Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

Abstract

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing. The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 hours for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Amotosalen is a bacterial multidrug efflux pump substrate potentially affecting its pathogen inactivation activity

“…Accordingly, two-fold serial dilutions of amotosalen were prepared in microwell format, and added to bacteria in standard cation-adjusted Mueller-Hinton broth under conditions recommended by the Clinical Laboratory Standards Institute (33). Microplates were exposed to 2 Joules/cm 2 of UVA, to approximate exposure during platelet pathogen inactivation (17), and incubated overnight to determine the MIC through absorbance measurements as previously described (34–37).…”

“…Filled 384-well plates were centrifuged at 1250 RCF for four minutes to ensure the entire inoculum was in continuity with the bottom of the well. Then two-fold doubling dilutions of stock solutions of amotosalen supplemented to 0.3% Tween-20 (Sigma-Aldrich), AMT, or 8-MOP were dispensed into microwells using the HP D300 digital dispensing system (HP, Inc. Palo Alto, CA), as previously described,(34–37) and the microplates were mixed for 5 minutes on a microplate shaker to ensure complete mixing of psoralens with the inoculum. Microplates were then exposed to ultraviolet light in a UV Stratalinker 1800 (Stratagene, La Jolla, CA), retrofitted with UV BL F8T5 CFL 12-inch UVA 365nm Blacklight Bulbs (Coolspider, Jiinyun, China) and calibrated according to manufacturer’s instructions with an UVA365 UV Light Meter (Amtast, Lakeland, FL).…”

“…After incubation at 35°C for 20 h, the A 600 of the microplate was measured using a TECAN M1000 microplate reader. Minimal inhibitory concentration was determined based on a growth inhibition A 600 cutoff of 0.06, consistent with our prior determination of absorbance cutoffs for accurate MIC determinations in 384-well plate format (35, 37, 81). Notably, the AdeABC pump was inactive in E. coli AG100X in Mueller-Hinton broth without the addition of divalent cations, consistent with prior use of magnesium supplementation when expressing this A. baumannii efflux pump in E. coli (82).…”

Discovery of small-molecule inhibitors of multidrug-resistance plasmid maintenance using a high-throughput screening approach

“…Serial twofold dilutions of meropenem and test compounds were dispensed into wells in orthogonal titrations using the D300 Digital Dispensing System. Plates were incubated for 24 h and combinatorial MICs determined based on A 600 measure using the TECAN M1000 as previously described (46,47).…”

We Cannot Do It Alone

“…Their laboratory network has adopted inkjet printing technology for this AST testing, originally described by Smith and Kirby and Brennan-Krohn and Kirby, that allows highly accurate and precise at-will set-up and testing of any desired antimicrobial alone or in combination with reference broth microdilution equivalent AST results. 10 , 11 , 12 , 13 , 14 The ALRN currently offers, for example, the combination AST of aztreonam + ceftazidime-avibactam. Furthermore, it has the capacity to characterize isolates via whole genome sequencing and other molecular testing.…”