TABLE 1.

Antibiotic susceptibilities and growth rates of S. pneumoniae pbp insertional mutants

Isolate	Moenomycin MIC (μg/ml)a	Doubling time (min)b
R6c	2	27.0 ± 0.5
R6 nanA::Eryr	2	27.5 ± 0.5
R6 pbp2a::Eryr-1d	0.25	26.0 ± 0.5
R6 pbp2a::Eryr-2d	0.13	27.5 ± 0.5
R6 pbp1a::Eryr	4	28.0 ± 0.5
R6 pbp1b::Eryr	2	27.5 ± 0.5
R6 pbp1a::Spcrpbp1b::Eryr	2	34.0 ± 0.5
R6 pbp1a::SpcrnanA::Eryr	2	28.0 ± 0.5

^aMICs were determined by the broth microdilution method. Antibiotic susceptibilities were determined by broth microdilution as recommended by the National Committee for Clinical Laboratory Standards (29) by using Mueller-Hinton II medium supplemented with 5% lysed horse blood. Other agents tested included vancomycin, penicillin G, cefazolin, and cefaclor (Eli Lilly and Co.), imipenem (Merck Sharp & Dohme), ramoplanin and teicoplanin (Gruppo Lepetit S.P.A., Milan, Italy), trimethoprim-sulfamethoxazole (Hoffmann-La Roche), and enduracidin, bacitracin, fosfomycin, chloramphenicol, tetracycline, rifampin, and ciprofloxacin (Sigma Chemical Co.). Moenomycin was prepared by fermentation and isolated at Lilly Research Laboratories. Optochin susceptibility was determined by disk diffusion. 

^bGrowth rates in Todd-Hewitt broth supplemented with 0.125% yeast extract and 1% IsoVitaleX. Cultures were set up by adding 10 μl of frozen stock to 210 μl of medium in microtiter trays. Then, 100 μl of mineral oil was layered in each well, and plates were incubated at 37°C in a Molecular Devices Thermomax plate reader programmed to take optical density (absorbance at 650 nm) measurements at 15-min intervals for 12 h. Values are means of calculated minimum doubling time ± standard errors. 

^cR6, S. pneumoniae (hex) R6. 

^dS. pneumoniae (hex) R6 pbp2a::Ery^r-1 appears to have a single copy of the Ery^r plasmid integrated into pbp2a, while S. pneumoniae (hex) R6 pbp2a::Ery^r-2 has two or more.