na sua patogenicidade. Sendo assim, o objetivo desse estudo foi realizar a análise transcricional dos genes envolvidos na formação e manutenção do biofilme (bap, abaI, ompA, bfmRS, csuAB, pgaA, pilZ, wspR, eal, eagg, IscRSU e csdA) de duas cepas clínicas de A. baumannii obtidas de hospitais de Porto Alegre, Brasil (AbH e AbC) e uma cepa controle ATCC 19606 (AbA) sob diferentes condições de crescimento. As cepas foram avaliadas quanto a sua capacidade de formar biofilme a 25 °C e 37 °C, crescendo em caldo Luria Bertani (LB), LB acrescido de 1% glicose (LB-G), LB acrescido de 10% sangue de carneiro (LB-SC) e urina pura (UP). A análise transcricional foi realizada por PCR quantitativo em tempo real. A cepas AbH, AbC e AbA foram fortes formadoras de biofilme em LB e LB-G a 25 e 37 °C. Em LB-SC, as cepas AbH e AbA foram fortes formadoras e AbC foi classificada fraca formadora de biofilme. Em UP, a cepa AbH foi moderadamente formadora, AbC e AbA foram fracas formadoras de biofilme. Os genes wspR, pgaA, ompA, bap, abaI de AbA, csdA de Ab He o operon IscRSU foram superexpressos em biofilme; os genes eagg de AbH, pilZ e csuAB foram inibidos e os genes bfmRS, eal, eaggde AbA, csdA de AbA e abaIde AbH não possuíram uma variação significativa na expressão durante a formação do biofilme.Acinetobacter baumannii is an opportunistic pathogen of high importance in the hospital environment due to its ability to develop resistance mechanisms as well as to adhere and form biofilms on different surface types. Due to the high relevance of infections caused by A. baumannii, knowing the mechanisms involved in its pathogenicity is of extreme importance. Thus, we aimed to perform a transcriptional analysis on genes involved in biofilm formation and maintenance (bap, abaI, ompA, bfmRS, csuAB, pgaA, pilZ, wspR, eal, eagg, IscRSU e csdA) of two clinically isolated A. baumannii strains obtained from hospitals in Porto Alegre city, Rio Grande do Sul state, southern Brazil (AbH and AbC), and of the reference strain ATCC 19606 (AbA), cultivated under different growth conditions. Biofilm formation ability was evaluated at 25 and 37 °C using LB broth (LB), LB + 1% glucose (LB-G), pure urine (PU), and LB + 10% sheep blood (LB-SB). Gene transcriptional profiling was performed by real time qPCR. Strains AbH, AbC and AbA were strong biofilm producers in LB and LB-G at 25 and 37 °C. In LB-SB, strains AbH and AbA were strong biofilm producers while AbC was a week biofilm producer. In PU, the strain AbH was a moderate biofilm producer, while strains AbC and AbA were weak biofilm producers. Genes wspR, pgaA, ompA, bap, abaI de AbA; csdA of AbH; and the operon IscRSU were all overexpressed in biofilms. On the other hand, genes eagg of AbH, pilZ and csuAB were suppressed, while genes bfmRS, eal and eagg of AbA; csdA of AbA; and abaI of AbH showed no significant variation in their expression during biofilm formation

Avaliação da Expressão Relativa dos Genes Envolvidos na Formação de Biofilme por Acinetobacter baumannii

