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Abstract(s)
género Arcobacter tem atualmente 18 espécies descritas, das quais se destaca a espécie A. butzleri, uma vez que esta é atualmente considerada um agente enteropatogénico zoonótico emergente tanto para animais como para humanos. Este microrganismo é geralmente detetado em produtos de origem alimentar e em água, possuindo também elevada resistência antimicrobiana a antibióticos e desinfetantes o que tem conduzido, nos últimos anos, à necessidade de desenvolver novas técnicas e investigar novos compostos para o controlo e, em última instância, erradicação desta bactéria. O resveratrol, principal composto da família das fitoalexinas, possui inúmeras propriedades bioativas, nomeadamente a atividade antimicrobiana contra fungos e bactérias Gram-positivas e Gram-negativas, tendo sido recentemente demonstrada a sua inibição sobre espécies de Arcobacter. De modo a fomentar a utilização deste composto em diversas aplicações, a sua baixa estabilidade e solubilidade aquosa têm vindo a ser ultrapassadas pela sua complexação e inclusão em diversos polímeros como, por exemplo, ciclodextrinas, sais biliares e microesferas poliméricas. De facto, a complexação do resveratrol com ciclodextrinas modificadas demonstrou um aumento considerável da solubilidade aquosa e da fotoestabilidade deste composto. Desta forma, o objetivo deste trabalho residiu no estudo da atividade antimicrobiana do resveratrol (RV), sob a forma de complexos de inclusão com a ciclodextrina modificada hidroxipropil-gama-ciclodextrina (HP-?-CD) contra uma estirpe de referência de A. butzleri (LMG 10828), dois isolados de origem animal (AB 17/11 e AB 36/11) e dois isolados de origem clínica (INSA 593 e INSA 776).
Primeiramente procedeu-se à determinação dos fatores de virulência, onde os dois isolados clínicos revelaram a presença dos genes cadF, tlyA, cj1349, hecA, pldA, ciaB e mviN; a estirpe AB 36/11, apresentou mais um gene, o hecB, enquanto as estirpes LMG 10828 e AB 17/11 além dos oito genes descritos anteriormente, apresentaram no seu genoma o gene irgA. A resistência das estirpes a 9 antibióticos comuns foi também determinada, observando-se para a estirpe de referência e para os isolados clínicos a resistência à ampicilina, à amoxicilina, à vancomicina, à cefoperazona, à piperacilina e ao trimetoprim. Para além de apresentar resistência a estes antibióticos, a estirpe AB 36/11 tem resistência à ciprofloxacina. A estirpe AB 17/11 foi de todas elas, aquela que apresentou maior suscetibilidade, uma vez que o seu perfil de resistência inclui apenas os antibióticos vancomicina, ciprofloxacina e trimetoprim. A suscetibilidade do microrganismo aos complexos de inclusão (CI) formados foi determinada pelo método de microdiluição, onde se observou uma concentração mínima inibitória (CMI) de 256 µg/mL para as estirpes LMG 10828 e INSA 776 e 64 µg/mL para AB 36/11, AB 17/11 e INSA 593. Nesta fase foi também avaliada a suscetibilidade na presença de RV livre, obtendo-se uma CMI de 100 µg/mL para as cinco estirpes. De modo a determinar a ação bacteriostática ou bactericida dos CIs foram realizadas curvas de morte para um isolado clínico (INSA 776) e um de origem animal (AB 36/11), o que permitiu concluir que os CIs possuem atividade bactericida dependente tanto do tempo de incubação como da concentração de agente antimicrobiano. Para concentrações de CI correspondentes a 2x e 4x a CMI a viabilidade celular é semelhante para os dois isolados, sendo que o isolado de origem humana demonstrou ser mais suscetível. Para o RV livre, o efeito bactericida ocorre de forma mais rápida para 1x, 2x e 4xCMI em ambos os isolados. O mecanismo de ação dos CI foi avaliado por citometria de fluxo para estes dois isolados, usando os fluorocromos SYTO® 40 para marcação de todas as células, bis-(ácido 1,3-dibutilbarbitúrico) trimetina oxonol (BOX) e 5-ciano-2,3-ditolil cloreto de tetrazólio (CTC), para avaliação de alterações a nível do potencial de membrana e atividade metabólica, respetivamente. Os resultados de citometria de fluxo demonstraram que, com o aumento da concentração de CI, ocorre a diminuição da atividade metabólica das células e, consequentemente, o aumento da despolarização da membrana das mesmas.
