Proteogenómica de isolados de Enterococcus spp. e Escherichia coli com a utilização do MALDI-TOF MS
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2016-01-26
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Os Enterococcus spp. e Escherichia coli são microrganismos comensais do trato
gastrintestinal da maioria dos animais e humanos, permitindo realizar estudos genéticos sobre
a ocorrência e disseminação da resistência aos antibióticos. Estas bactérias apresentam,
também, diferenças significativas no seu proteoma, representando bons organismos modelo
para a caracterização bacteriana por novas técnicas proteómicas, como o MALDI-TOF MS.
Estes dois aspetos estão relacionados com os objetivos deste trabalho: estudar a taxa de
resistência a antibióticos em isolados fecais de Enterococcus spp. de aves selvagens do
Arquipélago dos Açores e caracterizar, por meio de MALDI-TOF MS, isolados de E. coli e
enterococos com a mesma proveniência, a nível proteómico.
A resistência aos antibióticos é um problema emergente em saúde pública pelo que, a
avaliação da suscetibilidade aos antibióticos em “bactérias indicadoras”, atua como forma de
controlo da disseminação. As bactérias deste estudo são reservatórios de genes de resistência
que podem ser transmitidos para outras bactérias patogénicas ou comensais e, assim,
representar um problema à escala mundial. A emergência de estirpes de enterococos
resistentes à vancomicina (VRE) realça a inexistência de barreiras entre hospitais, população e animais. Os estudos sobre a incidência de enterococos resistentes em animais selvagens são
escassos, pelo que, a sua propagação oriunda de outros biossistemas é ainda pouco
compreendida. Porém, é reconhecido que os animais selvagens, incluindo as aves, albergam
bactérias que atuam como reservatórios de genes de resistência passíveis de ser disseminados.
A prevalência e os mecanismos implicados na resistência aos antibióticos foram
avaliados em 138 isolados Enterococcus spp. (59 Enterococcus faecalis, 40 E. faecium, 27 E.
durans e 12 E. hirae), de um total de 218 amostras fecais de aves dos Açores. Encontraram-se
percentagens elevadas de resistência nos enterococos para a tetraciclina (32,6%) e a
ciprofloxacina (19,6%), sendo que 46,4% dos isolados apresentaram resistência a pelo menos
um antibiótico. Pela técnica de PCR com primers específicos e corrida eletroforética dos
amplicões obtidos em gel de agarose, a presença dos genes erm(B), tet(M) e/ou tet(L), e
van(A) foi confirmada, nos isolados resistentes à eritromicina, tetraciclina e vancomicina,
respetivamente. Vários estudos estão de acordo com os resultados obtidos neste trabalho,
realizados noutras aves selvagens, como gaivotas e pombas. A informação conseguida é importante para fomentar o conhecimento sobre a disseminação de bactérias resistentes no ecossistema selvagem e as possíveis implicações resultantes da transferência para outros
animais ou humanos.
A técnica “Matrix Assisted Laser Desorption Ionization Time of Flight Mass
Spectrometry” (MALDI-TOF MS) é um método expedito e preciso na identificação de
bactérias intactas, em comparação com os métodos convencionais de microbiologia e/ou
biologia molecular. O “fingerprinting” de proteínas por MALDI pode, também, fornecer uma
solução para a caracterização das resistências a antibióticos presentes nas bactérias. A
caracterização e distinção de espécies bacterianas por esta técnica tem uma potencial
aplicação em laboratórios de diagnóstico.
Para a análise proteómica, via MALDI-TOF MS, proteínas solúveis foram extraídas
de células bacterianas intactas, provenientes de 60 isolados de Enterococcus spp. e 60
isolados de Escherichia coli, por um método expedito. Desde o crescimento da bactéria,
passando pela espetrometria de massa, até à análise bioinformática dos dados obtidos por
MALDI-TOF MS, foi possível comparar 1200 espetros de massa. No total, foram obtidos 215
picos distintos, entre eles o pico de massa m/z 4428 ± 3, encontrado exclusivamente em
isolados de enterococos, e outros dois picos (m/z 5379 ± 3 e 6253 ± 3) detetados apenas em
espetros dos isolados de E. coli, sugerindo que estes picos possam representar biomarcadores
específicos destes géneros bacterianos, respetivamente.