Acinetobacter baumannii is an opportunistic pathogen of high importance in the hospital environment due to its ability to develop resistance mechanisms as well as to adhere and form biofilms on different surface types. Due to the high relevance of infections caused by A. baumannii, knowing the mechanisms involved in its pathogenicity is of extreme importance. Thus, we aimed to perform a transcriptional analysis on genes involved in biofilm formation and maintenance (bap, abaI, ompA, bfmRS, csuAB, pgaA, pilZ, wspR, eal, eagg, IscRSU e csdA) of two clinically isolated A. baumannii strains obtained from hospitals in Porto Alegre city, Rio Grande do Sul state, southern Brazil (AbH and AbC), and of the reference strain ATCC 19606 (AbA), cultivated under different growth conditions. Biofilm formation ability was evaluated at 25 and 37 °C using LB broth (LB), LB + 1% glucose (LB-G), pure urine (PU), and LB + 10% sheep blood (LB-SB). Gene transcriptional profiling was performed by real time qPCR. Strains AbH, AbC and AbA were strong biofilm producers in LB and LB-G at 25 and 37 °C. In LB-SB, strains AbH and AbA were strong biofilm producers while AbC was a week biofilm producer. In PU, the strain AbH was a moderate biofilm producer, while strains AbC and AbA were weak biofilm producers. Genes wspR, pgaA, ompA, bap, abaI de AbA; csdA of AbH; and the operon IscRSU were all overexpressed in biofilms. On the other hand, genes eagg of AbH, pilZ and csuAB were suppressed, while genes bfmRS, eal and eagg of AbA; csdA of AbA; and abaI of AbH showed no significant variation in their expression during biofilm formation.Acinetobacter baumannii é um patógeno oportunista, de grande importância no ambiente hospitalar devido à sua capacidade de adquirir mecanismos de resistência, de aderir e formar biofilmes sob diferentes superfícies. Devido a relevância da infecção causadas pelo A. baumannii torna-se fundamental o conhecimento dos mecanismos envolvidos na sua patogenicidade. Sendo assim, o objetivo desse estudo foi realizar a análise transcricional dos genes envolvidos na formação e manutenção do biofilme (bap, abaI, ompA, bfmRS, csuAB, pgaA, pilZ, wspR, eal, eagg, IscRSU e csdA) de duas cepas clínicas de A. baumannii obtidas de hospitais de Porto Alegre, Brasil (AbH e AbC) e uma cepa controle ATCC 19606 (AbA) sob diferentes condições de crescimento. As cepas foram avaliadas quanto a sua capacidade de formar biofilme a 25°C e 37°C, crescendo em caldo Luria Bertani (LB), LB acrescido de 1% glicose (LB-G), LB acrescido de 10% sangue de carneiro (LB-SC) e urina pura (UP). A análise transcricional foi realizada por PCR quantitativo em tempo real. A cepas AbH, AbC e AbA foram fortes formadoras de biofilme em LB e LB-G à 25 e 37°C. Em LB-SC, as cepas AbH e AbA foram fortes formadoras e AbC foi classificada fraca formadora de biofilme. Em UP, a cepa AbH foi moderadamente formadora, AbC e AbA foram fracas formadoras de biofilme. Os genes wspR, pgaA, ompA, bap, abaI de AbA, csdA de AbH e o operon IscRSU foram superexpressos em biofilme; os genes eagg de AbH, pilZ e csuAB foram inibidos e os genes bfmRS, eal, eagg de AbA, csdA de AbA e abaI de AbH não possuíram uma variação significativa na expressão durante a formação do biofilme

Perfil de resistência antimicrobiana e análise de Enterococcus spp. isolados de alimentos em Porto Alegre, RS

