Surprising synergy of dual translation inhibition vs. Acinetobacter baumannii and other multidrug-resistant bacterial pathogens

Abstract

Background Multidrug-resistant (MDR) Acinetobacter baumannii infections have high mortality rates and few treatment options. Synergistic drug combinations may improve clinical outcome and reduce further emergence of resistance in MDR pathogens. Here we show an unexpected potent synergy of two translation inhibitors against the pathogen: commonly prescribed macrolide antibiotic azithromycin (AZM), widely ignored as a treatment alternative for invasive Gram-negative pathogens, and minocycline, among the current standard-of-care agents used for A. baumannii . Methods Media-dependent activities of AZM and MIN were evaluated in minimum inhibitory concentration assays and kinetic killing curves, alone or in combination, both in standard bacteriologic media (cation-adjusted Mueller-Hinton Broth) and more physiologic tissue culture media (RPMI), with variations of bicarbonate as a physiologic buffer. Synergy was calculated by fractional inhibitory concentration index (FICI). Therapeutic benefit of combining AZM and MIN was tested in a murine model of A. baumannii pneumonia. AZM + MIN synergism was probed mechanistically by bacterial cytological profiling (BCP), a quantitative fluorescence microscopy technique that identifies disrupted bacterial cellular pathways on a single cell level, and real-time kinetic measurement of translation inhibition via quantitative luminescence. AZM + MIN synergism was further evaluated vs. other contemporary high priority MDR bacterial pathogens. Findings Although two translation inhibitors are not expected to synergize, each drug complemented kinetic deficiencies of the other, speeding the initiation and extending the duration of translation inhibition as verified by FICI, BCP and kinetic luminescence markers. In an MDR A. baumannii pneumonia model, AZM + MIN combination therapy decreased lung bacterial burden and enhanced survival rates. Synergy between AZM and MIN was also detected vs. MDR strains of Gram-negative Klebsiella pneumoniae and Pseudomonas aeruginosa , and the leading Gram-positive pathogen methicillin-resistant Staphylococcus aureus . Interpretation As both agents are FDA approved with excellent safety profiles, clinical investigation of AZM and MIN combination regimens may immediately be contemplated for optimal treatment of A. baumannii and other MDR bacterial infections in humans. Fund National Institutes of Health U01 AI124326 (JP, GS, VN) and U54 HD090259 (GS, VN). IC was supported by the UCSD Research Training Program for Veterinarians T32 OD017863.

Genetic Determinants Enabling Medium-Dependent Adaptation to Nafcillin in Methicillin-Resistant Staphylococcus aureus

“…A murine pneumonia infection model was used to examine the virulence of mediumadapted strains in comparison to the pathogenic TCH1516 parental lineage. This model utilizes an intratracheal injection to establish a bacterial pneumonia and has been previously used to assess lung bacterial burdens (29). Surprisingly, despite RPMIϩ serving as a better mimic for physiological conditions, strains that were adapted to RPMIϩ did not have a virulence advantage within the host compared to those adapted to the standard laboratory growth medium (Fig.…”

Decomposition of transcriptional responses provides insights into differential antibiotic susceptibility

“…Antibiotic susceptibility was determined as described previously (Dillon et al, 2019) . Briefly, E. coli were cultured in the same media throughout (M9, R10LB, or CA-MHB) prior to the addition of antibiotics.…”

Characterizing the response of Acinetobacter baumannii ATCC 17978 to azithromycin in multiple in vitro growth conditions

“…High rates of resistance 2 , combined with a failing drug delivery pipeline 9 , necessitate a push to identify new efficacious therapeutical options for MDR A. baumannii infections. While pursuing potential new therapies we uncovered unappreciated activity for the macrolide azithromycin (AZM) against MDR A. baumannii 10,11 . Interestingly, enhanced AZM activity was found when A. baumannii was cultured in amended Roswell-Park memorial institute (RPMI+) tissue culture medium but not in the standard bacteriologic medium cation-adjusted Mueller-Hinton broth (CA-MHB) 10,11 .…”

“…Minimum inhibitory concentration (MIC) experiments were carried out as previously described 11 . Briefly, AZM (Fresenius Kabi) was serially diluted in the associated medium to generate working stocks of the drug.…”

“…Fluorescence microscopy was done as previously described with modifications 11,14,15,17 . In brief, at the 2.5 h timepoint, 20 µL cells were added to 1 µL dye mix containing 80 µg/mL DAPI, 10 µM SYTOX Green, and 200 µg/mL FM4-64 in 1x T-base.…”