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Article

Enhancing Antibacterial Efficacy: Combining Novel Bacterial Topoisomerase Inhibitors with Efflux Pump Inhibitors and Other Agents Against Gram-Negative Bacteria

by
Maša Zorman
1,2,
Maja Kokot
1,2,
Irena Zdovc
3,
Lidija Senerovic
4,
Mina Mandic
4,
Nace Zidar
2,
Andrej Emanuel Cotman
2,
Martina Durcik
2,
Lucija Peterlin Mašič
2,
Nikola Minovski
1,
Marko Anderluh
2 and
Martina Hrast Rambaher
2,*
1
Theory Department, Laboratory for Cheminformatics, National Institute of Chemistry, Hajdrihova 19, 1001 Ljubljana, Slovenia
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana, Aškerčeva Cesta 7, 1000 Ljubljana, Slovenia
3
Institute of Microbiology and Parasitology, Veterinary Faculty, University of Ljubljana, Gerbičeva 60, 1000 Ljubljana, Slovenia
4
Laboratory for Microbial Molecular Genetics and Ecology, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11042 Belgrade, Serbia
*
Author to whom correspondence should be addressed.
Antibiotics 2024, 13(11), 1081; https://doi.org/10.3390/antibiotics13111081
Submission received: 28 October 2024 / Revised: 10 November 2024 / Accepted: 12 November 2024 / Published: 13 November 2024
(This article belongs to the Special Issue Multitalented Synthetic Antimicrobial Compounds: From Design to Effect)
Browse Figures
Versions Notes

Abstract

:
Background: The novel bacterial topoisomerase inhibitors (NBTIs) developed in our laboratory show potent on-target enzyme inhibition but suffer from low activity against Gram-negative bacteria. Methods: With the aim of improving the antibacterial activity of our compounds against Gram-negative bacteria, we tested them in combination with different efflux pump inhibitors (EPIs), a strategy that showed promise in several other classes of antimicrobials. We also investigated the combined effect of NBTIs with ATP-competitive inhibitors of bacterial type II topoisomerases (ACIs), as well as the antibiofilm properties of our compounds and the combination with EPIs against early and mature Acietobacter baumannii biofilm. Results: Our results demonstrate that combinations of NBTIs with EPI Phenylalanine-arginyl-β-naphthylamide significantly reduce the corresponding NBTIs’ minimal inhibitory concentration values and show potentiation of A. baumannii biofilm inhibition as compared to NBTIs alone. Although combinations of NBITs and ACIs did not show synergistic effects, the FIC index value calculations revealed additive effects for all the combinations of a selected NBTI in combination with three ACIs in all the assayed Gram-negative bacteria from the ESKAPE group. Conclusions: These results show for the first time that combinations of NBTIs with either EPIs or a different class of the topoisomerase inhibitors may be a beneficial strategy to combat difficult-to-treat bacterial infections.

