Caracterização genética de factores de virulência e análise de perfis proteicos de estirpes de Enterococcus spp. isoladas de gaivotas (Larus cachinnans)
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2014-03-06
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A crescente detecção de bactérias resistentes a antibiótcos conduziu ao uso
prudente destes fármacos em medicina humana e veterinária, e à criação de uma rede de
sistemas para a sua vigilância. As bactérias comensais Enterococcus spp. são muitas
vezes eleitas como “indicadoras de resistência”, uma vez que estão presentes na
microflora intestinal animal, e por isso sujeitas à acção dos antibióticos. Estas bactérias
constituem um reservatório de genes, capazes de serem transferidos a outras bactérias
comensais ou mesmo patogénicas.
A interacção das bactérias com o hospedeiro é muitas vezes mediada pela
secreção de factores de virulência. As bactérias desenvolveram mecanismos para a
secreção destes factores para o interior das células do hospedeiro, de forma a potenciar a
colonização bacteriana. Estes conferem uma vantagem selectiva às bactérias que os
possuem.
Compreender os mecanismos de patogenicidade das bactérias é fundamental
para investigar doenças infecciosas, uma vez que, estes, muitas vezes estão pouco
evidenciados. A avaliação dos perfis proteicos de bactérias em situações de stress pode
representar uma abordagem integrada para o desenvolvimento de novas estratégias
terapêuticas e agentes antimicrobianos.
Este estudo teve como objectivos a caracterização, através de PCR com recurso
a primers específicos, de 12 genes codificadores de factores de virulência em 63 estirpes
de enterococos isolados de amostras fecais de gaivotas (Larus cachinnans) e a análise
dos perfis proteicos monodimensionais de 29 destes isolados. Foi ainda analisada a
produção de gelatinase e actividade hemolítica dos isolados.
O gene cylLL foi encontrado em 41 dos isolados, seguido do gene cpd, presente
em 39. Os genes gelE e cylM foram encontrados em 25 isolados, o gene hyl em 22, os
genes esp e ace foram detectados em 19, o gene cylB foi encontrado em 10 e o gene
cylA estava presente em apenas 5 estirpes. Nenhum isolado apresentou os genes agg, fsr
e cylLS. Apenas 4 isolados não apresentaram qualquer gene codificante de factor de
virulência. Foi detectado um número máximo de 8 genes em dois isolados (1 isolado
Enterococcus spp. apresentou os genes gelE, hyl, cpd, ace, cylA, cylB, cylLL e cylM e o
outro isolado pertencente à espécie E. faecium apresentou os genes gelE, hyl, esp, ace,
cylA, cylB, cylLL e cylM).
Nenhum dos isolados apresentou actividade β-hemolítica, sendo que também
nenhum apresentou a totalidade do operão cyl. Relativamente à produção de gelatinase,
foi observado que a grande maioria dos isolados apresentou actividade gelatinase
positiva. Nem todos os isolados onde foi detectado o gene gelE foram capazes de
degradar a gelatina.
Foram obtidos padrões proteicos através de electroforese monodimensional onde
foi possível distinguir bandas proteicas características das espécies estudadas. Foi
observada um elevado polimorfismo, sendo que os perfis proteicos de isolados
pertencentes a uma dada espécie eram muito similares entre si. Foi, no entanto, possível
determinar algumas bandas apenas presentes num dos isolados.
A análise proteómica de bactérias portadoras de genes de resistência a
antibióticos e de virulência é muito importante para a identificação de proteínas, com
vista à construção de mapas proteicos para comparação de proteomas e ao
desenvolvimento de novos antibióticos para combate às infecções multirresistentes,
actualmente de difícil terapêutica.
Complementarmente a monitorização da presença de genes codificantes de
factores de virulência, assim como da correlação destes com a multi-resistência a
antibióticos em estirpes de enterococos é importante para a análise dos padrões de
disseminação destes determinantes. Isto é particularmente importante em animais
migratórios, como as gaivotas, uma vez que ao percorrerem longas distâncias e entrarem
em contacto com humanos e outros animais, funcionam como um veículo de
transmissão destes genes.
