PMC6160733_01
"Real-time" high-throughput drug and combination testing against multidrug-resistant organisms (MDRO). A total of 8 drugs and 14 drug combinations (in 2,304 samples) were tested against three MDRO K. pneumoniae-KP11, E. coli-Ec1A and E. coli-Ec2B. (A-D) Growth inhibition of KP11, Ec1A and Ec2B by individual drugs (1 vehicle; 2 gentamicin; 3 colistin; 4 rifabutin; 5 imipenem; 6 ceftazidime; 7 meropenem; 8 tigecycline) and drug combinations (9 colistin+gentamicin+rifabutin, 10 colistin+imipenem+rifabutin, 11 meropenem+tigecycline+colistin, 12 meropenem+tigecycline+colistin+ceftazidime, 13 meropenem+tigecycline+colistin+rifabutin) in high-throughput bacterial assay. Colors represent drug potency (% of growth inhibition). Vehicle control was normalized as 0% inhibition and no bacterium was normalized as 100% inhibition. n = 4, mean +- SEM. (E) Growth inhibition of KP11 by combinations of colistin, gentamicin, and rifabutin in high-throughput bacterial assay. Vehicle (-), colistin at 0.5 mug/ml (+) or 2 mug/ml (++), gentamicin at 1 mug/ml (+) or 4 mug/ml (++), or rifabutin at 0.06 mug/ml were tested at indicated conditions. n = 3, mean +- SEM. (F) Growth inhibition of KP11 by colistin, gentamicin, and the combination of colistin and gentamicin in standard micro-broth assay. 100 mug/ml meropenem (-) or vehicle alone (+) were included as control. + was visual bacterial growth. - was no visual bacterial growth. Rep, repeat. n = 6.
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Each sample was tested in quadruplicate, amounting to a total of 2,304 samples which were run on three 1,536-well plates using clinically-relevant drug concentrations (Figures 1A-D). For the three-drug combinations, #9 (gentamicin+colistin+rifabutin) successfully suppressed > 90% growth of all three strains (Figure 1A). #10 (colistin+imipenem+rifabutin) and #11 (meropenem+tigecycline+colistin) were less effective, inhibiting 54-90% of all three strains. Next, we studied the three-drug combination (gentamicin+colistin+rifabutin) against KP11 at various drug concentrations (Figure 1E). The combination of 0.25 mug/ml colistin and 8 mug/ml gentamicin was inhibitory in all but one of six replicates (Figure 1F).