|Abstract or Summary
- Streptomycin resistance in both gram-positive and gram-negative
bacteria is usually caused by a single mutation in
the rpsL gene. The rpsL gene encodes the S12 protein of
the ribosomal complex. The rpsL genes of various bacteria
have consensus regions in their sequences. Primers were
designed from these consensus pockets and a fragment of the
rpsL gene was sequenced from S. gordonii using PCR based
methodologies. Using the Multiplex Restriction Sequence
PCR(mRS PCR), which used the known primer at one end and a
restriction site primer on the other, a gene walk was
conducted. In streptomycin resistant strains of S.
gordonii, namely GP204, SP204 and SP635, the AAA coding for
Lys56 was mutated to ACA, coding for Thr56. The lysine to
threonine transition, causing resistance to streptomycin
was identical to that expected from the literature.
The streptomycin resistance gene of S. pyogenes was
mapped using similar techniques. Streptomycin resistant
strains S43 ATCC, 543/192/4 and S43/192/30R were studied.
In streptomycin resistant S43 ATCC and S43/192/30R strains,
the lysine 56 changed to isoleucine and threonine
respectively. Surprisingly, the 192/4 had two mutations,
in each of the two hotspots in the rpsL gene where
mutations due to streptomycin resistance occur. It had the
amino acid 56, lysine, mutated to arginine and lysine 101
changed to asparagine. To check if this mutation was
stable in the host animal, S43/192/4 P8 (S43/192/4 passaged
eight times in mice) was sequenced and the sequence was
identical to the streptomycin resistant 192/4. Hence, the
lys101 mutation was stable and unlike the ancillary
mutations in E.coli and S. typhimurium, which are
compensated by new mutations.
The pathogenesis of S. pyogenes depends in part on the
ability of the pathogen to adhere to the epithelial cells
of the throat and the quantity of M protein. Pathogenesis
studies done on mice revealed the avirulence of S43/192/4smR
strain. To elucidate the reason for this avirulence, the
adherence properties and the production of M protein of the two strains S43/192/4smR and S43/192/30R were tested.
Qualitative immunoblot analysis of the M protein of 192/4
and 30R revealed no significant difference. Competition
ELISA was conducted to quantitate the M protein, and this
also did not show any significant difference in the M
protein levels. The adherence of 30R and 192/4 was measured
on human pharyngeal epithelial cell line. The adherence
properties of S43/192/4 SmR, was no different from other
strains in this experiment. Electron microscopy, using
immunogold to highlight the M protein on the cell surface
showed no differences.