results of a liquid culturing system ( bd bactec mgit 960 system ; bd diagnostics , sparks , md , us ) ( for details , see the technical appendix ) showed that all 5 beijing and 5 eai genotype strains were susceptible to isoniazid ( inh ) , rifampin ( rif ) , moxifloxacin ( mxf ) , and amikacin ( amk ) .
mics were determined by using the agar proportion method ( 11 ) , which showed that ranges were small for the beijing and eai genotype strains : inh , 0.0620.125 mg / l ; rif , 0.1251 mg / l ; mxf , 0.1250.5 mg / l ; and amk , 0.52 mg / l .
we determined the mutation frequencies of the beijing and eai genotype strains by using previously defined critical drug concentrations of 1 mg / l for inh , rif , and mxf and 5 mg / l for amk ( 11,12 ) ( for details , see the technical appendix ) .
the mutation frequencies of the beijing and eai genotype strains were similar for inh , mxf , and amk , but they were significantly different for rif ( 1.6  10 to 5.4  10 for beijing strains vs. 6.3  10 to 3.8  10 for eai strains ; p = 0.003 , unpaired mann - whitney test ) ( table 1 ; figure 1 ) . because rifamycin drugs are widely used to treat tb , the difference in the mutation frequencies of beijing and eai genotype strains for rif is a major finding .
frequency of rifampin ( rif)-resistant mutants in mycobacterium tuberculosis beijing and east - african / indian ( eai ) genotype strains ( 5 strains each ) originating from vietnam .
statistical analysis was performed by using an unpaired mann - whitney test . for beijing genotype strains ,
the increase in mutation frequency during exposure to rif could be due to described missense mutations in the mut genes ( 13 ) .
such mutations in the mut genes can change the dna repair mechanism ; as a consequence , the frequency of resistant mutant formation might increase . however , a direct correlation between the occurrence of particular mutations in mut genes and altered mutation frequency has not been proven .
furthermore , werngren and hoffner ( 14 ) found an equal mutation frequency for beijing ( 3.6  10 ) and non - beijing ( 4.4  10 ) genotypes . a possible explanation for the discrepancy in findings might be the concentration of rif used in the subculture plates . in our study ,
the critical concentration of 1 mg / l rif was used ( 11 ) , whereas werngren and hoffner used a concentration of 2 mg / l rif .
in addition , werngren and hoffner compared the beijing and non - beijing genotypes of several genotype families , whereas we compared beijing and eai genotype strains that were selected from the same tuberculosis - endemic area and during the same period .
we determined the time - kill kinetics of rif toward 2 strains with significantly different mutation frequencies : beijing-1585 ( 3.7  10 [ 3.0  10 and 4.3  10 , duplicates ] ) and eai-1627 ( 3.5  10 [ 2.8  10 and 4.1  10 , duplicates ] ) .
cultures with low and high densities of beijing-1585 and eai-1627 were investigated as described ( 15 ) .
rif showed strong time- and concentration - dependent activity toward low - density cultures of the 2 strains ( figure 2 ) .
low concentrations of rif were needed to achieve > 99% mycobacterial killing ; differences between beijing-1585 and eai-1627 were minor ( table 2 ) .
however , to achieve 100% killing , especially for beijing-1585 , rif concentrations had to be increased substantially ( table 2 ) .
compared with the low - density culture for beijing-1585 , a substantial increase in rif concentrations was needed to achieve 100% killing of the high - density culture ( table 2 ) .
this finding may be relevant in the clinical context because high - density mycobacteria populations are expected to exist in infected tissues of tb patients .
concentration- and time - dependent bactericidal effect of rifampin ( rif ) toward low - density cultures of mycobacterium tuberculosis be-1585 ( 5.1  10 cfu / ml ) ( a ) and m. tuberculosis eai-1627 ( 6.8  10 cfu / ml ) ( b ) .
after 1 , 3 , or 6 days of exposure , subcultures were performed on solid media to count cfus . *
accurate cfu counting could not be performed  because complete outgrowth of mycobacteria occurred on the sixth day of rif exposure , leading to aggregation .
* cultures were exposed to rif at 2-fold increasing concentrations for 6 days at 37c ; at indicated time - points , subcultures were performed on solid media for counting .
low , low - density culture ; high , high - density culture ; nd ,  not determined . 
rif - resistant mutants did not emerge in low - density cultures of beijing-1585 and eai-1627 .
however , rif - resistant mutants were selected at relatively high numbers from high - density beijing-1585 cultures compared with high - density eai-1627 cultures . in beijing-1585 cultures ,
exposure to rif concentrations of 232 mg / l selected resistant mutants only ; this was not observed in eai-1627 cultures .
analysis of rif - resistant beijing mutants showed the following altered rpob gene sequences : cacgac ( h526d ) , cactac ( h526y ) , and tcgttg ( s531l ) , as assessed by using the genotype mtbdrplus ( hain lifescience , nehren , germany ) assay ( for details , see the technical appendix ) . for 3 of the 4 anti - tb drugs ,
the difference in the range of mutant prevention concentrations for the beijing and eai genotype strains was small : inh , 128256 mg / l ; rif , 2561,024 mg / l ; and mxf , 28 mg / l .
we showed that the currently used anti - tb drug susceptibility assays do not discriminate between the in vitro susceptibility , as determined by the methods used in this study , of the m. tuberculosis beijing and eai genotype strains .
we also showed that the determination of mutation frequencies might be more informative than results of anti - tb drug susceptibility assays .
for rif , mutation frequencies in beijing genotype strains were high compared with those in eai genotype strains , and the selection of rif - resistant mutants among beijing strains , but not eai strains , occurred within a wide range of rif concentrations . in addition , the killing capacity of rif toward the beijing genotype is dependent on the density of mycobacteria : high concentrations of rif are required to achieve 100% killing of high - density beijing genotype populations but not of high - density eai genotype populations .
these in vitro characteristics might contribute to the less favorable treatment outcome of beijing genotype tb infections and their significant association with drug resistance .
our findings demonstrate the need for anti - tb drug treatments that will prevent resistance among m. tuberculosis beijing genotype tb cases , and they suggest that the development of genotype - specific tb therapy might be justified .
detailed methods for liquid culturing system ( bd bactec mgit 960 system ; bd diagnostics , sparks , md , us ) used to show drug susceptibility of mycobacterium tuberculosis beijing and east - african / indian genotype strains .
 to determine differences in the ability of mycobacterium tuberculosis strains to withstand antituberculosis drug treatment , we compared the activity of antituberculosis drugs against susceptible beijing and east - african / indian genotype m. tuberculosis strains . 
 beijing genotype strains showed high rates of mutation within a wide range of drug concentrations , possibly explaining this genotype s association with multidrug - resistant tuberculosis . 
The Study
Conclusions
Supplementary Material