Microbiologia básica de Leptospira spp.: ferramenta para a otimização dos estudos em leptospirose

Abstract

Leptospirosis is a zoonosis with a high incidence worldwide, caused by spirochetes of the genus Leptospira. The maintenance of the strains and the in vitro growth of the microorganism are still a difficult and time-consuming processes, there is an evident need to optimize laboratory culture, and this is essential to improve studies in leptospirosis. The objective of this work was to evaluate the in vitro growth parameters of Leptospira spp. and the virulence corresponding to each parameter in an animal model for acute leptospirosis. L. interrogans strains L1-130 and RCA and L. kirschneri strain UFPel-61H were cultured under different conditions in vitro, using the commercial EMJH culture medium (Difco) and EMJH homemade supplemented with rabbit serum (EMJH++) together with different temperatures (28 °C and 37 °C), initial containers and inocula (100 to 104) to evaluate the growth dynamics of the bacteria. Leptospires from different stages of bacterial growth, with high in vitro passages (25, 50 and 75 passages) and adapted to the mammalian host were used to evaluate their impact on virulence in the hamster model of acute infection. All the in vitro conditions tested were positive for leptospiral growth, except for the commercial EMJH culture medium at 37 °C. The highest bacterial densities and best duplication time rates were obtained with this medium at 28 °C, especially when associated with orbital agitation. The EMJH++ medium was efficient to permit growth from a low inoculation (from 1 leptospira), especially for the L. interrogans strains. In vivo experiments demonstrated that commercial EMJH culture medium at 28 °C is more suitable for maintaining the stability and virulence of leptospires at the different bacterial growth stages. Bacteria with high in vitro passages had virulence doses of 1 leptospira, demonstrating that the organisms maintained their virulence. Host-adapted bacteria presented a virulence dose of 3 leptospires, even with EMJH++ at 37 ºC, which did not present good results in vitro in this condition. In conclusion, the in vitro conditions that the bacteria undergo can directly influence virulence, demonstrating the need to understand the dynamics of in vitro growth of the microorganism. The standardization of leptospiral cultivation techniques will allow for improved reproducibility of experiments involving pathogenic leptospires.