Journal: Emerging Microbes & Infections

Article Title: Rapid antimicrobial susceptibility test for identification of new therapeutics and drug combinations against multidrug-resistant bacteria

doi: 10.1038/emi.2016.123

Figure Lengend Snippet: List of bacterial strains used in this study

Article Snippet: ECB2 , E. cloacae isolate , College of American Pathologists Breakpoint Implementation Toolkit, 2012.

Techniques:

Journal: Emerging Microbes & Infections

Article Title: Rapid antimicrobial susceptibility test for identification of new therapeutics and drug combinations against multidrug-resistant bacteria

doi: 10.1038/emi.2016.123

Figure Lengend Snippet: Development and validation of automated high-throughput bacterial growth assay (HIGA). ( A ) Diagram of standard susceptibility assay and high-throughput antibiotic screening method timelines. ( B ) Endpoint growth assay for K . pneumoniae (KPNIH301), A. baumannii (ABNIH144), P. aeruginosa (PANIH338), C. freundii (CFB10), E. cloacae (ECB2) and E. coli (ECOB11); n =32. Bars represent mean, and error bars represent the SD. ( C ) Growth curve of K. pneumoniae KPNIH301 in 1536-well plate. Frozen KPNIH301 were diluted to different starting ratios and incubated at 37 °C. Data points represent the mean, and error bars represent the SD; n =3. ( D ) Time course of KPNIH301 growth in the presence of 46 μM ciprofloxacin or 0.46% dimethylsulphoxide (DMSO). Data points represent individual experiments; n =3. * X axis is not linear. ( E ) Dose–response curves for ciprofloxacin. KPNIH301 was incubated with varying concentrations of ciprofloxacin at 37 °C for 6, 8, 10 and 20 h. The data points represent the mean, and error bars represent the SD; n =3. ( F ) Concentration–response curves for ciprofloxacin in 96-well, 384-well, 1536-well absorbance assay (AB) and ATP content luminescence assay (LM). KPNIH301 was incubated with different concentrations of ciprofloxacin for 8 h at 37 °C before detection at OD 600 . The data points represent the mean, and error bars represent the SD; n =3. ( G ) Scatter plot of the results from a DMSO plate screening. The wells in column 1 of the 1536-well assay plates contained 46 μM ciprofloxacin as a positive control (0% viability); the wells in column 3 contained varying doses of ciprofloxacin at 1:3 serial dilutions from top to bottom. The wells in the rest of plate contained DMSO as a negative control (100% viability). The signal-to-basal ratio (S/B) in this plate was 10.6-fold, with a coefficient of variation (CV) of 8% and a Z′ factor of 0.61.

Article Snippet: ECB2 , E. cloacae isolate , College of American Pathologists Breakpoint Implementation Toolkit, 2012.

Techniques: Biomarker Discovery, High Throughput Screening Assay, Growth Assay, Drug Susceptibility Assay, Incubation, Concentration Assay, Luminescence Assay, Positive Control, Negative Control

Journal: Emerging Microbes & Infections

Article Title: Rapid antimicrobial susceptibility test for identification of new therapeutics and drug combinations against multidrug-resistant bacteria