Este estudo foi o primeiro a fazer uma separação clara com recurso ao MALDI entre
isolados de espécies diferentes do género enterococos com origem animal. Tal foi alcançado
com o processamento e análise estatística, via R e MassUp. Este trabalho provou a correta
separação dos isolados de espécies diferentes, atráves da análise de componentes principais
(PCA), clustering e medidas estatísticas.
Os resultados da análise proteómica neste estudo vêm reforçar a capacidade da
espectrometria de massa, com ionização assistida por laser, na caracterização bacteriana por
célula intacta, tendo detetado picos de massa específicos dos dois géneros estudados e picos
de massa caracterizadores das diferentes espécies do género enterococos. No futuro, é
passível que, com uma optimização do protocolo para a obtenção de proteínas intracelulares e
o desenvolvimento das ferramentas bioinformáticas, ocorra a deteção de picos de massa característicos de resistências antimicrobianas.
Enterococcus spp. and Escherichia coli are commensal microorganisms of the gastrointestinal tract of most animals and humans, enabling genetic studies on the occurrence and spread of antibiotic resistance. These bacteria also show significant differences on their proteomes, representing good model organisms for bacterial characterization by new proteomic techniques such as MALDI-TOF MS. These two aspects are related with the objectives of this work: study the rate of antibiotic resistance in fecal isolates of Enterococcus spp. from wild birds of Azores Archipelago and characterize at the proteomic level isolates of E. coli and enterococci from the same origin, by MALDI-TOF MS. Antibiotic resistance is an emerging problem in public health and therefore, the evaluation of the susceptibility to antibiotics in "indicator bacteria" acts as a mean of controlling this spread. Bacteria of this study are reservoirs of resistance genes that can be transmitted to other pathogenic bacteria and thus represents a worldwide problem. The emergence of vancomycin-resistant enterococci strains (VRE) highlights the lack of barriers between hospitals, people and animals. Studies on resistant enterococci incidence in wild animals are scarce, so its propagation originating from others biosystems is still poorly understood. However, it's recognized that wildlife animals, including birds, act as reservoirs of resistance genes wich may be disseminated into the environment. The prevalence and mechanisms involved in antibiotic resistance were evaluated in 138 Enterococcus spp. isolates (59 Enterococcus faecalis, 40 E. faecium, 27 E. durans, 12 E. hirae), from a total of 218 fecal samples from azorean birds. High percentages of resistance were found in enterococci resistant to tetracycline (32.6%) and ciprofloxacin (19.6%), whereas 46.4% of the isolates were resistant to at least one antibiotic. Using PCR with specific primers and amplicons obtained by electrophoretic run in agarose gel, the presence of erm(B), tet(M) and/or tet(L), and van(A) genes were confirmed in isolates resistant to erythromycin, tetracycline and vancomycin, respectively. Several studies conducted in other wild birds such as gulls and pigeons, are in agreement with the results obtained in this work. The information gained is important to raise awareness of the spread of resistant bacteria in the wild ecosystem and the implications resulting from the transfer to other animals or humans. The Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) is a speedy and accurate method for identification of intact bacteria compared with convetional microbiology and/or molecular biology. This protein fingerprinting by MALDI can also provide a solution to the caracterization of antibiotic resistance present in bacteria. The distinguishing and characterization of bacterial species by this technique are potentialy applicable in diagnostic laboratories. For the proteomic analysis via MALDI-TOF MS, soluble proteins were extracted from intact bacterial cells, from 60 isolates of Enterococcus spp. and 60 isolates of Escherichia coli by an expedient method. Since the growth of bacteria, throug mass spectrometry, to bioinformatic analysis of the data obtained by MALDI-TOF MS spectra it was possible to compare 1200 mass spectra. A total of 215 distinct peaks were obtained, including the mass peak m/z 4428 ± 3, exclusively found in isolates of enterococci and two other peaks (m/z 5379 ± 3 e 6253 ± 3) only detected in the spectra of E. coli isolates, suggesting that these peaks can represent specific biomarkers to these bacterial genera, respectively. This was the first study that made a clear separation, by MALDI, between isolates from different species of the genus enterococci from animal source. This was achieved with processing and statistical analysis, via R and MassUp. This work proved the correct separation of isolates of different species through principal component analysis (PCA), clustering and statistical measures. The results of this proteomic analysis confirmed the ability of the mass spectrometryassisted laser ionization for characterizing whole bacterial cells, sensing specific mass peaks of the two kinds of genera studied and mass peaks that characterize the different species of enterococci. In the future, it is likely that with an optimized protocol for obtaining intracellular proteins and the development of bioinformatic tools, the detection of characteristic mass peaks associated to antimicrobial resistance will occur.
