Avaliação da redução e resistência bacteriana via múltiplos ciclos de terapia fotodinâmica.
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Data
2018-12-17
Autores
Zago, Lucas Henrique de Paula
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Universidade Estadual Paulista (Unesp)
Resumo
A doença periodontal é um processo patológico infecto-inflamatório, que pode causar eventual perda do elemento dental. O principal tratamento realizado é a raspagem e alisamento radicular (RAR), que embora seja efetiva, tem como fator limitante a dificuldade técnica de intervir em biofilmes de áreas de difícil acesso. As associações com antibióticos podem ocasionar efeitos colaterais e em bactérias resistentes podem ser ineficazes. Desta forma, a terapia fotodinâmica antimicrobiana (TFDa) surge como uma opção adjuvante, para o tratamento das doenças periodontais. Este trabalho investigou, in vitro, o potencial de redução e desenvolvimento de resistência bacteriana, em um pool clínico resistente ao metronidazol, frente a múltiplos ciclos de TFDa, em fase planctônica e biofilme, utilizando azul de metileno, clorina-e6 e curcumina, como fotossensibilizadores (FS). Para realização dos ciclos de indução de resistência, foi necessário encontrar as condições sub ótimas da TFDa, através da quantificação das colônias formadas por mililitro de amostra (UFC/mL). Os seguintes parâmetros foram encontrados em fase planctônica: 50 µg/mL de azul de metileno e dose de luz de 120 J/cm2 com redução de 1,67 log10, clorina-e6 e curcumina obtiveram reduções de 1,91 log10 e 1,76 log10, respectivamente, com dose de luz de 25 J/cm2 e 100 µg/mL de FS. Posteriormente o pool de bactérias foi sujeito a múltiplas exposições de TFDa, seguido de um período de recuperação, em que células bacterianas sobreviventes foram submetidas novamente ao mesmo tratamento, e assim sucessivamente até que se observasse ou não o desenvolvimento de resistência ou até o limite de 15 ciclos. Para o azul de metileno e clorina-e6, houve eliminação total dos micro-organismos ao fim do 5° ciclo e para curcumina ao fim do 3° ciclo. Em biofilme, a dose sub ótima de TFDa com azul de metileno foi de 150 µg/mL + 150 J/cm2 com uma redução de 2,53 log10. Para clorina-e6 e curcumina a dose sub ótima foi de 150 J/cm2 associada a 100 µg/mL de cada FS, com as respectivas reduções: 1,47 log10; 1,88 log10. Foram aplicados 15 ciclos de TFDa, que apesar de não reduzirem por completo a carga microbiana, foram capazes de promover um aumento significativo na morte bacteriana. Os resultados indicam que a TFDa pode ser uma abordagem adjuvante promissora no tratamento da doença periodontal.
Periodontal disease is an infectious-inflammatory pathological process, which can cause eventual loss of the dental element. The main treatment is scaling and root planing (RAR), which, although effective, has as a limiting factor the technical difficulty of intervening in biofilms of difficult access areas. Associations with antibiotics can cause side effects and in resistant bacteria they may be ineffective. In this way, antimicrobial photodynamic therapy (aPDT) appears as an adjuvant option for the treatment of periodontal diseases. This study investigated, in vitro, the potential for reduction and development of bacterial resistance in a clinical pool, resistant to metronidazole, against multiple cycles of aPDT in planktonic phase and biofilm using methylene blue, chlorine-e6 and curcumin as photosensitizers (PS). For resistance induction cycles, it was necessary to find the sub optimal conditions of the aPDT, by quantifying the colonies formed per milliliter of sample (CFU/mL). The following parameters were found in the planktonic phase: 50 μg/mL methylene blue and light dose of 120 J/cm2 with a reduction of 1.67 log10, chlorine-e6 and curcumina obtained reductions of 1.91 log10 and 1.76 log10, respectively, with light dose of 25 J/cm2 and 100 μg/mL PS. Subsequently, the pool of bacteria was subjected to multiple exposures of aPDT, followed by a recovery period, in which surviving bacterial cells were resuspended in the same treatment, and so on until resistance development was observed or up to the limit of 15 cycles. For methylene blue and chlorine-e6, the microorganisms were completely eliminated at the end of the 5th cycle and for curcumin at the end of the third cycle. In biofilm, the sub optimal dose of methylene blue aPDT was 150 μg/mL + 150 J/cm2 with a reduction of 2.53 log10. For chlorine-e6 and curcumin the sub optimal dose was 150 J/cm2 associated with 100 μg/mL of each PS, with respective reductions: 1.47 log10; 1.88 log10. Fifteen cycles of aPDT were applied, which although not completely reducing the microbial load, were able to promote a significant increase in bacterial death. The results indicate that aPDT may be a promising adjuvant approach in the treatment of periodontal disease.
Periodontal disease is an infectious-inflammatory pathological process, which can cause eventual loss of the dental element. The main treatment is scaling and root planing (RAR), which, although effective, has as a limiting factor the technical difficulty of intervening in biofilms of difficult access areas. Associations with antibiotics can cause side effects and in resistant bacteria they may be ineffective. In this way, antimicrobial photodynamic therapy (aPDT) appears as an adjuvant option for the treatment of periodontal diseases. This study investigated, in vitro, the potential for reduction and development of bacterial resistance in a clinical pool, resistant to metronidazole, against multiple cycles of aPDT in planktonic phase and biofilm using methylene blue, chlorine-e6 and curcumin as photosensitizers (PS). For resistance induction cycles, it was necessary to find the sub optimal conditions of the aPDT, by quantifying the colonies formed per milliliter of sample (CFU/mL). The following parameters were found in the planktonic phase: 50 μg/mL methylene blue and light dose of 120 J/cm2 with a reduction of 1.67 log10, chlorine-e6 and curcumina obtained reductions of 1.91 log10 and 1.76 log10, respectively, with light dose of 25 J/cm2 and 100 μg/mL PS. Subsequently, the pool of bacteria was subjected to multiple exposures of aPDT, followed by a recovery period, in which surviving bacterial cells were resuspended in the same treatment, and so on until resistance development was observed or up to the limit of 15 cycles. For methylene blue and chlorine-e6, the microorganisms were completely eliminated at the end of the 5th cycle and for curcumin at the end of the third cycle. In biofilm, the sub optimal dose of methylene blue aPDT was 150 μg/mL + 150 J/cm2 with a reduction of 2.53 log10. For chlorine-e6 and curcumin the sub optimal dose was 150 J/cm2 associated with 100 μg/mL of each PS, with respective reductions: 1.47 log10; 1.88 log10. Fifteen cycles of aPDT were applied, which although not completely reducing the microbial load, were able to promote a significant increase in bacterial death. The results indicate that aPDT may be a promising adjuvant approach in the treatment of periodontal disease.
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Palavras-chave
Terapia fotodinâmica, resistência bacteriana, doença periodontal, fotossensibilizador, photodynamic therapy, bacterial resistance, periodontal disease, photosensitizer