doi: 10.1038/emi.2016.123

Figure Lengend Snippet: Broad-spectrum bactericidal effects of three-drug combinations. Fifteen three-drug targeted drug combinations (TDCs) were tested at their IC 90 concentrations against 10 multidrug-resistant strains, including K. pneumoniae (KPNIH776 and KPNIH892), A. baumannii (ABNIH144, ABNIH233 and ABNIH333), P. aeruginosa (PANIH338 and PANIH668), C. freundii (CFB10), E. cloacae (ECB2) and E. coli (ECOB11). ( A ) Heatmap of bactericidal effects of 15 TDCs against 10 MDR bacteria; 0% viability (red), 20% viability (white), 50% viability (blue) and 100% viability (black). Comb1 (rifabutin—0.052 μM, polymyxin B—1 μM and zidovudine—1 μM); Comb2 (rifabutin—0.056 μM, polymyxin B—1 μM and trimethoprim—4 μM); Comb3 (rifabutin—0.056 μM, polymyxin B—1 μM and aztreonam—4 μM); Comb4 (rifabutin—0.06 μM, polymyxin B—1 μM and ceftazidime—15 μM); Comb4* (rifabutin—0.06 μM, polymyxin B—1 μM and ceftazidime—4 μM); Comb5 (rifabutin—0.09 μM, polymyxin B—1 μM and imipenem—16 μM); Comb5* (rifabutin—0.09 μM, polymyxin B—1 μM and imipenem—8 μM); Comb6 (rifabutin—0.052 μM, colistin—2.1 μM and zidovudine—1 μM); Comb7 (rifabutin—0.056 μM, colistin—2.1 μM and trimethoprim—4 μM); Comb8 (rifabutin—0.056 μM, colistin—2.1 μM and aztreonam—4 μM); Comb9 (rifabutin—0.06 μM, colistin—2.1 μM and ceftazidime—15 μM); Comb9* (rifabutin—0.06 μM, colistin—2.1 μM and ceftazidime—8 μM); Comb9# (rifabutin—0.06 μM, colistin—2.1 μM and ceftazidime—4 μM); Comb10 (rifabutin—0.09 μM, colistin—2.1 μM and imipenem—16 μM); Comb10* (rifabutin—0.09 μM, colistin—2.1 μM and imipenem—8 μM); Comb11 (colistin—1.2 μM, auranofin—1 μM and imipenem—16 μM); Comb11* (colistin—1.2 μM, auranofin—1 μM and imipenem—8 μM); Comb12 (colistin—1.9 μM, auranofin—1 μM and rifabutin—0.2 μM); Comb13 (colistin—1.7 μM, auranofin—1 μM and ceftazidime—15 μM); Comb13* (colistin—1.7 μM, auranofin—1 μM and ceftazidime—8 μM); Comb13# (colistin—1.7 μM, auranofin—1 μM and ceftazidime—4 μM); Comb14 (colistin—2.1 μM, auranofin—1 μM and zidovudine—1 μM); Comb15 (polymyxin B—1.8 μM, auranofin—1 μM and ceftazidime—15 μM); Comb15* (polymyxin B—1.8 μM, auranofin—1 μM and ceftazidime—8 μM); Comb15# (polymyxin B—1.8 μM, auranofin—1 μM and ceftazidime—4 μM); n =4; * and # represent the same drugs are in combination but with different concentrations. ( B ) Top three TDCs and dimethylsulphoxide (DMSO) control were plotted as % normalized viability of different MDR isolates: (i) colistin—1.96 μg/mL, auranofin—0.68 μg/mL and ceftazidime—8.20 μg/mL (green) (Comb13 in A; (ii) colistin—2.19 μg/mL, auranofin—0.68 μg/mL and rifabutin—0.17 μg/mL (blue) (Comb12 in A; (iii) rifabutin—0.08 μg/mL, colistin—2.43 μg/mL and imipenem—4.80 μg/mL (purple) (Comb10 in A; n =4. Bar graph represent mean, and error bars represent the SEM. ( C ) Clinical breakpoints , and drug concentrations in three-drug TDCs. Bars represent drug concentrations of colistin (black), auranofin (green), ceftazidime (blue), rifabutin (purple) and imipenem (orange), and corresponding colored dashed lines represent individual drug susceptibility breakpoints. Breakpoints were selected for auranofin and rifabutin based on the selected literature. Imipenem and ceftazadime breakpoints were based on CLSI guidelines for Enterobacteriaceae. Imipenem breakpoint is 2 μg/mL and ceftazadime breakpoint is 8 μg/mL for Acinetobacter spp. and Pseudomonas spp.

Article Snippet: ECB2 , E. cloacae isolate , College of American Pathologists Breakpoint Implementation Toolkit, 2012.

Techniques: Bacteria, Control