Enterococcus spp. and Escherichia coli are commensal microorganisms of the gastrointestinal tract of most animals and humans, enabling genetic studies on the occurrence and spread of antibiotic resistance. These bacteria also show significant differences on their proteomes, representing good model organisms for bacterial characterization by new proteomic techniques such as MALDI-TOF MS. These two aspects are related with the objectives of this work: study the rate of antibiotic resistance in fecal isolates of Enterococcus spp. from wild birds of Azores Archipelago and characterize at the proteomic level isolates of E. coli and enterococci from the same origin, by MALDI-TOF MS. Antibiotic resistance is an emerging problem in public health and therefore, the evaluation of the susceptibility to antibiotics in "indicator bacteria" acts as a mean of controlling this spread. Bacteria of this study are reservoirs of resistance genes that can be transmitted to other pathogenic bacteria and thus represents a worldwide problem. The emergence of vancomycin-resistant enterococci strains (VRE) highlights the lack of barriers between hospitals, people and animals. Studies on resistant enterococci incidence in wild animals are scarce, so its propagation originating from others biosystems is still poorly understood. However, it's recognized that wildlife animals, including birds, act as reservoirs of resistance genes wich may be disseminated into the environment. The prevalence and mechanisms involved in antibiotic resistance were evaluated in 138 Enterococcus spp. isolates (59 Enterococcus faecalis, 40 E. faecium, 27 E. durans, 12 E. hirae), from a total of 218 fecal samples from azorean birds. High percentages of resistance were found in enterococci resistant to tetracycline (32.6%) and ciprofloxacin (19.6%), whereas 46.4% of the isolates were resistant to at least one antibiotic. Using PCR with specific primers and amplicons obtained by electrophoretic run in agarose gel, the presence of erm(B), tet(M) and/or tet(L), and van(A) genes were confirmed in isolates resistant to erythromycin, tetracycline and vancomycin, respectively. Several studies conducted in other wild birds such as gulls and pigeons, are in agreement with the results obtained in this work. The information gained is important to raise awareness of the spread of resistant bacteria in the wild ecosystem and the implications resulting from the transfer to other animals or humans. The Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) is a speedy and accurate method for identification of intact bacteria compared with convetional microbiology and/or molecular biology. This protein fingerprinting by MALDI can also provide a solution to the caracterization of antibiotic resistance present in bacteria. The distinguishing and characterization of bacterial species by this technique are potentialy applicable in diagnostic laboratories. For the proteomic analysis via MALDI-TOF MS, soluble proteins were extracted from intact bacterial cells, from 60 isolates of Enterococcus spp. and 60 isolates of Escherichia coli by an expedient method. Since the growth of bacteria, throug mass spectrometry, to bioinformatic analysis of the data obtained by MALDI-TOF MS spectra it was possible to compare 1200 mass spectra. A total of 215 distinct peaks were obtained, including the mass peak m/z 4428 ± 3, exclusively found in isolates of enterococci and two other peaks (m/z 5379 ± 3 e 6253 ± 3) only detected in the spectra of E. coli isolates, suggesting that these peaks can represent specific biomarkers to these bacterial genera, respectively. This was the first study that made a clear separation, by MALDI, between isolates from different species of the genus enterococci from animal source. This was achieved with processing and statistical analysis, via R and MassUp. This work proved the correct separation of isolates of different species through principal component analysis (PCA), clustering and statistical measures. The results of this proteomic analysis confirmed the ability of the mass spectrometryassisted laser ionization for characterizing whole bacterial cells, sensing specific mass peaks of the two kinds of genera studied and mass peaks that characterize the different species of enterococci. In the future, it is likely that with an optimized protocol for obtaining intracellular proteins and the development of bioinformatic tools, the detection of characteristic mass peaks associated to antimicrobial resistance will occur.
Descrição
Dissertação de Mestrado em Genética Molecular Comparativa e Tecnológica
Palavras-chave
Microrganismos , Resistência a antibióticos , Espectrometria de massas por ionização e dessorção a laser assistida por matriz (MALDI-TOF/MS)