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Establishment and validation of a 384-well antibacterial assay amenable for high-throughput screening and combination testing
Journal of Microbiological Methods 118 (2015) 173–175
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Establishment and validation of a 384-well antibacterial assay amenable for high-throughput screening and combination testing Arti Mishra a, Svetlana V. Dobritsa a,b, Marie-Laure Crouch b, John Rabenstein b, Joycelyn Xiang Yi Lee a, Saravanakumar Dhakshinamoorthy a,⁎ a b
Biology and Pharmacology, Albany Molecular Research, Inc., The Galen, #05-01, 61 Science Park Road, 117525, Singapore Lead Discovery, Albany Molecular Research, Inc., 22215 26th Avenue SE, Bothell, WA 98021, United States
a r t i c l e
i n f o
Article history: Received 7 September 2015 Received in revised form 29 September 2015 Accepted 29 September 2015 Available online 1 October 2015
a b s t r a c t A 384-well-based antibacterial assay amenable for high-throughput screening and combination testing is described. The assay uses 100–500 nL of test compounds and tolerates up to 2.5% dimethyl sulfoxide concentrations. It can be used for screening compound libraries and testing combinatory/synergistic/antagonistic effects of antibiotics, small molecules, and natural product extracts. © 2015 Elsevier B.V. All rights reserved.
Keywords: Antibacterial assay Combination testing DMSO tolerance HTS
The rise and spread of multiple drug resistance among many bacterial strains have resulted in enhanced screening efforts to identify novel chemical entities active against wild-type and drug-resistant human pathogens (Nicasio et al., 2008; Kumarasamy et al., 2010). Screening for new antibacterial drug candidates requires a rapid, robust and cost-effective in vitro susceptibility assay. In the present study, a bacterial growth inhibition assay amenable for high-throughput screening (HTS) and combination testing was optimized, validated, and most importantly miniaturized to a 384-well plate format and automated. Assay validation included assessments of assay performance and dimethyl sulfoxide (DMSO) tolerance. Also, the minimum inhibitory concentration (MIC) values obtained were confirmed to be within the standard ranges published by the Clinical and Laboratory Standards Institute (CLSI, 2012). The bacterial strains used for the study were Staphylococcus aureus ATCC 29213, Salmonella typhimurium ATCC 14028, Klebsiella pneumoniae ATCC 4352, Acinetobacter baumanii BAA-1605, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922. The strains were propagated in BBL™ Mueller–Hinton II broth (MHB, CationAdjusted) or on BBL™ Mueller-Hinton II agar (Becton, Dickinson and Company, USA). Fresh glycerol (10%) stocks were made for each strain and stored at − 80 °C. Bacterial titers based on colony-forming units (CFUs) were determined using standard procedures (Miles et al., 1938). DMSO tolerance was determined for each bacterium by growing ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (S. Dhakshinamoorthy).
http://dx.doi.org/10.1016/j.mimet.2015.09.019 0167-7012/© 2015 Elsevier B.V. All rights reserved.
cells in MHB supplemented with increasing concentrations of DMSO (0.01–5%). All bacterial strains showed tolerance to DMSO up to a concentration of 2.5% (data not shown). The standard broth microdilution method for the determination of MIC values (CLSI, 2012) was modified to allow the use of 384-well
Table 1 Assay performance parameters for different bacterial strains. Strain
A.baumanii BAA 1605 E. coli ATCC 25922 K. pneumoniae ATCC 4352 P. aeruginosa ATCC 27853 S. typhimurium ATCC 14028 S. aureus ATCC 29213
Assay Day
1 2 1 2 1 2 1 2 1 2 1 2
Statistical assay parameters Positive control
Negative control
Assay criteria
Absorbance (mean ± SD)
CV (%)
Absorbance (mean ± SD)
CV (%)
S/Ba
Z′b
1.47 ± 0.04 1.57 ± 0.04 1.23 ± 0.06 1.57 ± 0.04 0.91 ± 0.04 0.97 ± 0.05 1.66 ± 0.02 1.67 ± 0.15 1.31 ± 0.06 1.23 ± 0.07 0.77 ± 0.03 0.81 ± 0.05
3.00 2.27 5.10 2.37 4.14 7.50 0.91 0.94 4.14 5.47 6.26 6.62
0.17 ± 0.01 0.17 ± 0.01 0.17 ± 0.01 0.17 ± 0.01 0.16 ± 0.01 0.17 ± 0.01 0.19 ± 0.01 0.17 ± 0.01 0.18 ± 0.01 0.17 ± 0.03 0.17 ± 0.01 0.16 ± 0.01
6.45 6.70 7.24 6.70 5.01 9.52 7.23 8.41 8.44 9.69 6.57 7.30
8.82 9.03 7.36 7.40 5.60 6.36 8.64 9.91 7.32 7.46 4.22 4.42
0.87 0.90 0.79 0.79 0.80 0.68 0.94 0.94 0.83 0.77 0.69 0.68
a S/B — signal-to-background ratio (Mean absorbance for positive control/Mean absorbance for negative control). b Z′ was calculated using 1 − [(3SD of positive control + 3SD of negative control) / (Mean absorbance for positive control − Mean absorbance for negative control] (Zhang et al., 1999).