Dado que a formação de biofilmes por parte dos microrganismos está associada com a sua virulência, colonização e resistência a antibióticos, torna-se importante a sua monitorização bem como o estudo de formas que inibam o seu desenvolvimento. Assim sendo, foi também evidenciada a ação dos CIs e do RV livre na formação e na promoção da dispersão de biofilmes pré-formados, com elevada redução de biomassa de biofilme para as concentrações de 1x, 2x e 4x a CMI.
De forma geral, os resultados demonstram que os CIs apresentam atividade antimicrobiana sobre os isolados de A. butzleri tanto em células planctónicas como em biofilmes, sugerindo a aplicação destas formulações como soluções de origem natural para a indústria alimentar contra o desenvolvimento de Arcobacter spp.
Currently, the genus Arcobacter has 18 described species, with the most relevant being A. butzleri since it is now considered an emerging zoonotic enteropathogenic agent for both animals and humans. This microorganism is usually detected in food products and water environments and also presents a high antimicrobial resistance to antimicrobials and disinfectants which has led, in recent years, to the need to develop new techniques and to search new compounds for the control and, even, eradication of this bacterium. Resveratrol, the main compound of the phytoalexins family, has innumerous bioactive properties, including antimicrobial activity against fungi and Gram-positive and Gram-negative bacteria, and recently its inhibition over Arcobacter species was demonstrated. In order to promote the use of this compound in various applications, its low stability and water solubility have been overcome by its complexation and inclusion in various polymers such as cyclodextrins, bile salts and polymeric microspheres. In fact, resveratrol complexation with modified cyclodextrins showed a considerable increase in the aqueous solubility and photostability of this compound. Thus, the main goal of this work was the evaluation of resveratrol antimicrobial activity, inclusion complexes with modified cyclodextrin hydroxypropyl-gamma-cyclodextrin (HP-?-CD) against an A. butzleri reference strain (LMG 10828), two animal origin (AB 17/11 and AB 36/11) and two clinical origin (INSA 593 and INSA 776) isolates. The first step of the study was the determination of the virulence factors where both clinical isolates presented the cadF, tlyA, cj1349, hecA, pldA, ciaB and mviN genes, the strain AB 36/11 presented one more gene, hecB while the strains LMG 10828 and AB 17/11 presented in their genome the gen irgA besides the eight ones already described for the other strains. The antimicrobial resistance profile to 9 common antibiotics was also evaluated and the results showed that both the reference strain and the clinical isolates were resistant to ampicillin, amoxicillin, vancomycin, cefoperazone, piperacillin and trimethoprim. Besides being resistant to all antibiotics previously mentioned, the strain AB 36/11 also presented resistance to ciprofloxacin. The strain AB 17/11 was the one that exhibited the highest susceptibility since its resistance profile only included vancomycin, ciprofloxacin and trimethoprim. This microrganism’s susceptibility to the inclusion complexes (ICs) was determined by the microdilution method where minimum inhibitory concentrations (MIC) of resveratrol contained in the IC of 256 µg/mL for strains LMG 10828 and INSA 776 and 64 µg/mL for AB 36/11, AB 17/11 and INSA 593 were obtained. At this stage, the susceptibility of all strains to free RV was also assessed, obtaining a MIC of 100 µg/mL for the five strains studied. In order to determine the bacteriostatic or bactericidal action of ICs, time-kill curves were performed for a clinical (INSA 776) and an animal origin (AB 36/11) isolates, which showed that the observed bactericidal activity of the ICs was dependent on both incubation time and antimicrobial agent concentration. For IC concentrations corresponding to 2x and 4x MIC, cell viability is similar for both isolates, with the clinical origin isolate exhibiting a higher susceptibility when exposed to concentrations equal to MIC values. For free RV, the bactericidal effect occurs more quickly for 1x, 2x e 4xMIC in both isolates. The ICs mechanism of action was evaluated by flow cytometry for these two isolates, using SYTO® 40 fluorochrome for total cell staining and bis-(1,3-dibutylbarbituric acid)trimethine oxonol (BOX) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) for the evaluation of cell membrane potential and metabolic activity, respectively. Flow cytometry results showed that increasing IC concentration led to increased reduction of the metabolic activity of cells which, in turn, resulted in increased cell membrane depolarization. A microorganism’s ability to form biofilms is commonly associated with its increased virulence, colonization and resistance to antibiotics, thus being of extreme relevance to monitor these biofilms’ formation while studying new ways to prevent or inhibit their development. Given this, this work also evidenced ICs and free RV action on biofilm formation as well as dispersion of pre-formed biofilms showing a high reduction in the biofilm’s biomass for concentrations of 1x, 2x and 4x CMI. Overall, the results obtained in this work showed that the ICs exhibited antimicrobial activity over A. butzleri reference strain and isolates both in planktonic cells and biofilms, suggesting the application of these natural-origin formulations in the food industry to prevent Arcobacter spp. development.
