Chapter 7
Isolation, Purification, and Characterization of Leptophages
Olivier Schiettekatte and Pascale Bourhy
Abstract
To date, only three bacteriophages of leptospires—leptophages—are known. Nonetheless, numerous
prophages have been found in the genus, especially in the genomes of pathogenic species. Thus, some
laboratories attempt to isolate leptophage particles from environmental samples or following mitomycin C
induction of bacterial cultures. Here, we propose multiple procedures to isolate, purify, and characterize
bacteriophages, based on protocols used for LE3 and LE4 characterization.
Key words Leptophages, Polyethylene glycol precipitation, Cesium chloride purification, TEM
1 Introduction
Three bacteriophages of leptospires have been isolated as of yet and
are named “leptophages” [1]. They have been sequenced [2, 3] and
morphologically characterized [1, 3]. In the last 30 years, no other
leptophage has been isolated, but numerous prophages have been
found in the genus, especially in the genomes of pathogenic species
[3, 4], suggesting a role in pathogenicity acquisition [5]. Thus,
some laboratories have attempted to isolate additional phage particles by mitomycin C inductions [6] or from environmental samples, using the method of Saint Girons et al. [1].
We propose, herein, a combination of methods for the extraction of leptophages from environmental samples and induced cultures as well as their isolation, purification, and phenotypic
characterization. These protocols are based on classical bacteriophage purification methods—i.e., polyethylene glycol precipitation, plating, and TEM observations—considering Leptospira
spp.-specific medium requirements and slow growth.
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_7, © Springer Science+Business Media, LLC, part of Springer Nature 2020
67
Isolation, Purification, and Characterization of Leptophages
Olivier Schiettekatte and Pascale Bourhy
Abstract
To date, only three bacteriophages of leptospires—leptophages—are known. Nonetheless, numerous
prophages have been found in the genus, especially in the genomes of pathogenic species. Thus, some
laboratories attempt to isolate leptophage particles from environmental samples or following mitomycin C
induction of bacterial cultures. Here, we propose multiple procedures to isolate, purify, and characterize
bacteriophages, based on protocols used for LE3 and LE4 characterization.
Key words Leptophages, Polyethylene glycol precipitation, Cesium chloride purification, TEM
1 Introduction
Three bacteriophages of leptospires have been isolated as of yet and
are named “leptophages” [1]. They have been sequenced [2, 3] and
morphologically characterized [1, 3]. In the last 30 years, no other
leptophage has been isolated, but numerous prophages have been
found in the genus, especially in the genomes of pathogenic species
[3, 4], suggesting a role in pathogenicity acquisition [5]. Thus,
some laboratories have attempted to isolate additional phage particles by mitomycin C inductions [6] or from environmental samples, using the method of Saint Girons et al. [1].
We propose, herein, a combination of methods for the extraction of leptophages from environmental samples and induced cultures as well as their isolation, purification, and phenotypic
characterization. These protocols are based on classical bacteriophage purification methods—i.e., polyethylene glycol precipitation, plating, and TEM observations—considering Leptospira
spp.-specific medium requirements and slow growth.
Nobuo Koizumi and Mathieu Picardeau (eds.), Leptospira spp.: Methods and Protocols, Methods in Molecular Biology, vol. 2134,
https://doi.org/10.1007/978-1-0716-0459-5_7, © Springer Science+Business Media, LLC, part of Springer Nature 2020
67