174
A. Mishra et al. / Journal of Microbiological Methods 118 (2015) 173–175
Fig. 1. Assay parameters calculated for different strains after 14–24 h of incubation: A) Z′ values and B) S/B ratios.
microplates. The antibiotics tested included amikacin, azithromycin, ceftriaxone, chloramphenicol, levofloxacin, and tetracycline (Sigma-Aldrich, USA). Antibiotic stock solutions were prepared in DMSO and source plates (Corning #3656, USA) containing two-fold serial dilutions of each antibiotic were prepared using a BioMek FX liquid handler (Beckman Coulter, USA). Assay plates (Corning #3680, USA) were prepared from source plates by capillary transfer of 500 nL per well using a HummingBird Plus liquid handling instrument (Digilab, Marlborough, MA). Bacterial inocula of 5 × 105 CFU/mL were used and inoculum densities were verified for each assay using CFU counts. Bacterial cultures (50 μl/well) were dispensed using Multidrop™ combi reagent dispenser (Thermo Scientific, USA). A total of 13 antibiotic concentrations (from 0.03 to 128 μg/mL) were tested. The final concentration of DMSO in the assay was kept at 1%. Each assay plate contained 64 positive control wells and 32 negative control wells. The negative control wells received the assay medium only, and the positive control wells contained bacterial cultures without antibiotics but with DMSO. Following cell dispensing, plates were incubated for 20 h at 37 °C, and bacterial growth was measured by absorbance at 600 nm using Analyst GT Multimode Reader (Molecular Devices, Sunnyvale CA). Assay performance was monitored using a combination of the Z′ score, coefficient of variation (CV) and signal-to-background (S/B) ratios. The Z′ scores of ≥0.5 (Zhang et al., 1999) the CV values of ≤10% for both positive and negative growth controls (Iversen et al., 2012), and the signal-to-background (S/B) ratio of N2 were chosen as assay acceptance criteria (Zhang et al., 1999). We have used CV b 10% instead of CV ≤ 20%, recommended by Iversen et al. (2012) to make the acceptance criteria more stringent and suitable for bacterial HTS campaign. The assay performance parameters obtained for the bacterial strains after 20 h of incubation are listed in Table 1. The Z′ scores were greater
than 0.5 (0.68 to 0.94), and the CV values for the positive and negative controls were b10%. The S/B values were N2 (4.2–9.9), indicating good separation between the positive and negative growth controls. The data was also found to be consistent between the two experimental days, confirming the robustness of the assay. The MIC data for different antibiotics/strains were within the expected ranges as reported by the CLSI (Table 1, supplemental data). The assay performance for all strains was also evaluated using incubation periods of 14 to 24 h. All strains maintained a good assay window between 18 and 24 h of incubation, with Z′ N 0.5 and S/B N 2 (Fig. 1, A and B). Moreover, the MIC values obtained for the different antibiotics at different time points between 18 and 24 h of incubation were comparable (data not shown). This suggests that the assay is robust and stable between 18 and 24 h of incubation, which allows reading multiple assay plates in a staggered manner during the HTS campaign. Combinations of different concentrations of amikacin and ceftriaxone were also tested against P. aeruginosa and A. baumanii in 384-well plates using a checkerboard technique (Zusman et al., 2013). The combination experiments were carried out at a final DMSO concentration of 2%. In the combination testing, P. aeruginosa was more susceptible to the combination than to a single agent (Fig. 2A) (Prosser et al., 1991), while A. baumanii showed no difference (Fig. 2B). Combination of amikacin and ceftriaxone showed a synergistic effect at some of the concentrations tested against P. aeruginosa (Fig. 2A, highlighted in yellow) (Prosser et al., 1991). The described bacterial growth inhibition assay is easy, rapid, costeffective and highly robust. The entire procedure from preparation of plates, test compound stamping, bacterial inoculation, and plate reading to data analysis takes 20–24 h. The plate map can easily be modified to accommodate compounds in single concentration (maximum 320
A. Mishra et al. / Journal of Microbiological Methods 118 (2015) 173–175
175
Fig. 2. Percentage growth inhibition shown by P. aeruginosa ATCC 27853 (A) and A. baumanii BAA-1605 (B) in the presence of different concentrations of amikacin and ceftriaxone tested in duplicate.