Graphical Abstract

1. Introduction

Antibiotic resistance is one of the greatest threats to global health, with 1.27 million deaths being directly linked to it according to a 2019 estimate [1]. Staggeringly, around three-quarters of antimicrobial-resistance-associated deaths were attributable to only six pathogen species—Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa—some of which belong to the multidrug-resistant ESKAPE pathogen group [2,3]. While some bacteria are intrinsically resistant to certain antibiotics due to inherent structural or functional characteristics, such as low bacterial cell wall permeability, constitutive expression of efflux pumps, and biofilm formation, others can acquire resistance to antibiotics due to either horizontal gene transfer, e.g., plasmid-mediated transmission of resistance genes, or genetic mutations that lead to target modifications or modifications of antibiotic-modifying enzymes [4,5,6,7,8]. Gram-negative bacteria are more susceptible to antibiotics compared to Gram-positive ones due to inherent structural differences, such as low permeability due to the presence of an outer membrane and drug efflux via specific efflux pumps [9,10]. Additionally, bacterial biofilms offer bacteria an improved living environment, wherein the resistance seems to depend on multicellular strategies that may differ from the mechanisms of resistance in free-floating (planktonic) bacteria [11].
Multidrug efflux systems are able to pump out various substrates, including several classes of antibiotics [12,13]. The overexpression of a specific efflux pump in bacteria commonly leads to the development of resistance as the intracellular concentration of the antibiotic decreases, making the bacteria less sensitive to the active substance. The minimum inhibitory concentration (MIC) of those strains overexpressing efflux pumps is usually two to eight times greater than that of a susceptible strain of the same species but can also be much greater [14,15,16,17]. The concomitant use of efflux pump inhibitors (EPIs) with already known antibiotics is an interesting approach to improve the clinical efficacy of antibiotics, but this is not clinically validated [18]. EPIs can potentially enhance antibiotic efficacy by reducing the intrinsic bacterial resistance to antibiotics, reversing the acquired resistance associated with efflux pump overexpression, and reducing the frequency of emergence of resistant mutant strains [19,20,21,22].
Phenylalanine-arginyl-β-naphthylamide (PAβN, Figure 1) is a broad-spectrum competitive EPI and the first compound to reduce the intrinsic resistance of P. aeruginosa to levofloxacin [23,24,25]. Although PAβN caused a reduction in levofloxacin MIC in the wild-type P. aeruginosa, as well as in strains overexpressing efflux pumps, its unfavorable pharmacokinetic and toxicological profiles prevent its clinical use [19,21,22]. 1-Naphthylmethyl-piperazine (NMP, Figure 1) is one of the most potent unsubstituted arylpiperazines, with the ability to increase the intracellular concentration of several antibiotics in A. baumannii and several Enterobacteriaceae but not in P. aeruginosa [26]. In contrast to PAβN, the (low) intrinsic antibacterial activity of NMP and its cellular accumulation are not enhanced in cells with inactivated efflux pumps [27]. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Figure 1) is an example of a proton-motive force inhibitor that disrupts the proton-motive force of the membrane, thereby reducing the bacterial cell viability and promoting cell death [28]. Unfortunately, it is highly noxious and cytotoxic. Reserpine (Figure 1), an inhibitor of vesicular monoamine transporters and blocker of transmembrane calcium entry, is routinely used to evaluate the efflux pump activity of Gram-positive bacteria; however, the concentrations necessary to block bacterial efflux are neurotoxic [28]. Berberine (Figure 1), a naturally occurring product, also exhibits activity against efflux pumps; nevertheless, problems with its synthesis, purification, stability, and solubility, as well as its potential toxicity, have reduced the interest in its further development as an EPI [21,29].
Bacteria occur either as free-living cells or as aggregates of cells surrounded by a self-produced polymer matrix that adhere to each other and/or to a surface, called biofilms [30]. Biofilm-growing microorganisms have different specific characteristics compared to planktonic microorganisms, e.g., growth rate and gene transcription, and may exhibit as much as a 1000-fold increase in antibiotic resistance against conventional antibacterials [6,7]. It is speculated that the main driving forces of antimicrobial resistance in biofilms are slowed diffusion of antibacterials into the biofilm matrix, the altered chemical microenvironment within the biofilm, and cell differentiation similar to spore formation [11]. This evidence does not, however, exclude the possibility that conventional resistance mechanisms, such as drug pumps, are expressed in biofilms and contribute to antibiotic resistance in biofilms [11]. Some EPIs have been shown to inhibit biofilm formation in Gram-negative bacteria that showed upregulated expression of efflux pumps [28]. Other mechanisms of action of antibiofilm molecules include inhibition of quorum sensing, inhibition of adhesion, and disruption of various extracellular targets involved in various signaling pathways, all of which may be achieved by a variety of different structural classes of compounds [31].
Bacterial type II topoisomerases are a well-validated class of targets for antibacterial chemotherapy. Although two classes of topoisomerase inhibitors, fluoroquinolones and aminocoumarins, have reached the drug market, aminocoumarins have since been withdrawn due to their ineffectiveness, and fluoroquinolones are increasingly losing efficacy due to emerging resistant bacterial strains. Novel bacterial topoisomerase inhibitors (NBTIs) are a new class of inhibitors that bind to a separate binding site on the enzyme and show no cross-resistance with either fluoroquinolones or aminocoumarins [32,33,34]. ATP-competitive inhibitors of bacterial type II topoisomerases (ACIs), which act at the same site as aminocoumarins, are also being developed and show improved antibacterial properties [35]. Both NBTIs and ACIs exhibit excellent antibacterial activity against Gram-positive bacteria, but the activity of the current inhibitors against Gram-negative bacteria in the planktonic form and biofilms is not optimal due to problems with membrane permeability and efflux from the cell [36,37,38,39,40]. The reasons for their ineffectiveness in biofilms are likely even more complex.
Combining antimicrobial agents with EPIs is a promising strategy aimed at improving and maintaining the antibacterial potency of antibacterials. This was confirmed to work in other classes of antibacterial agents but never in NBTIs [19,22,41,42]. The coadministration of EPIs and NBTIs is expected to increase the intracellular concentration of NBTIs that are substrates of efflux pumps, decrease the intrinsic bacterial resistance to antibiotics, reverse the acquired resistance associated with efflux pump overexpression, and reduce the frequency of emergence of resistant mutant strains [29]. All these aspects prompted us to further investigate the use of NBTIs in combination with various EPIs in Gram-negative bacteria. Another well-known strategy for combating the ineffectiveness of individual drugs in the treatment of bacterial infections is the coadministration of two or more antibacterials [43]. The main reasons for this are broad-spectrum coverage, treatment of polymicrobial infections, prevention of a high mutation rate of the bacteria due to the use of a single antibiotic, dose reduction and corresponding reduction in dose-related toxicity, and antimicrobial synergistic activity [44]. With the aim of improving the antibacterial activity of our compounds, we have investigated the potential synergistic effect of NBTIs and selected ACIs (Figure 2) in different Gram-negative bacteria. Furthermore, the ability of an antibacterial to inhibit both bacteria found in planktonic form as well as bacteria in biofilms could expand its overall functionality and broaden its use. Since some EPIs are known biofilm disruptors, we investigated the antibiofilm effects of NBTIs alone, as well as NBTIs in combination with EPIs.

