Abstract
A clone of Hungarian pneumococcal strains has recently been isolated with notably high levels of β-lactam resistance (penicillin MIC, 16 μg/mL; cefotaxime MIC, 4 μg/mL). The role that each penicillin-binding
protein (PBP) plays in the development of resistance in these strains was investigated via transformation of susceptible strain R6 with
pbp
DNA from resistant strain 3191. Transformation of strain
R6 with
pbp2X
DNA resulted in transformants with penicillin and cefotaxime MICs of 0.06 and 0.25 μg/mL, respectively. Further introduction of
pbp2B
and
1A
DNA increased penicillin
MICs to 0.25 and 4 μg/mL, respectively. Transformation of strain R6 with a combination of
pbp2X
and
pbp1A
DNA produced R6
3191/2X/1A
strains with an unexpected low cefotaxime
MIC of 0.5 μg/mL. This low-level of cefotaxime resistance was surprisingly increased from 0.5 to 2 μg/mL in R6
3191/2X/2B/1A
strains. This suggests the involvement of altered PBP 2B in cefotaxime
resistance. Therefore, within this particular setting of resistance, the environmental presence of selectors for altered PBP 2B (penicillin or piperacillin) are required for the development of high-level
cefotaxime resistance. The MICs of R6
3191/2X/2B/1A
strains never reached the MICs of the donor strain. Full MICs of the donor were eventually reached by transforming R6
3191/2X/2B
with
chromosomal
3191
DNA. Resultant transformants revealed the introduction of altered PBP 1A, while unaltered PBPs 1B, 2A, and 3 proved that these PBPs were not involved in resistance. A non-PBP
resistance determinant has therefore made up the difference in resistance between R6
3191/2X/2B/1A
and donor strain 3191. Beta-lactamase activity and efflux systems have so far been eliminated
as causes of resistance. This resistance determinant represents a novel mechanism for β-lactam resistance in clinical isolates of pneumococci, operates at the highest level of resistance, and remains
to be identified.