Enterococcus spp são microrganismos que colonizam o trato gastrintestinal de humanos. Estes microrganismos podem ser também encontrados em alimentos, onde possuem um papel benéfico durante a maturação de determinados produtos fermentados. Enterococos eram consideradas bactérias de baixa virulência; no entanto, estes microrganismos, recentemente, assumiram uma importância clínica, sendo considerados patógenos oportunistas para humanos. Esta característica dualista do microrganismo se tornou um motivo de discussão constante entre a classe científica no mundo todo. O objetivo desse estudo foi determinar a distribuição e o perfil de resistência antimicrobiana de Enterococcus spp. isolados de diferentes fontes de alimentos da cidade de Porto Alegre, RS, Brasil.Linhagens isoladas de vegetais, carne e produtos lácteos foram submetidas a análises morfológicas, bioquímicas e de identificação molecular utilizando a técnica de PCR e as linhagens positivas foram analisadas quanto à susceptibilidade a 12 antimicrobianos utilizados na clínica ou na pecuária. Ainda, estas amostras foram analisadas quanto à diversidade genotípica pela utilização da técnica de RAPD-PCR. A análise estatística dos perfis de RAPD-PCR obtidos para as amostras verificou uma presença de 42 padrões de banda diferenciados e 6 perfis diferentes de RAPD-PCR. Relacionando os testes fenotípicos clássicos previamente realizados, somente um perfil agrupou todas as linhagens de mesma espécie. Os dados aqui descritos sugerem atenção especial aos microrganismos provenientes de todos os três grupos de alimentos utilizados como fonte de isolamento, principalmente devido à presença de linhagens com altos níveis de resistência a antimicrobianos, incluindo aqueles de maior relevância clínica como agentamicina, estreptomicina, ampicilina e vancomicina. Além disso, análises dos dados de RAPD-PCR demonstraram uma variabilidade genética entre as linhagens de enterococos provenientes de diferentes fontes de alimentos.Enterococcus spp. are commensal microorganisms, which colonize the gastrointestinal tract in humans. These microorganisms are natural microbiotic compounds in foods, playing a beneficial role during the maturation of some fermented products. Enterococci presents, generally, relative low virulence. However, these organisms have recently assumed major importance in clinical microbiology as well. They are considered emerging pathogens for humans, with Enterococcus faecalis causing human enterococcal infections at high rates, and these dualistic characteristics have turned a subject of constant debate worldwide.The aim of the present study was to determine the species distribution and the antimicrobial resistance profiles of enterococci isolated from several food in Porto Alegre, RS, Brazil. Strains isolated from vegetables, meat and dairy products were submitted to morphological, biochemical and molecular identification using PCR technique, and positive strains were submitted to antimicrobial susceptibility tests using twelve clinical and agricultural antimicrobians. Furthermore, we investigated the genotypic diversity of enterococci strains. Randomly amplified polymorphic DNA (RAPD)-PCR was used to study the genetic variability, which distinguished closely related isolates. After numerical analyses of the RAPD-PCR profiles obtained with M13 primer, 42 different patterns were obtained. Six different RAPD profiles were recorded for the enterococcal isolates. According to previous species determination by phenotypical tests, only one cluster associated all strains from the same species. Our data suggest a special attention to strains isolated from all the three groups of food in analysis, especially due to the presence of enterococci resistant to high level and a wide range of clinical antimicrobians thatinclude gentamycin, streptomycin, ampicillin and vancomycin. Results of the present study suggest that there is some genotypic variability within strains of enterococci deriving from several food sources in Southern Brazil

