Re-sensitization of Multidrug-Resistant and Colistin-Resistant Gram-Negative Bacteria to Colistin by Povarov/Doebner-Derived Compounds

Abstract

Colistin, typically viewed as the antibiotic of last resort to treat infections caused by multidrug-resistant (MDR) Gram-negative bacteria, had fallen out of favor due to toxicity issues. The recent increase in clinical usage of colistin has resulted in colistin-resistant isolates becoming more common. To counter this threat, we have investigated previously reported compounds, HSD07 and HSD17, and developed 13 compounds with more desirable drug-like properties for colistin sensitization against 16 colistin-resistant bacterial strains, three of which harbor the plasmid-borne mobile colistin resistance (mcr-1). Lead compound HSD1624, which has a lower LogD_pH7.4 (2.46) compared to HSD07 (>5.58), reduces the minimum inhibitory concentration (MIC) of colistin against Pseudomonas aeruginosa strain TRPA161 to 0.03 μg/mL from 1024 μg/mL (34,000-fold reduction). Checkerboard assays revealed that HSD1624 and analogues are also synergistic with colistin against colistin-resistant strains of Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae. Preliminary mechanism of action studies indicate that HSD1624 exerts its action differently depending on the bacterial species. Time-kill studies suggested that HSD1624 in combination with 0.5 μg/mL colistin was bactericidal to extended-spectrum beta-lactamase (ESBL)-producing E. coli, as well as to E. coli harboring mcr-1, while against P. aeruginosa TRPA161, the combination was bacteriostatic. Mechanistically, HSD1624 increased membrane permeability in K. pneumoniae harboring a plasmid containing the mcr-1 gene but did not increase radical oxygen species (ROS), while a combination of 15 μM HSD1624 and 0.5 μg/mL colistin significantly increased ROS in P. aeruginosa TRPA161. HSD1624 was not toxic to mammalian red blood cells (up to 226 μM).

Keywords: Gram-negative; antibiotic potentiation; colistin-resistant bacteria; multidrug-resistant.

Figure 1.

Incorporation of polar groups into first-generation antibacterial agents to improve activity against Gram-negative bacteria and reduce c Log P.

Figure 2.

Second generation compounds bearing polar groups with reduced c Log P^a.

Figure 3.

Time-kill analysis showing (A) HSD1624 + low dose colistin bactericidal activity to ESBL-producing and colistin-resistant E. coli CDC-AR 0346; (B) HSD1625 + low dose colistin bactericidal activity to ESBL-producing and colistin-resistant E. coli CDC-AR 0346. Graphs made using Origin (Pro), Version 2020. OriginLab Corporation, Northampton, MA, USA.

Figure 4.

Time-kill curve for compound HSD1624 and colistin. Green: Untreated TRPA 161. Blue: 15 μM HSD1624. Red: 0.5 μg/mL colistin. Pink: 0.5 μg/mL colistin + 15 μM HSD1624. Data ± SD from at least four replicates. Note: the dashed line represents the LOD.

Figure 5.

Radical oxygen species assay. (A) DCFH-DA fluorescence assay of P. aeruginosa TRPA161 cells treated with (1) 0.15% hydrogen peroxide, (2) 0.5 μ/mL colistin, (3) 15 μM HSD1624, and (4) 0.5 μg/mL colistin + 15 μM HSD1624. (B) DCFH-DA fluorescence assay of K. pneumoniae F2210291^mcr-1 cells treated with (1) 0.15% hydrogen peroxide, (2) 1 μg/mL colistin, (3) 30 μM HSD1624, and (4) 1 μg/mL colistin + 30 μM HSD1624. Percentages shown indicate the average increase in fluorescence compared to the bacterial control; *indicates a p-value <0.05; ***indicates a p-value of <0.001 compared to untreated controls subjected to fluorescent dye treatment. Fluorescence of DCFH-DA was normalized by the OD600 of the well to account for potential differences due to growth rates; measurements were obtained simultaneously. Note: error bars represent the standard error.