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Detecção e identificação rápidas dos principais contaminantes microbiológicos em fármacos por espetroscopia de infra-vermelho
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Resumo(s)
A espetroscopia de infravermelho com transformada de Fourier com refletância total atenuada (FTIR-ATR) combinada com a análise multivariada de dados tem vindo a ser proposta como um método para a discriminação de bactérias patogénicas a diferentes níveis taxonómicos (p.e., espécie, sub-espécie e serotipo). O controlo microbiológico na indústria farmacêutica é um fator extremamente importante para garantir a segurança e eficácia de um medicamento. Durante a análise microbiológica de um produto, pode observar-se crescimento de microrganismos provenientes de várias fontes e é necessário assegurar a qualidade do produto fazendo a sua deteção e identificação. O principal objetivo deste trabalho é avaliar o potencial da espetroscopia FTIR-ATR combinada com técnicas quimiométricas para diminuir o tempo de identificação de microrganismos em medicamentos (formas sólidas). Esta metodologia foi testada com Escherichia coli, Staphylococcus aureus e Pseudomonas aeruginosa. Foram utilizados 52 isolados destas bactérias recolhidos do ambiente e de fármacos não estéreis. Estes foram identificados pelo método VITEK implementado na Generis Farmacêutica SA e em seguida analisados por FTIR-ATR. Após o tratamento espectral foram construidos modelos de análise de componentes principais (PCA) e mínimos quadrados parciais de tipologia discriminante (PLS-DA). O método FTIR-ATR permitiu distinguir corretamente bactérias gram-negativas de gram-positivas, assim como discriminar espécies do género Staphylococcus. Quanto ao género Pseudomonas, apesar de se ter considerado um número muito reduzido de isolados, foi possível identificar o correto agrupamento de cada espécie nos modelos PCA e de agrupamento hierárquico. Relativamente ao género Escherichia, não se verificou a presença de microrganismos correspondentes. Dos dois grupos de microrganismos em estudo (Staphylococcus e Pseudomonas) foi possível observar agrupamentos correspondentes aos principais contaminantes patogénicos. Este resultado confirma o potencial do método FTIR-ATR neste campo de utilização mostrando a sua aplicabilidade num laboratório microbiológico de um laboratório farmacêutico.
Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy combined with multivariate data analysis has been proposed as a taxonomic method used to classify and distinguish pathogenic bacteria at multiple levels (for example, species, subspecies and serotype). The microbial contamination control in the pharmaceutical industry is a key factor to be considered in order to assure the safety and efficacy of a drug. During microbiological analysis of a drug, the growth of several microorganisms can be observed. Therefore it is crucial their identification and classification to safeguard the quality of the product. The aim of this work is to propose and describe a fastest method based on FTIR spectroscopy and chemometric techniques to be used in the microbiological analysis of a drug (solid form). This methodology was applied to distinguish different microorganisms, namely Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The fifty-two bacterial isolates used in this methodology were collected from the environment and from the microbiological analysis of non-sterile drugs. They were identified according to the reference method (VITEK) used in Generis Farmacêutica SA, and the spectral analysis of each isolate was performed by ATR-FTIR spectroscopy. PCA and PLS-DA models were correspondingly constructed, and the two methods were compared. The results obtained revealed that both methods are in agreement. The FTIR method correctly distinguished gram-negative from gram-positive bacteria. It was also able to distinguish species belonging to the Staphylococcus genus. Regarding the Pseudomonas genus although it was considered a reduced number of samples, it was possible to identified accurately group each species when performing the PCA and HCA analysis. Concerning the Escherichia genus, no microorganisms were found. For the two groups of microorganisms considered in this study, well-defined clusters of the main pathogens were identified: S.aureus and P.aerugionosa. Overall, the results of this study demonstrate that ATR-FTIR spectroscopy combined with chemometrics serves as a valid method to be considered in a microbial laboratory of a pharmaceutical company.
Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy combined with multivariate data analysis has been proposed as a taxonomic method used to classify and distinguish pathogenic bacteria at multiple levels (for example, species, subspecies and serotype). The microbial contamination control in the pharmaceutical industry is a key factor to be considered in order to assure the safety and efficacy of a drug. During microbiological analysis of a drug, the growth of several microorganisms can be observed. Therefore it is crucial their identification and classification to safeguard the quality of the product. The aim of this work is to propose and describe a fastest method based on FTIR spectroscopy and chemometric techniques to be used in the microbiological analysis of a drug (solid form). This methodology was applied to distinguish different microorganisms, namely Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. The fifty-two bacterial isolates used in this methodology were collected from the environment and from the microbiological analysis of non-sterile drugs. They were identified according to the reference method (VITEK) used in Generis Farmacêutica SA, and the spectral analysis of each isolate was performed by ATR-FTIR spectroscopy. PCA and PLS-DA models were correspondingly constructed, and the two methods were compared. The results obtained revealed that both methods are in agreement. The FTIR method correctly distinguished gram-negative from gram-positive bacteria. It was also able to distinguish species belonging to the Staphylococcus genus. Regarding the Pseudomonas genus although it was considered a reduced number of samples, it was possible to identified accurately group each species when performing the PCA and HCA analysis. Concerning the Escherichia genus, no microorganisms were found. For the two groups of microorganisms considered in this study, well-defined clusters of the main pathogens were identified: S.aureus and P.aerugionosa. Overall, the results of this study demonstrate that ATR-FTIR spectroscopy combined with chemometrics serves as a valid method to be considered in a microbial laboratory of a pharmaceutical company.
Descrição
Tese de mestrado, Engenharia Farmacêutica, Universidade de Lisboa, Faculdade de Farmácia, 2017
Palavras-chave
FTIR-ATR contaminação microbiológica Staphylococcus VITEK análise multivariada Teses de mestrado - 2017