Perfil de resistência antimicrobiana e análise de Enterococcus spp. isolados de alimentos em Porto Alegre, RS

Enterococcus spp são microrganismos que colonizam o trato gastrintestinal de humanos. Estes microrganismos podem ser também encontrados em alimentos, onde possuem um papel benéfico durante a maturação de determinados produtos fermentados. Enterococos eram consideradas bactérias de baixa virulência; no entanto, estes microrganismos, recentemente, assumiram uma importância clínica, sendo considerados patógenos oportunistas para humanos. Esta característica dualista do microrganismo se tornou um motivo de discussão constante entre a classe científica no mundo todo. O objetivo desse estudo foi determinar a distribuição e o perfil de resistência antimicrobiana de Enterococcus spp. isolados de diferentes fontes de alimentos da cidade de Porto Alegre, RS, Brasil.Linhagens isoladas de vegetais, carne e produtos lácteos foram submetidas a análises morfológicas, bioquímicas e de identificação molecular utilizando a técnica de PCR e as linhagens positivas foram analisadas quanto à susceptibilidade a 12 antimicrobianos utilizados na clínica ou na pecuária. Ainda, estas amostras foram analisadas quanto à diversidade genotípica pela utilização da técnica de RAPD-PCR. A análise estatística dos perfis de RAPD-PCR obtidos para as amostras verificou uma presença de 42 padrões de banda diferenciados e 6 perfis diferentes de RAPD-PCR. Relacionando os testes fenotípicos clássicos previamente realizados, somente um perfil agrupou todas as linhagens de mesma espécie. Os dados aqui descritos sugerem atenção especial aos microrganismos provenientes de todos os três grupos de alimentos utilizados como fonte de isolamento, principalmente devido à presença de linhagens com altos níveis de resistência a antimicrobianos, incluindo aqueles de maior relevância clínica como agentamicina, estreptomicina, ampicilina e vancomicina. Além disso, análises dos dados de RAPD-PCR demonstraram uma variabilidade genética entre as linhagens de enterococos provenientes de diferentes fontes de alimentos.Enterococcus spp. are commensal microorganisms, which colonize the gastrointestinal tract in humans. These microorganisms are natural microbiotic compounds in foods, playing a beneficial role during the maturation of some fermented products. Enterococci presents, generally, relative low virulence. However, these organisms have recently assumed major importance in clinical microbiology as well. They are considered emerging pathogens for humans, with Enterococcus faecalis causing human enterococcal infections at high rates, and these dualistic characteristics have turned a subject of constant debate worldwide.The aim of the present study was to determine the species distribution and the antimicrobial resistance profiles of enterococci isolated from several food in Porto Alegre, RS, Brazil. Strains isolated from vegetables, meat and dairy products were submitted to morphological, biochemical and molecular identification using PCR technique, and positive strains were submitted to antimicrobial susceptibility tests using twelve clinical and agricultural antimicrobians. Furthermore, we investigated the genotypic diversity of enterococci strains. Randomly amplified polymorphic DNA (RAPD)-PCR was used to study the genetic variability, which distinguished closely related isolates. After numerical analyses of the RAPD-PCR profiles obtained with M13 primer, 42 different patterns were obtained. Six different RAPD profiles were recorded for the enterococcal isolates. According to previous species determination by phenotypical tests, only one cluster associated all strains from the same species. Our data suggest a special attention to strains isolated from all the three groups of food in analysis, especially due to the presence of enterococci resistant to high level and a wide range of clinical antimicrobians thatinclude gentamycin, streptomycin, ampicillin and vancomycin. Results of the present study suggest that there is some genotypic variability within strains of enterococci deriving from several food sources in Southern Brazil

