Antibiotic Resistance Markers for Genetic Manipulations of Leptospira spp.

We measured the frequency of appearance of spontaneous mutants resistant to gentamicin, kanamycin, streptomycin, and spectinomycin in saprophytic and pathogenic Leptospira strains. The mutations responsible for the spontaneous resistance to streptomycin and spectinomycin were identified in the rpsL and rrs genes, respectively. We also generated a gentamicin resistance cassette that allows the use of a third selectable marker in leptospires. These results may facilitate further advances in gene transfer systems in Leptospira spp.Our understanding of leptospiral pathogenesis depends on reliable genetic tools for fully characterizing genes of interest. Significant advances in genetics of Leptospira spp. have been made over the last few years (8, 11). For generating antibiotic resistance genetic markers, our group focused on antibiotics other than those used therapeutically. We therefore excluded the use of β-lactams, as they are used to treat leptospirosis, which is an emerging disease with more 500,000 severe cases occurring annually (8). Plasmid DNA can be introduced into Leptospira by electroporation (2, 21) or conjugation (16). In 1990, Saint Girons et al. used the replication origin of the LE1 leptophage (22) to generate a plasmid that was able to replicate autonomously in both the saprophyte Leptospira biflexa and Escherichia coli (21). They used resistance to kanamycin (Kan), which was conferred by a gene from the Gram-positive bacterium Enterococcus faecalis, as a genetic marker to select for introduced DNA. Another marker, a spectinomycin (Spc) resistance cassette from Staphylococcus aureus, was also used as a selectable marker in Leptospira spp. (1). Further studies have used Spc and Kan markers to screen for transformants resulting from plasmid replication or chromosomal integration in leptospires (8, 11). As the proportion of allelic-exchange mutants is low and as chromosomal integration generally occurs through a single recombination event, a plasmid containing the rpsL wild-type gene as a counterselectable marker in a streptomycin (Str)-resistant strain of L. biflexa (due to a mutation in rpsL) was also used to eliminate clones harboring the plasmid and/or clones that have integrated the plasmid through a single-crossover event (9, 17, 20).