Currently, the genus Arcobacter has 18 described species, with the most relevant being A. butzleri since it is now considered an emerging zoonotic enteropathogenic agent for both animals and humans. This microorganism is usually detected in food products and water environments and also presents a high antimicrobial resistance to antimicrobials and disinfectants which has led, in recent years, to the need to develop new techniques and to search new compounds for the control and, even, eradication of this bacterium. Resveratrol, the main compound of the phytoalexins family, has innumerous bioactive properties, including antimicrobial activity against fungi and Gram-positive and Gram-negative bacteria, and recently its inhibition over Arcobacter species was demonstrated. In order to promote the use of this compound in various applications, its low stability and water solubility have been overcome by its complexation and inclusion in various polymers such as cyclodextrins, bile salts and polymeric microspheres. In fact, resveratrol complexation with modified cyclodextrins showed a considerable increase in the aqueous solubility and photostability of this compound. Thus, the main goal of this work was the evaluation of resveratrol antimicrobial activity, inclusion complexes with modified cyclodextrin hydroxypropyl-gamma-cyclodextrin (HP-?-CD) against an A. butzleri reference strain (LMG 10828), two animal origin (AB 17/11 and AB 36/11) and two clinical origin (INSA 593 and INSA 776) isolates. The first step of the study was the determination of the virulence factors where both clinical isolates presented the cadF, tlyA, cj1349, hecA, pldA, ciaB and mviN genes, the strain AB 36/11 presented one more gene, hecB while the strains LMG 10828 and AB 17/11 presented in their genome the gen irgA besides the eight ones already described for the other strains. The antimicrobial resistance profile to 9 common antibiotics was also evaluated and the results showed that both the reference strain and the clinical isolates were resistant to ampicillin, amoxicillin, vancomycin, cefoperazone, piperacillin and trimethoprim. Besides being resistant to all antibiotics previously mentioned, the strain AB 36/11 also presented resistance to ciprofloxacin. The strain AB 17/11 was the one that exhibited the highest susceptibility since its resistance profile only included vancomycin, ciprofloxacin and trimethoprim. This microrganism’s susceptibility to the inclusion complexes (ICs) was determined by the microdilution method where minimum inhibitory concentrations (MIC) of resveratrol contained in the IC of 256 µg/mL for strains LMG 10828 and INSA 776 and 64 µg/mL for AB 36/11, AB 17/11 and INSA 593 were obtained. At this stage, the susceptibility of all strains to free RV was also assessed, obtaining a MIC of 100 µg/mL for the five strains studied. In order to determine the bacteriostatic or bactericidal action of ICs, time-kill curves were performed for a clinical (INSA 776) and an animal origin (AB 36/11) isolates, which showed that the observed bactericidal activity of the ICs was dependent on both incubation time and antimicrobial agent concentration. For IC concentrations corresponding to 2x and 4x MIC, cell viability is similar for both isolates, with the clinical origin isolate exhibiting a higher susceptibility when exposed to concentrations equal to MIC values. For free RV, the bactericidal effect occurs more quickly for 1x, 2x e 4xMIC in both isolates. The ICs mechanism of action was evaluated by flow cytometry for these two isolates, using SYTO® 40 fluorochrome for total cell staining and bis-(1,3-dibutylbarbituric acid)trimethine oxonol (BOX) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) for the evaluation of cell membrane potential and metabolic activity, respectively. Flow cytometry results showed that increasing IC concentration led to increased reduction of the metabolic activity of cells which, in turn, resulted in increased cell membrane depolarization. A microorganism’s ability to form biofilms is commonly associated with its increased virulence, colonization and resistance to antibiotics, thus being of extreme relevance to monitor these biofilms’ formation while studying new ways to prevent or inhibit their development. Given this, this work also evidenced ICs and free RV action on biofilm formation as well as dispersion of pre-formed biofilms showing a high reduction in the biofilm’s biomass for concentrations of 1x, 2x and 4x CMI. Overall, the results obtained in this work showed that the ICs exhibited antimicrobial activity over A. butzleri reference strain and isolates both in planktonic cells and biofilms, suggesting the application of these natural-origin formulations in the food industry to prevent Arcobacter spp. development.
Description
Keywords
Arcobacter Butzleri Atividade Antimicrobiana Biofilme Ciclodextrina Complexo de Inclusão Resveratrol