Caracterização da maquinaria SUF responsável pela formação e associação dos cofatores [Fe-S] em Enterococcus faecalis

Cofatores ferro-enxofre são grupos prostéticos inorgânicos ubíquos e evolutivamente ancestrais, cuja formação é dependente de complexas maquinarias protéicas. Três sistemas de formação distintos já foram determinados, denominados sistemas NIF, ISC e SUF. Apesar de bem descritos em diversos organismos, estas maquinarias são pouco caracterizadas no filo Firmicutes, o qual agrupa diversas bactérias patogênicas, e onde Enterococcus faecalis aparece como um representante clinicamente relevante. O objetivo deste estudo foi identificar a maquinaria biossintética de formação dos cofatores [Fe-S] de E. faecalis mediante análises de bioinformática, determinação das regiões promotoras do operon e de elementos cis-atuantes, padrão de expressão gênica, caracterização bioquímica dos elementos encontrados e comparação entre as maquinarias de associação do cofator [Fe-S] presente em Proteobacteria e Firmicutes através da capacidade de complementação deste sistema nos sistemas ISC e SUF de Azotobacter vinelandii e Escherichia coli, respectivamente. Metodologias de bioinformática permitiram identificar representantes da maquinaria SUF de formação dos cofatores [Fe-S], previamente identificado em Proteobacteria, apresentando os genes sufB, sufC, sufD e sufS e a presença de sufU, o único representante homólogo do sistema ISC, codificando possível proteína arcabouço, no lugar de sufA; da mesma forma, sufE e sufR não foram identificadas. A alta conservação deste sistema foi verificada em Firmicutes através de análises filogenéticas. Análise de sequências primária e estrutural de SufU verificaram um padrão estrutural similar à IscU. Modelagem molecular de SufU de E. faecalis apresentou dados de alta flexibilidade na região do sítio ativo, bem como a presença de região específica em Firmicutes, denominada região Gram-positiva (GPR), possivelmente envolvida em interações com outros fatores e/ou reguladores. SufU e o complexo SufSU são capazes de reconstituir cofactor [4Fe-4S], apresentando-se portanto como a proteína arcabouço do sistema. A enzima SufS purificada apresenta PLP ligado como cofator e atividade de cisteína desulfurase. Esta enzima apresenta um residuo catalítico essencial de cisteína na posição 365 , e necessita SufU como ativador, onde outro residuo de cisteína (128) atua como aceptor do enxofre durante a reação de transpersulfuração. SufC apresenta atividade ATPase, porém em nível reduzido em comparação ao homólogo de E. coli; SufD apresenta alta similaridade com homólogo de proteobactérias. Por outro lado, SufB não apresenta os resíduos de cisteína previamente descritos como importantes na formação dos cofatores [Fe-S] em outros organismos, assim sua função no sistema ainda deve ser determinada. Experimentos in vivo demonstraram a conservação específica de sistemas biossintéticos dos cofatores [Fe-S], onde o operon SUF de E. faecalis não foi capaz de complementar os sistemas ISC de Proteobacteria, porém complementou sistema SUF de E. coli, tornando viáveis mutantes de ambos os operons sufABCDSE e iscRSU-hscBA-fdx.Iron-sulfur clusters are ubiquitous and evolutionary ancient inorganic prosthetic groups, which biosynthesis depends on complex protein machineries. Three distinct assembly systems involved in the maturation of cellular Fe-S proteins have been determined, designated the NIF, ISC and SUF systems. Although well described in several organisms, these machineries are poorly understood in the Firmicutes phylum, which groups several pathological bacteria, where Enterococcus faecalis rises as a clinical relevant representative. The aim of this study was to identify the E. faecalis [Fe-S] cluster biosynthetic machinery through bioinformatics analysis, determination of operon promoter regions and cis-acting elements, relative genetic expression pattern, biochemical characterization of putative elements, and comparison of Proteobacteria and Firmicutes machineries through the ability of complementing Azotobacter vinelandii and Escherichia coli ISC and SUF systems, respectively. Bioinformatics methods enabled us to identify representatives of the SUF machinery for [Fe-S] cluster biosynthesis, previously verified in Proteobacteria showing conserved sufB, sufC, sufD and sufS genes and the presence of sufU, the only ISC homolog representative, coding for putative scaffold protein, instead of sufA; neither sufE nor sufR are present. High conservancy of this system for Firmicutes bacteria was verified through phylogenetic analysis. Primary sequences and structural analysis of the SufU protein demonstrated its structural-like pattern to the scaffold protein IscU. E. faecalis SufU molecular modeling showed high flexibility over the active site regions, and demonstrated the existence of a specific region in Firmicutes, the Gram positive region (GPR), a possible candidate for interaction with other factors and/or regulators. SufU is able to reconstitute a [4Fe-4S] cluster, such as the complex SufSU, arising as the scaffold protein in the system. Purified SufS corresponds to a PLP containing enzyme with cysteine desulfurase activity. It encloses a catalytically essential cysteine residue at position 365, and requires SufU as activator, where another cysteine residue (128) works as a proximal sulfur acceptor site for transpersulfurization reaction. SufC presents ATPase activity, though in a reduced level, when compared to the Escherichia coli homolog; SufD also shares high similarity with proteobacterial SufD. On the other hand, SufB does not present cysteine residues previously described as important involved in the [Fe-S] cluster formation process of other organisms, therefor its function in the system still have to be determined. In vivo experiments enabled us to dfemonstrate the conservancy of specific [Fe-S] cluster biosynthetic systems, where E. faecalis SUF operon was not able to complement Proteobacteria ISC systems, but complemented E. coli SUF system, turning viable mutants of both sufABCDSE and iscRSU-hscBA-fdx operons

