Susceptibilidade antimicrobiana in vitro de biofilme e forma planctônica deLeptospira Interrogans obtidos de cães naturalmente infectados

Abstract

CARVALHO, R. R. M. In vitro antimicrobial susceptibility of biofilm and planktonic form of Leptospira interrogans, obtained from naturally infected dogs. 2022. 76p. Dissertação (Mestre em Ciência Animal nos Trópicos) - Escola de Medicina Veterinária e Zootecnia - Universidade Federal da Bahia, 2022. Leptospirosis is a zoonosis caused by spirochetes from the genus Leptospira, considered to have great impact on public health. Leptospira spp. are able to form biofilms, however, despite their importance, there are still few studies involving isolation of autochthonous strains, with subsequent evaluation of antimicrobial sensitivity in planktonic and biofilm forms. The objective of this study was to evaluate the capacity of in vitro biofilm formation from a reference strain (L. interrogans, sv. Copenhageni L1 130 - L20) and from recently isolated strains of L. interrogans (C20, C29, C51, C82), with subsequent evaluation of antimicrobial susceptibility in planktonic and biofilm forms. In vitro biofilm production was tested wih incubation at 30°C, under static conditions, and subsequent observation in scanning electron microscopy (SEM). The semi quantification of biofilm production was performed by spectrophotometry in polystyrene plates at different observational points (1, 7, 14 and 21 days). All strains were efficient for in vitro biofilm formation up until the 21st day of observation. Semi quantification revealed that both L1 130 and the recently isolated strains (C20, C29, C51 and C82) presented similar optical density (OD) values until the 3rd day of incubation. On the 5th day ODs varied from a minimum of 0.176 (C20) to a maximum of 0.236 (C51). Increase in biofilm biomass was observed from the 7th day onward, with maximum optical densities on the 21st day (L1 130 OD = 0.441±0.02, C20 OD = 0.437±0.02, C29 OD = 0.309±0.01, C51 OD = 0.503±0.02, C82 OD = 0.353±0.02). Through SEM visualization, it was possible to observe formation of mature biofilm for all strains on the 7th day of incubation. On days 14 and 21, the biofilms presented themselves as morphologically more developed and cohesive structures, maintaining the characteristics of a mature biofilm, but with greater deposition of extracellular matrix. On the 21st day, the images demonstrated the presence of several interconnected channels and branched filaments of extracellular matrix formed at various points. Four antimicrobials (amoxicillin, ampicillin, doxycycline, and ciprofloxacin) were used in the antimicrobial susceptibility tests, with final concentrations ranging from 0.02µg/mL to 1,600µg/mL. In the planktonic form, the strains showed sensitivity to all drugs with MICs90 of ≤0.02 µg/mL for amoxicillin and ampicillin, 0.2 µg/mL for doxycycline, and 0.1 µg/mL for ciprofloxacin. These strains on biofilm were considerably more resistant compared to their planktonic form, with MIC90 of 1,600 µg/mL for amoxicillin, 800 µg/mL for ampicillin, and >1,600 µg/mL for doxycycline and ciprofloxacin. Within the range of antimicrobial concentrations, the strains in the biofilm form were 13 to 16 times more resistant than the planktonic forms. Some isolates showed MICs higher than the maximum dilution used in this study (1,600ug/ml). The strains in this study were obtained from samples of naturally infected dogs, who might act as reservoirs and sentinels for infection in humans. The isolation of biofilm-forming strains, with a potential antimicrobial resistance profile, demonstrates important information for genomic investigations that aim to elucidate the molecular mechanisms responsible for such characteristics. These approaches will allow the development of refined treatment and control strategies focused on the epidemiological reality of the disease.