Dados do Trabalho

Título

Lyophilization as a strategy for storage of Yersinia pestis strains from an unique bacterial collection in Brazil (Fiocruz-CYP)

Introdução

The Yersinia pestis Collection (Fiocruz-CYP) has 907 strains isolated since 1966 from human cases of plague, rodents, small mammals, and fleas and other hematophagous in the various foci of Northeast Brazil during epidemic periods. The cultures are currently kept in peptone agar tubes with rubber caps, under refrigeration (4 to 10°C), however, in a preliminary study to evaluate the viability of the subcultures in the collection, it was observed that the recovery rates were relatively low, being inversely related to the conservation time. As an attempt to preserve the Collection, alternative stocking methods were tested, however, the need to use a deep freezer makes maintaining cultures difficult and expensive. Thus, lyophilization represents an alternative for the maintenance of strains and the viability of the Collection.

Objetivo(s)

We sought to present a simple lyophilization protocol strategy for long-term storage of Y. pestis strains from Fiocruz-CYP, replacing the methodologies currently used.

Material e Métodos

Four different Y. pestis strains were growth by standard protocol and mixed (1:1; v/v) with saturated trehalose solution, being lyophilized under vacuum at -40 °C for 8 hours. The reactivation test of the lyophilized strains was performed by aseptically adding 500 µL of LB to each vial and, immediately, 250 µL was transferred to a 3 mL LB broth tube and plated (100 to 10 µL) on LB agar by Drigalski-spatula technique. The cultures were incubated at 28 °C and visually inspected for bacterial growth analysis and colony count after 72 hours.

Resultados e Conclusão

All strains were viable after the lyophilization process, showing characteristic growth in LB broth and plate colony counts that ranged from 500 to >1000 CFU/mL, confirmed by the specific bacteriophage test for Y. pestis. Two isolates showed additional fungal growth, probably due to environmental contamination during the lyophilization process. As an alternative, it is suggested to use LB/Cefsulodine-Irgasan-Novobiocin-Nistatin in the resuspension and inoculum/plating steps. The success in the recovery rates of Y. pestis after lyophilization shows that it is a safe technique for the conservation of the Collection, as it preserves the genetic material, allows storage at room temperature and guarantees the viability of the bacteria for several years.

Palavras-chave

Yersinia pestis; plague; storage; lyophilization.