Investigating the Antimicrobial Potential of 560 Compounds from the Pandemic Response Box and COVID Box against Resistant Gram-Negative Bacteria

Abstract

Antimicrobial resistance (AMR) has emerged as a significant threat to public health, particularly in infections caused by critically important Gram-negative bacteria. The development of novel antibiotics has its limitations, and therefore it is crucial to explore alternative strategies to effectively combat infections with resistant pathogens. In this context, the present study investigated the antibacterial potency of 560 compounds against the multidrug-resistant (MDR) strains of Klebsiella pneumoniae and Serratia marcescens. The evaluated compounds were selected from the Pandemic Response Box (PRB) and COVID Box (CB) and subjected to assays to determine the inhibitory concentration (IC), minimum bactericidal concentration (MBC), and biofilm formation. Further, the effects of these compounds on membrane integrity were assessed through protein quantification. Several of the evaluated compounds, including fusidic acid, MMV1580853, and MMV1634399, exhibited a significant reduction in biofilm formation and growth in K. pneumoniae. Trimethoprim exhibited potential against S. marcescens. The IC values of the compounds indicated significant microbial growth inhibition at various concentrations. These findings underscore the potency of the existing antibiotics and novel compounds in combating the MDR strains of bacteria. The importance of reconsidering the known antibiotics and utilizing drug repositioning strategies to address the increasing risk of AMR is highlighted.

Keywords: Gram-negative bacteria; antimicrobial resistance; drug screening; multidrug-resistant resistance.

Figure 1

Initial screening results for the boxes. (A) Among the PRB compounds evaluated against CPR-Kp, 38 exhibited inhibition percentages above 80%, with five exhibiting exceptionally high inhibition rates of above 90%: fusidic acid, MMV1580853, and MMV1634399. Only two PRB compounds exhibited an inhibition percentage above 80%: gepotidacin and MMV1634402 for CR-Kp, and against CR-Sm, trimethoprim exhibited a remarkable inhibition percentage of 82.14%. (B) Among the CB compounds evaluated against CPR-Kp, four compounds, oxyclozanide, doxorubicin, simeprevir, and niclosamide, exhibited inhibition rates above 80%.

Figure 2

Comparative analysis of the antibacterial activities of PRB and the CB compounds. Dose–response analysis of the growth of K. pneumoniae and S. marcescens. (a) ICs were determined for the compounds from PRB with inhibitory activity against CPR-Kp: fusidic acid, MMV1580853, MMV1634399, nadifloxacin, pibenzimol, clofazimine, MMV1593541, and erythromycin. (b) ICs were determined for the CB compounds that inhibited the growth of CPR-Kp: doxycyline, tetracycline, oxyclozanide, doxorubicin, and niclosamide. (c) ICs were determined for the compounds from PRB that inhibited the growth of CR-Kp: gepotidacin and MMV1634402. (d) ICs were determined for the compound from PRB that inhibited the growth of CR-Sm: trimethoprim. All tests were performed in duplicate. Each point represents the mean of duplicate determinations.

Figure 3

Elimination time kinetics of different antibacterial agents against the growth curves of selected bacterial strains under the effect of different compounds from PRB and CB. (a) Survival curve (over 24 h) for CPR-Kp when treated with the compounds from PRB: MMV1634399, MMV1580853, fusidic acid, nadifloxacin, MMV1480967, eberconazole, gepotidacin, erythromycin, pibenzimol, eravacycline, and MMV1580852. (b) Survival curve (over 24 h) for CPR-Kp when treated with the compounds from CB: doxycycline tetracycline, doxorubicin, oxyclozanide, and niclosamide. (c) Survival curve (over 24 h) for CR-Kp when treated with the PRB compounds: gepotidacin and MMV1634402. (d) Survival curve (over 24 h) for CR-Sm when treated with the compounds from PRB: trimethoprim. All tests were performed in duplicate. Each point represents the average of duplicate determinations.

Figure 4

Inhibition of biofilm formation by PRB and CB compounds. (a) CPR-Kp treated with different PRB compounds exhibiting variable levels of biofilm formation, with only a few compounds demonstrated to significantly reduce biofilm formation, namely, MMV 1634399, MMV 1580853, fusidic acid, nadifloxacin, gepotidacin, erythromycin, pibenzimol MMV 1593537, eravacycline, and MMV1580852. (b) The CB compounds doxycycline, tetracycline, doxorubicin, oxyclozanide, and niclosamide exhibited inhibitory effects on biofilm formation in CPR-Kp compared to the positive control. (c) Gepotidacin and MMV1634402, among the PRB compounds evaluated for biofilm formation against CR-Kp, exhibited low efficacy in biofilm inhibition, with values close to those noted for the positive control. (d) Trimethoprim from PRB did not exhibit a significant difference in its inhibitory effect on biofilm formation in CR-Sm compared to the positive control. All tests were performed in triplicate. Each point represents the average of triplicate determinations. Untreated controls were compared using a one-way analysis of variance (ANOVA) followed by the Kruskal–Wallis test. * p value < 0.5; ns: not significant.

Figure 5

The number of proteins released over time, reflecting the integrity of the cell membrane when the bacterial strains were exposed to the compounds from PRB and CB, are depicted. (a) Extravasation of CPR-Kp proteins; (b) extravasation of CPR-Kp proteins; (c) extravasation of CR-Kp proteins; (d) extravasation of CR-Sm proteins. The data points represent the averages of the protein concentration values measured at 0, 1, 2, and 4 h post-treatment, providing insights into the temporal dynamics of bacterial membrane damage. All tests were performed in duplicate. Each point represents the average of duplicate determinations.