compounds per plate) or in dose–response series. As little as 100– 500 nL of an antibiotic/test compound per well can be used for this assay, and the assay tolerates a wide range of DMSO concentrations (up to 2.5%). Overall, the assay described herein is suitable for HTS campaigns using small molecule or natural product libraries and for testing combinatory/synergistic/antagonistic effects of antibiotics, small molecules and natural product extracts. We thank our colleagues Michele Luche and Dr. Grant Carr for useful discussions and suggestions. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mimet.2015.09.019. References CLSI, 2012. Performance Standards for Antimicrobial Testing. 23rd Informat. Suppl., M100-S22 32 (No. 3. CLSI). Iversen, P.W., Beck, B., Chen, Y.F., Dere, W., Devanarayan, V., Eastwood, B.J., Farmen, M.W., Iturria, S.J., Montrose, C., Moore, R.A., Weidner, J.R., Sittampalam, G.S., 2012. HTS Assay Validation. In: Sittampalam, G.S., Coussens, N.P., Nelson, H., et al. (Eds.), Assay Guidance Manual [Internet]. Eli Lilly & Company and the National Center for
Advancing Translational Sciences, Bethesda (MD) May 1 [Updated 2012 Oct 1]. (2004-. Available from: http://www.ncbi.nlm.nih.gov/books/NBK83783/). Kumarasamy, K.K., Toleman, M.A., Walsh, T.R., Bagaria, J., Butt, F., Balakrishnan, R., Chaudhary, U., Doumith, M., Giske, C.G., Irfan, S., Krishnan, P., Kumar, A.V., Maharjan, S., Mushtaq, S., Noorie, T., Paterson, D.L., Pearson, A., Perry, C., Pike, R., Rao, B., Ray, U., Sarma, J.B., Sharma, M., Sheridan, E., Thirunarayan, M.A., Turton, J., Upadhyay, S., Warner, M., Welfare, W., Livermore, D.M., Woodford, N., 2010. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect. Dis. 10, 597–602. http:// dx.doi.org/10.1016/S1473-3099(10)70143-2. Miles, A.A., Misra, S.S., Irwin, J.O., 1938. The estimation of the bactericidal power of the blood. J. Hyg. 38, 732–749. Nicasio, A.M., Kuti, J.L., Nicolau, D.P., 2008. The current state of multidrug-resistant Gramnegative bacilli in North America. Pharmacotherapy 28, 235–249. Prosser, B.L.T., Taylor, D.B., Cleeland, R., 1991. In vitro activity of ceftriaxone and amikacin against Pseudomonas aeruginosa. Chemotherapy 37, 93–97. Zhang, J., Chung, T., Oldenburg, K.R., 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73. Zusman, O., Avni, T., Leibovici, L., Adler, A., Friberg, L., Stergiopoulou, T., Carmeli, Y., Paul, M., 2013. Systematic review and meta-analysis of in vitro synergy of polymyxins and carbapenems. Antimicrob. Agents Chemother. 57, 5104–5111. http://dx.doi.org/10. 1128/AAC.01230-13.