Structural studies of the Enterococcus faecalis SufU [Fe-S] cluster protein

Background: Iron-sulfur clusters are ubiquitous and evolutionarily ancient inorganic prosthetic groups, the biosynthesis of which depends on complex protein machineries. Three distinct assembly systems involved in the maturation of cellular Fe-S proteins have been determined, designated the NIF, ISC and SUF systems. Although well described in several organisms, these machineries are poorly understood in Gram-positive bacteria. Within the Firmicutes phylum, the Enterococcus spp. genus have recently assumed importance in clinical microbiology being considered as emerging pathogens for humans, wherein Enterococcus faecalis represents the major species associated with nosocomial infections. The aim of this study was to carry out a phylogenetic analysis in Enterococcus faecalis V583 and a structural and conformational characterisation of it SufU protein. Results: BLAST searches of the Enterococcus genome revealed a series of genes with sequence similarity to the Escherichia coli SUF machinery of [Fe-S] cluster biosynthesis, namely sufB, sufC, sufD and SufS. In addition, the E. coli IscU ortholog SufU was found to be the scaffold protein of Enterococcus spp., containing all features considered essential for its biological activity, including conserved amino acid residues involved in substrate and/or co-factor binding (Cys50,76,138 and Asp52) and, phylogenetic analyses showed a close relationship with orthologues from other Gram-positive bacteria. Molecular dynamics for structural determinations and molecular modeling using E. faecalis SufU primary sequence protein over the PDB:1su0 crystallographic model from Streptococcus pyogenes were carried out with a subsequent 50 ns molecular dynamic trajectory. This presented a stable model, showing secondary structure modifications near the active site and conserved cysteine residues. Molecular modeling using Haemophilus influenzae IscU primary sequence over the PDB:1su0 crystal followed by a MD trajectory was performed to analyse differences in the C-terminus region of Gram-positive SufU and Gram-negative orthologous proteins, in which several modifications in secondary structure were observed. Conclusion: The data describe the identification of the SUF machinery for [Fe-S] cluster biosynthesis present in the Firmicutes genome, showing conserved sufB, sufC, sufD and sufS genes and the presence of the sufU gene coding for scaffold protein, instead of sufA; neither sufE nor sufR are present. Primary sequences and structural analysis of the SufU protein demonstrated its structural-like pattern to the scaffold protein IscU nearby on the ISC machinery. E. faecalis SufU molecular modeling showed high flexibility over the active site regions, and demonstrated the existence of a specific region in Firmicutes denoting the Gram positive region (GPR), suggested asa possible candidate for interaction with other factors and/or regulators

Structural studies of the Enterococcus faecalis SufU [Fe-S] cluster protein

Background: Iron-sulfur clusters are ubiquitous and evolutionarily ancient inorganic prosthetic groups, the biosynthesis of which depends on complex protein machineries. Three distinct assembly systems involved in the maturation of cellular Fe-S proteins have been determined, designated the NIF, ISC and SUF systems. Although well described in several organisms, these machineries are poorly understood in Gram-positive bacteria. Within the Firmicutes phylum, the Enterococcus spp. genus have recently assumed importance in clinical microbiology being considered as emerging pathogens for humans, wherein Enterococcus faecalis represents the major species associated with nosocomial infections. The aim of this study was to carry out a phylogenetic analysis in Enterococcus faecalis V583 and a structural and conformational characterisation of it SufU protein. Results: BLAST searches of the Enterococcus genome revealed a series of genes with sequence similarity to the Escherichia coli SUF machinery of [Fe-S] cluster biosynthesis, namely sufB, sufC, sufD and SufS. In addition, the E. coli IscU ortholog SufU was found to be the scaffold protein of Enterococcus spp., containing all features considered essential for its biological activity, including conserved amino acid residues involved in substrate and/or co-factor binding (Cys50,76,138 and Asp52) and, phylogenetic analyses showed a close relationship with orthologues from other Gram-positive bacteria. Molecular dynamics for structural determinations and molecular modeling using E. faecalis SufU primary sequence protein over the PDB:1su0 crystallographic model from Streptococcus pyogenes were carried out with a subsequent 50 ns molecular dynamic trajectory. This presented a stable model, showing secondary structure modifications near the active site and conserved cysteine residues. Molecular modeling using Haemophilus influenzae IscU primary sequence over the PDB:1su0 crystal followed by a MD trajectory was performed to analyse differences in the C-terminus region of Gram-positive SufU and Gram-negative orthologous proteins, in which several modifications in secondary structure were observed. Conclusion: The data describe the identification of the SUF machinery for [Fe-S] cluster biosynthesis present in the Firmicutes genome, showing conserved sufB, sufC, sufD and sufS genes and the presence of the sufU gene coding for scaffold protein, instead of sufA; neither sufE nor sufR are present. Primary sequences and structural analysis of the SufU protein demonstrated its structural-like pattern to the scaffold protein IscU nearby on the ISC machinery. E. faecalis SufU molecular modeling showed high flexibility over the active site regions, and demonstrated the existence of a specific region in Firmicutes denoting the Gram positive region (GPR), suggested asa possible candidate for interaction with other factors and/or regulators

Análise de rota de transmissão de Enterococcus spp. em infecções nosocomiais a partir de alimentos

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