Table 4 Minimal inhibitory concentrations to penicillin of Streptococcus pneumoniae mutants and transformants

R6

Wild type

CCRI1974

S. pneumoniae, sensitive clinical isolate

R6M1

R6 clone selected in vitro for PG resistance

R6M2

R6 clone selected in vitro for PG resistance

1974M1

1974 clone selected in vitro for PG resistance

1974M2

1974 clone selected in vitro for PG resistance

CP1250

S. pneumoniae that contains a spontaneous mutation in the rpsL gene that confers resistance to SM

R6^2x-M1

R6-WT transformed with pbp2x PCR fragments from R6M1 (contains all three missense mutations present in PBP2x of R6M1)

R6^2x-M2

R6-WT transformed with pbp2x PCR fragments from R6M2 (contains just three mutations - Q281P, A369V, R384G - out of five mutations present in PBP2x of R6M2)

R6^2x2b-M1

R6^2x-M1 transformed with pbp2b PCR fragments from R6M1 so all missense mutations present in PBP2b of R6M1 transformed into this transformant

R6^2x2b-M2

R6^2x-M2 transformed with pbp2b PCR fragments from R6M2 so all missense mutations present in PBP2b of R6M2 transformed into this transformant

R6^{2x2b-M2, 1a::Janus}

R6^2x2b-M2 knocked out in pbp1a by Janus cassette, KM^RSM^S

R6^{SmR, 2x2b1a-M2,}

R6^{2x2b-M2, 1a::Janus} subjected to second step transformation with pbp1a of the Janus cassette with pbp1a of R6M2, KM^SSM^R

R6^{SmR, 2x2b1a,spr1178-M2,}

R6^2x2b1a-M2 transformed with spr1178 PCR fragments from R6M2 (contains a Q28* nonsense mutation in spr1178), SM^R

R6^{2x2b,spr1254-M2}

R6^2x2b-M2 transformed with pstB PCR fragment from R6M2 (contains a T205I mutation in PstB)

R6^{2x2b, spr1254, spr1178-M2}

R6^2x2b-M2 transformed with spr1178 and pstB PCR fragments from R6M2 (contains the substitutions Q28* in spr1178 and T205I in PstB)

R6M1^SmR,1a-WT

R6M1 co-transformed with a pbp1a PCR fragment from R6-WT and a rpsL streptomycin resistance marker, SM^R

R6^{ko in spr1178}

R6-WT knocked out in spr1178, CM^R

R6^{ko in spr1254}

R6-WT knocked out in spr1254, KM^R

R6^{spr1888::janus}

R6-WT knocked out in spr1888 by Janus cassette, KM^RSM^S