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
Note
Establishment and validation of a 384-well antibacterial assay amenable for high-throughput screening and combination testing Arti Mishra a, Svetlana V. Dobritsa a,b, Marie-Laure Crouch b, John Rabenstein b, Joycelyn Xiang Yi Lee a, Saravanakumar Dhakshinamoorthy a,⁎ a b
Biology and Pharmacology, Albany Molecular Research, Inc., The Galen, #05-01, 61 Science Park Road, 117525, Singapore Lead Discovery, Albany Molecular Research, Inc., 22215 26th Avenue SE, Bothell, WA 98021, United States
a r t i c l e
i n f o
Article history: Received 7 September 2015 Received in revised form 29 September 2015 Accepted 29 September 2015 Available online 1 October 2015
a b s t r a c t A 384-well-based antibacterial assay amenable for high-throughput screening and combination testing is described. The assay uses 100–500 nL of test compounds and tolerates up to 2.5% dimethyl sulfoxide concentrations. It can be used for screening compound libraries and testing combinatory/synergistic/antagonistic effects of antibiotics, small molecules, and natural product extracts. © 2015 Elsevier B.V. All rights reserved.
Keywords: Antibacterial assay Combination testing DMSO tolerance HTS
The rise and spread of multiple drug resistance among many bacterial strains have resulted in enhanced screening efforts to identify novel chemical entities active against wild-type and drug-resistant human pathogens (Nicasio et al., 2008; Kumarasamy et al., 2010). Screening for new antibacterial drug candidates requires a rapid, robust and cost-effective in vitro susceptibility assay. In the present study, a bacterial growth inhibition assay amenable for high-throughput screening (HTS) and combination testing was optimized, validated, and most importantly miniaturized to a 384-well plate format and automated. Assay validation included assessments of assay performance and dimethyl sulfoxide (DMSO) tolerance. Also, the minimum inhibitory concentration (MIC) values obtained were confirmed to be within the standard ranges published by the Clinical and Laboratory Standards Institute (CLSI, 2012). The bacterial strains used for the study were Staphylococcus aureus ATCC 29213, Salmonella typhimurium ATCC 14028, Klebsiella pneumoniae ATCC 4352, Acinetobacter baumanii BAA-1605, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 25922. The strains were propagated in BBL™ Mueller–Hinton II broth (MHB, CationAdjusted) or on BBL™ Mueller-Hinton II agar (Becton, Dickinson and Company, USA). Fresh glycerol (10%) stocks were made for each strain and stored at − 80 °C. Bacterial titers based on colony-forming units (CFUs) were determined using standard procedures (Miles et al., 1938). DMSO tolerance was determined for each bacterium by growing ⁎ Corresponding author. E-mail addresses: [email protected], [email protected] (S. Dhakshinamoorthy).
http://dx.doi.org/10.1016/j.mimet.2015.09.019 0167-7012/© 2015 Elsevier B.V. All rights reserved.
cells in MHB supplemented with increasing concentrations of DMSO (0.01–5%). All bacterial strains showed tolerance to DMSO up to a concentration of 2.5% (data not shown). The standard broth microdilution method for the determination of MIC values (CLSI, 2012) was modified to allow the use of 384-well
Table 1 Assay performance parameters for different bacterial strains. Strain
A.baumanii BAA 1605 E. coli ATCC 25922 K. pneumoniae ATCC 4352 P. aeruginosa ATCC 27853 S. typhimurium ATCC 14028 S. aureus ATCC 29213
Assay Day
1 2 1 2 1 2 1 2 1 2 1 2
Statistical assay parameters Positive control
Negative control
Assay criteria
Absorbance (mean ± SD)
CV (%)
Absorbance (mean ± SD)
CV (%)
S/Ba
Z′b
1.47 ± 0.04 1.57 ± 0.04 1.23 ± 0.06 1.57 ± 0.04 0.91 ± 0.04 0.97 ± 0.05 1.66 ± 0.02 1.67 ± 0.15 1.31 ± 0.06 1.23 ± 0.07 0.77 ± 0.03 0.81 ± 0.05
3.00 2.27 5.10 2.37 4.14 7.50 0.91 0.94 4.14 5.47 6.26 6.62
0.17 ± 0.01 0.17 ± 0.01 0.17 ± 0.01 0.17 ± 0.01 0.16 ± 0.01 0.17 ± 0.01 0.19 ± 0.01 0.17 ± 0.01 0.18 ± 0.01 0.17 ± 0.03 0.17 ± 0.01 0.16 ± 0.01
6.45 6.70 7.24 6.70 5.01 9.52 7.23 8.41 8.44 9.69 6.57 7.30
8.82 9.03 7.36 7.40 5.60 6.36 8.64 9.91 7.32 7.46 4.22 4.42
0.87 0.90 0.79 0.79 0.80 0.68 0.94 0.94 0.83 0.77 0.69 0.68
a S/B — signal-to-background ratio (Mean absorbance for positive control/Mean absorbance for negative control). b Z′ was calculated using 1 − [(3SD of positive control + 3SD of negative control) / (Mean absorbance for positive control − Mean absorbance for negative control] (Zhang et al., 1999).
