Dados do Trabalho

Título

Lyophilization: A promising approach for preserving Yersinia pestis strains of an exceptional bacterial collection from Brazil (Fiocruz-CYP)

Introdução

The Fiocruz-CYP Yersinia pestis Collection comprises 917 strains isolated since 1966 from human plague cases, rodents, small mammals, fleas, and other blood-feeding organisms in different epidemic areas of Northeast Brazil. The cultures are currently kept in peptone agar tubes with rubber caps, under refrigeration (4 to 10°C). A preliminary study assessing the viability of subcultures within the Collection revealed a notable decrease in recovery rates, which were found to be inversely correlated with the duration of conservation. In an effort to preserve the Collection, alternative storage methods are being explored, however, the requirement of utilizing a deep freezer poses challenges in maintaining cultures due to its cost and complexity. Lyophilization offers an alternative approach to maintain strains and ensure the viability of the Collection.

Objetivo (s)

To introduce a straightforward lyophilization protocol as a strategy for the long-term storage of Y. pestis strains from Fiocruz-CYP.

Material e Métodos

The non-virulent Y. pestis EV76 strain was 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 lyophilized cultures were separated in four groups and stocked under -80, -20, 4-8ºC and at room temperature during one year. The reactivation test of the lyophilized samples was performed quarterly by aseptically adding 500 µL of LB to each vial and, immediately, 250 µL was transferred to a 3 mL CYP broth tube and plated 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 CYP broth and cell count that ranged from 3.9 × 107 to 2.2 × 108 CFU/mL among the different evaluated storage temperatures. The lyophilization has proven to be a safe technique for the preservation of the tested strains, as it preserves the genetic material, allows storage at room temperature and guarantees the viability of the bacteria for several years and it will be used for the maintenance and preservation of Fiocruz-CYP Collection.

Palavras-chave

Yersinia pestis; plague; storage; lyophilization.

Agradecimentos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (400734/2019-2).