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Desenvolvimento e otimização de um modelo de infeção em Galleria mellonella para ensaios de patogenicidade
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RESUMO
A larva de Galleria mellonella representa um novo modelo de infeção invertebrado com potencial aplicação em diversas áreas de investigação. Até à data, G. mellonella tem sido usada em estudos de avaliação do potencial de virulência de agentes patogénicos, de interação hospedeiro-agente patogénico e ensaios de farmacotoxicidade. A sua aplicabilidade tem sido demonstrada para alguns dos agentes patogénicos de maior relevância clínica, incluindo Pseudomonas aeruginosa e Staphylococcus aureus.
Os objetivos principais deste trabalho foram a implementação de uma colónia de G. mellonella no GHTM/IHMT-NOVA, e a otimização de protocolos de infeção para ensaios de patogenicidade para quatro agentes patogénicos bacterianos relevantes: P. aeruginosa, S. aureus, Neisseria gonorrhoeae e Escherichia coli.
Adquiriram-se larvas de G. mellonella na última fase larval a uma casa comercial especializada em animais exóticos e a espécie foi confirmada por sequenciação do gene COI. As larvas foram criadas numa sala dedicada no Insectário do IHMT-NOVA, na ausência de luz, a 27-28ºC e a 60-80% de humidade relativa, com uma dieta rica em nutrientes baseada em farinha de milho, levedura de cerveja, farinha de soja, leite em pó, mel, glicerol e cera de abelha. O ciclo de vida de G. mellonella foi acompanhado e a taxa de oviposição foi determinada. Após otimização de condições de pré-incubação (aclimatização a 37 ºC na ausência de comida), foram conduzidos ensaios de infeção com a estirpe de referência S. aureus ATCC25923 de forma a comparar a suscetibilidade à infeção entre as larvas comerciais e as larvas criadas in-house. O modelo de infeção de G. mellonella foi usado com as larvas in-house para determinar o potencial de virulência das estirpes de referência P. aeruginosa ATCC27583, N. gonorrhoeae ATCC49226, E. coli ATCC25922 e estirpes clínicas de S. aureus.
Foi estabelecida no GHTM/IHMT-NOVA uma colónia de G. mellonella. A G. mellonella criada in-house apresentou um ciclo de vida entre 31 a 34 dias. Em média, cada fêmea colocou 1794 ovos, com uma taxa de eclosão de ~34%. Em comparação com as larvas comerciais, as larvas criadas in-house, mostraram ser mais saudáveis e menos suscetíveis à infeção por S. aureus ATCC25923. A pré-incubação das larvas por 48 horas a 37ºC, sendo deprivadas de comida nas últimas 24 horas, mostrou ser a melhor condição de aclimatização. A realização de ensaios de infeção dos quatro agentes patogénicos permitiu, a inóculos equivalentes, diferenciá-los de acordo com o seu potencial de virulência da seguinte forma: P. aeruginosa >> S. aureus > E. coli > N. gonorrhoeae.
Este trabalho representa a expansão dos modelos de infeção animal disponíveis no GHTM/IHMT-NOVA e a sua aplicabilidade para estudar agentes patogénicos bacterianos relevantes que são o foco de linhas de investigação em curso no IHMT-NOVA. Trabalho futuro poderá incidir na utilização deste modelo para o estudo de agentes fúngicos e parasitas, assim como estudos de farmacotoxicidade.
