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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).
Susceptibility testing of both saprophytic (L. biflexa serovar Patoc strain Patoc 1) and pathogenic (L. interrogans serovar Copenhageni strain Fiocruz L1-130, L. interrogans serogroup Canicola strain Kito, and L. interrogans serovar Lai strain 56601) Leptospira strains to aminoglycosides and Spc was performed to determine the MICs (Table (Table1).1). We then examined the frequency of appearance of spontaneous mutants resistant to gentamicin (Gen), Kan, Spc, and Str (Table (Table2).2). Spontaneous mutations resulting in Str and Spc resistance arose in vitro at frequencies of approximately 10−9 to 10−10 (Table (Table2).2). The development of Spc resistance in Leptospira occurs at the level of detection in transformation assays in which 109 transformants are plated. The two copies of rrs (18) were amplified and sequenced in wild-type and Str- and Spc-resistant mutant strains (Table (Table3).3). The rrs sequences of three independent L. interrogans Spc-resistant isolates indicated that all had a C-to-U substitution in the two rrs copies at position 1192 (using E. coli as the reference for numbering). In contrast, the alleles identified in four L. biflexa Spc-resistant variants contained a G-to-U substitution in the 16S rRNA gene at position 1064 (Fig. (Fig.11 B). The expression of the resistant phenotype may therefore require a homogeneous population of cellular antibiotic-resistant ribosomes (19). MICs for these Spc-resistant mutants with transversion G1064C and transition C1192U were greater than 100 μg/ml, whereas MICs between 2 and 4 μg/ml were observed for wild-type parents. Mutations at these positions confer Spc resistance in E. coli (12), Neisseria spp. (7), and Nicotiana tabacum chloroplasts (5).
Frequencies of spontaneous mutation in rpsL, which encodes the ribosomal protein S12, were on the order of 10−9 to 10−10 (Table (Table2),2), similar to the mutation frequency of rpsL in E. coli (4 × 10−10) (10) and that in Borrelia burgdorferi (10−9) (3). The rate of spontaneous resistance to Str for strains Patoc and Kito was about 10 times higher than that for strains Fiocruz L1-130 and 56601 (Table (Table2).2). Str-resistant mutants (MIC of >100 μg/ml) had mutations in codons 43 and 88 (AAA to AGA) of the rpsL gene, resulting in an amino acid change of lysine to arginine, formerly identified as being responsible for resistance to Str in other bacteria (6, 15). Although the Etest has not been validated for testing antimicrobial susceptibility in leptospires, this method also provides an indication of the in vitro susceptibility of L. biflexa strains (Fig. (Fig.1C1C).
L. interrogans flgB and hsp10 promoters were amplified with the FlgA/FlgC and HspA/HspC primer pairs (Table (Table3),3), respectively, and were inserted into the PvuII restriction site of the E. coli-L. biflexa shuttle vector pSLe94 (1), generating pSLe94PF and pSLe94PH, respectively. The promoterless Gen resistance marker was amplified by PCR with primers GTBam5 and GTXho3 (Table (Table3)3) from pBSV2G (4). The amplified product was then digested with BamHI and XhoI and inserted into the corresponding sites of pSLe94PF and pSLe94PH, resulting in pSLE94PfGenta and pSLe94PhGenta, respectively (Fig. (Fig.2).2). The plasmids were then transferred into L. biflexa through electroporation (11). The MIC for L. biflexa transformants expressing the Gen cassette via the two L. interrogans promoters was ~20 μg/ml (versus 4 μg/ml for the L. biflexa wild-type strain) in both liquid and solid media; we then typically selected for transformants on solid media at a concentration of 10 μg/ml. At this concentration, spontaneous Gen-resistant mutants appeared at a frequency of 10−9 in L. biflexa.
We also used the Himar1 transposon to deliver the Gen resistance cassette into the pathogen L. interrogans. The Gen resistance cassette was amplified from pSLE94PfGenta with FgAsc and GTAsc3 (Table (Table3).3). The PCR products were then purified, digested with AscI, and inserted into the AscI restriction site of the Kan-resistant transposon carried by pMKL (2). After a 6-week incubation, we selected transformants for further identification of the site of transposon insertion, as previously described (14). The MIC of Gen for an L. interrogans serovar Lai transformant that contained an insertion at position 3064024 in the intergenic region in the large chromosome (CI) was >50 μg/ml (versus 4 μg/ml for the wild-type strain) in liquid media, therefore demonstrating that this cassette confers resistance to Gen in pathogenic Leptospira.
We thank P. Stewart for the generous gift of the gentamicin-resistant plasmid and S. Brémont for susceptibility testing of Leptospira.
Published ahead of print on 28 May 2010.