The increasing detection of antibiotic resistant bacteria has led to the prudent use of these drugs in medicine and veterinary, and to the creation of a network for its surveillance. The commensal bacteria of the genus Enterococcus spp. are often chosen as resistance indicators, since they are present in the animal intestinal microflora, and therefore are subject to the action of antibiotics. These bacteria constitute a gene reservoir, capable of being transferred to other commensal and also pathogenic bacteria. The bacteria-host interaction is often mediated by the secretion of virulence factors. The bacteria have developed mechanisms for the secretion of these factors on to the interior of the host cells, in a way to expand bacterial colonization. These factors give a selective advantage to the bacteria that possess them. Understanding the mechanisms of bacterial pathogenicity is fundamental to further investigate infeccious diseases, since these mechanism are, on many cases, poorly understood. The analysis of protein profiles of bacteria in stress situations can represent an integrated approach for the development of new therapeutic strategies and antimicrobial agents. The goals of the present study were the characterization of 12 virulence factor genes, using PCR with specific primers, in 63 isolates of Enterococcus spp., obtained from fecal samples of seagulls (Larus cachinnans), and the analysis of the monodimensional protein profiles of 29 of these isolates. It was also analyzed the production of gelatinase and the haemolytic activity of the isolates. The cylLL gene was found in 41 of the isolates, followed by the cpd gene, present in 39. The gelE and cylM genes were found in 25 of the isolates, the hyl gene was found in 22, the esp e ace genes were detected in 19, the cylB gene was founf in 10 and the cylA gene was present in only 5 of the cases. None of the isolated presented the agg, fsr and cylLS genes. Only 4 isolates didn’t present any virulence factor gene. It was detected a maximum number of 8 genes in two isolates (1 Enterococcus spp. presented the gelE, hyl, cpd, ace, cylA, cylB, cylLL and cylM genes, and the other isolated of the E. faecium species presented the gelE, hyl, esp, ace, cylA, cylB, cylLL and cylM genes). None of the isolates displayed β-haemolytic activity or had the totality of the cyl operon present. In relation to the production of gelatinase, it was observed that the great majority of the isolates presented positive gelatinase activity. Some of the isolates where the gelE gene was detected weren’t capable of degrading gelatin. Throught proteomic analysis, reproducible monodimensional protein profiles were obtained, where it was possible to distinguish protein bands characteristic of the species studied. It was observed a great polymorphism of bands, and the protein profiles of isolates of a certain species were very similar between themselves. It was also possible to pinpoint some bands present in only one isolate. The proteomic analysis of bacteria carrying resistance and virulence genes, is very important for the identification of proteins, to later build protein maps for proteome comparison, and also for the development of new antibiotics for treatment of multiresistant infections, currently which have problematic therapeutics. Complementarily, the monitorization of the presence of virulence factor genes, as well as the correlation between these and antibiotic multi-resistance in enterococci isolates it’s important for the determination of the dissemination patterns of these determinants. This is particularly important in migratory animals, as in the case of seagulls, since they travel long distances and contact humans and other animals during migration, functioning as a vehicle of transmission of these genes.
The increasing detection of antibiotic resistant bacteria has led to the prudent use of these drugs in medicine and veterinary, and to the creation of a network for its surveillance. The commensal bacteria of the genus Enterococcus spp. are often chosen as resistance indicators, since they are present in the animal intestinal microflora, and therefore are subject to the action of antibiotics. These bacteria constitute a gene reservoir, capable of being transferred to other commensal and also pathogenic bacteria. The bacteria-host interaction is often mediated by the secretion of virulence factors. The bacteria have developed mechanisms for the secretion of these factors on to the interior of the host cells, in a way to expand bacterial colonization. These factors give a selective advantage to the bacteria that possess them. Understanding the mechanisms of bacterial pathogenicity is fundamental to further investigate infeccious diseases, since these mechanism are, on many cases, poorly understood. The analysis of protein profiles of bacteria in stress situations can represent an integrated approach for the development of new therapeutic strategies and antimicrobial agents. The goals of the present study were the characterization of 12 virulence factor genes, using PCR with specific primers, in 63 isolates of Enterococcus spp., obtained from fecal samples of seagulls (Larus cachinnans), and the analysis of the monodimensional protein profiles of 29 of these isolates. It was also analyzed the production of gelatinase and the haemolytic activity of the isolates. The cylLL gene was found in 41 of the isolates, followed by the cpd gene, present in 39. The gelE and cylM genes were found in 25 of the isolates, the hyl gene was found in 22, the esp e ace genes were detected in 19, the cylB gene was founf in 10 and the cylA gene was present in only 5 of the cases. None of the isolated presented the agg, fsr and cylLS genes. Only 4 isolates didn’t present any virulence factor gene. It was detected a maximum number of 8 genes in two isolates (1 Enterococcus spp. presented the gelE, hyl, cpd, ace, cylA, cylB, cylLL and cylM genes, and the other isolated of the E. faecium species presented the gelE, hyl, esp, ace, cylA, cylB, cylLL and cylM genes). None of the isolates displayed β-haemolytic activity or had the totality of the cyl operon present. In relation to the production of gelatinase, it was observed that the great majority of the isolates presented positive gelatinase activity. Some of the isolates where the gelE gene was detected weren’t capable of degrading gelatin. Throught proteomic analysis, reproducible monodimensional protein profiles were obtained, where it was possible to distinguish protein bands characteristic of the species studied. It was observed a great polymorphism of bands, and the protein profiles of isolates of a certain species were very similar between themselves. It was also possible to pinpoint some bands present in only one isolate. The proteomic analysis of bacteria carrying resistance and virulence genes, is very important for the identification of proteins, to later build protein maps for proteome comparison, and also for the development of new antibiotics for treatment of multiresistant infections, currently which have problematic therapeutics. Complementarily, the monitorization of the presence of virulence factor genes, as well as the correlation between these and antibiotic multi-resistance in enterococci isolates it’s important for the determination of the dissemination patterns of these determinants. This is particularly important in migratory animals, as in the case of seagulls, since they travel long distances and contact humans and other animals during migration, functioning as a vehicle of transmission of these genes.
Descrição
Dissertação de Mestrado em Genética Molecular, Comparativa e Tecnológica
Palavras-chave
Gaivota , Enterococcus , Antibiótico , Electroforese (Monodimensional) , Fatores de virulência