Palavras-chave: Galleria mellonella; modelo de infeção invertebrado
ABSTRACT Galleria mellonella larvae represents a novel invertebrate infection model with potential for application in diverse research fields. To date, it has been used in studies on assessment of pathogens virulence potential, host-pathogen interaction, and pharmaco-toxicological studies. Its usefulness has been proven for some of the most clinically relevant pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus. The main objectives of this work were the implementation of a G. mellonella colony at GHTM/IHMT-NOVA and the optimization infection protocols for pathogenicity assays of four relevant bacterial pathogens: P. aeruginosa, S. aureus, Neisseria gonorrhoeae and Escherichia coli. G. mellonella at the last larval stage were acquired from a commercial pet house specialized in exotic animals and the species was confirmed by COI sequencing. The larvae were reared in a designated room at the Insectary infrastructures of IHMT-NOVA, in the dark, at 27-28 °C and 60-80% relative humidity with a nutrient rich diet consisting of corn meal, dried yeast, soy flour, powder milk, honey, glycerol and beewax blocks. The G. mellonella life cycle was followed and the egg productivity determined. After optimizing pre-incubation conditions (acclimatization to 37ºC and absence of food), infection assays were conducted with the reference strain S. aureus ATCC25923 to compare the susceptibility to infection between commercial and in-house reared larvae. The G. mellonella infection model was then applied with in-house larvae to assess with the virulence potential of the reference strains P. aeruginosa ATCC27583, N. gonorrhoeae ATCC49226, E. coli ATCC25922 and S. aureus clinical strains. A G. mellonella colony was established at GHTM/IHMT-NOVA. In-house reared G. mellonella presents a life cycle of 31-34 days. In average, each female laid 1 794 eggs and a rate of egg hatching of ~34% was observed. Compared to the commercially acquired, in-house reared larvae were healthier and less susceptible to infection by S. aureus ATCC25923. Pre-incubation of the larvae for 48 hours at 37 ºC, withdrawing the food at the last 24 hours was shown to be the best acclimatization conditions. After infection assays, at equivalent inoculums, the four bacterial pathogens tested could be differentiated and ranked according to their virulence potential, as follows: P. aeruginosa >> S. aureus > E. coli > N. gonorrhoeae. This work represents a build-up on the animal infection models available at GHTM/IHMT-NOVA and its applicability to study relevant bacterial pathogens that are the focus of research studies ongoing at IHMT-NOVA. Future work could focus on expanding this model to fungal and parasite pathogens, as well as additional functional assays, such as pharmaco-toxicological studies.
ABSTRACT Galleria mellonella larvae represents a novel invertebrate infection model with potential for application in diverse research fields. To date, it has been used in studies on assessment of pathogens virulence potential, host-pathogen interaction, and pharmaco-toxicological studies. Its usefulness has been proven for some of the most clinically relevant pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus. The main objectives of this work were the implementation of a G. mellonella colony at GHTM/IHMT-NOVA and the optimization infection protocols for pathogenicity assays of four relevant bacterial pathogens: P. aeruginosa, S. aureus, Neisseria gonorrhoeae and Escherichia coli. G. mellonella at the last larval stage were acquired from a commercial pet house specialized in exotic animals and the species was confirmed by COI sequencing. The larvae were reared in a designated room at the Insectary infrastructures of IHMT-NOVA, in the dark, at 27-28 °C and 60-80% relative humidity with a nutrient rich diet consisting of corn meal, dried yeast, soy flour, powder milk, honey, glycerol and beewax blocks. The G. mellonella life cycle was followed and the egg productivity determined. After optimizing pre-incubation conditions (acclimatization to 37ºC and absence of food), infection assays were conducted with the reference strain S. aureus ATCC25923 to compare the susceptibility to infection between commercial and in-house reared larvae. The G. mellonella infection model was then applied with in-house larvae to assess with the virulence potential of the reference strains P. aeruginosa ATCC27583, N. gonorrhoeae ATCC49226, E. coli ATCC25922 and S. aureus clinical strains. A G. mellonella colony was established at GHTM/IHMT-NOVA. In-house reared G. mellonella presents a life cycle of 31-34 days. In average, each female laid 1 794 eggs and a rate of egg hatching of ~34% was observed. Compared to the commercially acquired, in-house reared larvae were healthier and less susceptible to infection by S. aureus ATCC25923. Pre-incubation of the larvae for 48 hours at 37 ºC, withdrawing the food at the last 24 hours was shown to be the best acclimatization conditions. After infection assays, at equivalent inoculums, the four bacterial pathogens tested could be differentiated and ranked according to their virulence potential, as follows: P. aeruginosa >> S. aureus > E. coli > N. gonorrhoeae. This work represents a build-up on the animal infection models available at GHTM/IHMT-NOVA and its applicability to study relevant bacterial pathogens that are the focus of research studies ongoing at IHMT-NOVA. Future work could focus on expanding this model to fungal and parasite pathogens, as well as additional functional assays, such as pharmaco-toxicological studies.
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Biologia molecular Microbiologia Virologia Galleria mellonella Modelo de infeção invertebrado Agentes patogénicos bacterianos Virulência