174
A. Mishra et al. / Journal of Microbiological Methods 118 (2015) 173–175
Fig. 1. Assay parameters calculated for different strains after 14–24 h of incubation: A) Z′ values and B) S/B ratios.
microplates. The antibiotics tested included amikacin, azithromycin, ceftriaxone, chloramphenicol, levofloxacin, and tetracycline (Sigma-Aldrich, USA). Antibiotic stock solutions were prepared in DMSO and source plates (Corning #3656, USA) containing two-fold serial dilutions of each antibiotic were prepared using a BioMek FX liquid handler (Beckman Coulter, USA). Assay plates (Corning #3680, USA) were prepared from source plates by capillary transfer of 500 nL per well using a HummingBird Plus liquid handling instrument (Digilab, Marlborough, MA). Bacterial inocula of 5 × 105 CFU/mL were used and inoculum densities were verified for each assay using CFU counts. Bacterial cultures (50 μl/well) were dispensed using Multidrop™ combi reagent dispenser (Thermo Scientific, USA). A total of 13 antibiotic concentrations (from 0.03 to 128 μg/mL) were tested. The final concentration of DMSO in the assay was kept at 1%. Each assay plate contained 64 positive control wells and 32 negative control wells. The negative control wells received the assay medium only, and the positive control wells contained bacterial cultures without antibiotics but with DMSO. Following cell dispensing, plates were incubated for 20 h at 37 °C, and bacterial growth was measured by absorbance at 600 nm using Analyst GT Multimode Reader (Molecular Devices, Sunnyvale CA). Assay performance was monitored using a combination of the Z′ score, coefficient of variation (CV) and signal-to-background (S/B) ratios. The Z′ scores of ≥0.5 (Zhang et al., 1999) the CV values of ≤10% for both positive and negative growth controls (Iversen et al., 2012), and the signal-to-background (S/B) ratio of N2 were chosen as assay acceptance criteria (Zhang et al., 1999). We have used CV b 10% instead of CV ≤ 20%, recommended by Iversen et al. (2012) to make the acceptance criteria more stringent and suitable for bacterial HTS campaign. The assay performance parameters obtained for the bacterial strains after 20 h of incubation are listed in Table 1. The Z′ scores were greater
than 0.5 (0.68 to 0.94), and the CV values for the positive and negative controls were b10%. The S/B values were N2 (4.2–9.9), indicating good separation between the positive and negative growth controls. The data was also found to be consistent between the two experimental days, confirming the robustness of the assay. The MIC data for different antibiotics/strains were within the expected ranges as reported by the CLSI (Table 1, supplemental data). The assay performance for all strains was also evaluated using incubation periods of 14 to 24 h. All strains maintained a good assay window between 18 and 24 h of incubation, with Z′ N 0.5 and S/B N 2 (Fig. 1, A and B). Moreover, the MIC values obtained for the different antibiotics at different time points between 18 and 24 h of incubation were comparable (data not shown). This suggests that the assay is robust and stable between 18 and 24 h of incubation, which allows reading multiple assay plates in a staggered manner during the HTS campaign. Combinations of different concentrations of amikacin and ceftriaxone were also tested against P. aeruginosa and A. baumanii in 384-well plates using a checkerboard technique (Zusman et al., 2013). The combination experiments were carried out at a final DMSO concentration of 2%. In the combination testing, P. aeruginosa was more susceptible to the combination than to a single agent (Fig. 2A) (Prosser et al., 1991), while A. baumanii showed no difference (Fig. 2B). Combination of amikacin and ceftriaxone showed a synergistic effect at some of the concentrations tested against P. aeruginosa (Fig. 2A, highlighted in yellow) (Prosser et al., 1991). The described bacterial growth inhibition assay is easy, rapid, costeffective and highly robust. The entire procedure from preparation of plates, test compound stamping, bacterial inoculation, and plate reading to data analysis takes 20–24 h. The plate map can easily be modified to accommodate compounds in single concentration (maximum 320
A. Mishra et al. / Journal of Microbiological Methods 118 (2015) 173–175
175
Fig. 2. Percentage growth inhibition shown by P. aeruginosa ATCC 27853 (A) and A. baumanii BAA-1605 (B) in the presence of different concentrations of amikacin and ceftriaxone tested in duplicate.
compounds per plate) or in dose–response series. As little as 100– 500 nL of an antibiotic/test compound per well can be used for this assay, and the assay tolerates a wide range of DMSO concentrations (up to 2.5%). Overall, the assay described herein is suitable for HTS campaigns using small molecule or natural product libraries and for testing combinatory/synergistic/antagonistic effects of antibiotics, small molecules and natural product extracts. We thank our colleagues Michele Luche and Dr. Grant Carr for useful discussions and suggestions. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.mimet.2015.09.019. References CLSI, 2012. Performance Standards for Antimicrobial Testing. 23rd Informat. Suppl., M100-S22 32 (No. 3. CLSI). Iversen, P.W., Beck, B., Chen, Y.F., Dere, W., Devanarayan, V., Eastwood, B.J., Farmen, M.W., Iturria, S.J., Montrose, C., Moore, R.A., Weidner, J.R., Sittampalam, G.S., 2012. HTS Assay Validation. In: Sittampalam, G.S., Coussens, N.P., Nelson, H., et al. (Eds.), Assay Guidance Manual [Internet]. Eli Lilly & Company and the National Center for
Advancing Translational Sciences, Bethesda (MD) May 1 [Updated 2012 Oct 1]. (2004-. Available from: http://www.ncbi.nlm.nih.gov/books/NBK83783/). Kumarasamy, K.K., Toleman, M.A., Walsh, T.R., Bagaria, J., Butt, F., Balakrishnan, R., Chaudhary, U., Doumith, M., Giske, C.G., Irfan, S., Krishnan, P., Kumar, A.V., Maharjan, S., Mushtaq, S., Noorie, T., Paterson, D.L., Pearson, A., Perry, C., Pike, R., Rao, B., Ray, U., Sarma, J.B., Sharma, M., Sheridan, E., Thirunarayan, M.A., Turton, J., Upadhyay, S., Warner, M., Welfare, W., Livermore, D.M., Woodford, N., 2010. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect. Dis. 10, 597–602. http:// dx.doi.org/10.1016/S1473-3099(10)70143-2. Miles, A.A., Misra, S.S., Irwin, J.O., 1938. The estimation of the bactericidal power of the blood. J. Hyg. 38, 732–749. Nicasio, A.M., Kuti, J.L., Nicolau, D.P., 2008. The current state of multidrug-resistant Gramnegative bacilli in North America. Pharmacotherapy 28, 235–249. Prosser, B.L.T., Taylor, D.B., Cleeland, R., 1991. In vitro activity of ceftriaxone and amikacin against Pseudomonas aeruginosa. Chemotherapy 37, 93–97. Zhang, J., Chung, T., Oldenburg, K.R., 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73. Zusman, O., Avni, T., Leibovici, L., Adler, A., Friberg, L., Stergiopoulou, T., Carmeli, Y., Paul, M., 2013. Systematic review and meta-analysis of in vitro synergy of polymyxins and carbapenems. Antimicrob. Agents Chemother. 57, 5104–5111. http://dx.doi.org/10. 1128/AAC.01230-13.