2. Results

2.1. Intrinsic Antibacterial Activity of NBTIs

The intrinsic antibacterial activities of the presented compounds were evaluated against E. coli, P. aeruginosa, K. pneumoniae, and A. baumannii and are shown in Table 1. All the EPIs, except for 3, exhibited intrinsic antibacterial activities higher than the tested concentrations. Moreover, 3 displayed weak–moderate antibacterial activity in E. coli, K. pneumoniae, and P. aeruginosa (MIC = 32, 64, and 128 µg/mL, respectively).
Compounds 6 and 7 showed strong antibacterial activity against E. coli and A. baumannii (MIC = 0.5 and 1 µg/mL for 6 and 7, respectively); however, they exhibited weaker antibacterial activity with 8–16-fold higher MIC values against P. aeruginosa and K. pneumoniae. Weak intrinsic antibacterial activity of 8 was observed in all the tested strains. The NBTIs were also tested against E. coli D22, which has increased membrane permeability compared to the wild-type E. coli and E. coli N43 strain, which lacks the AcrA component of a major multidrug resistance pump. The intrinsic antibacterial activities in both strains were significantly lower than in the wild-type E. coli.
Additionally, the three ACIs were evaluated via the checkerboard assay against E. coli, P. aeruginosa, K. pneumoniae, and A. baumannii (Table 1). Compound 10 exhibited relatively good antibacterial activity against all the tested strains (MIC ≤ 2 µg/mL in all the strains), whereas 9 and 11 showed moderate inhibition of E. coli, P. aeruginosa, and A. baumannii and weaker activity against K. pneumoniae.

2.2. EPI Potentiation of NBTI Antibacterial Activity in Various Gram-Negative Bacteria

To assess whether any EPIs potentiated the activity of NBTIs against different Gram-negative bacteria, the MICs of the three investigated NBTIs were determined for E. coli, P. aeruginosa, K. pneumoniae, and A. baumannii in the presence and absence of different concentrations of EPIs.
In the presence of PAβN, 1, there was a significant improvement in the antibacterial activity of the NBTIs. In E. coli and K. pneumoniae, a PAβN concentration of 4–8 µg/mL resulted in an 8-fold improvement in the MIC for compounds 6 and 7 (Figures S1 and S2). In P. aeruginosa, which is known as a problematic Gram-negative bacterium, an 8-fold improvement was achieved with a PAβN concentration of 16 µg/mL (Table 2) for both compounds, 6 and 7. Exact values for 8 could not be determined due to the weak antibacterial activity of 8 alone (MIC > 128 µg/mL). For P. aeruginosa and K. pneumonia, however, at least an 8-fold reduction in MIC was observed with the addition of 32 µg/mL of PAβN. For the combination of 8 and PAβN, no correlation was observed in E. coli and A. baumannii, and the MIC could not be determined as the turbidity readout did not yield the expected linear increase in response to the increased concentration of each compound (Figure S3). No significant effects of 2, 3, 4, and 5 were observed regarding the NBTIs (Figures S4–S12) in any bacterial species tested.

2.3. ACI and NBTI Checkerboard Assay Results

The effect of the combination of 6 and an ACI on the antibacterial potency in comparison to their individual activities was determined for E. coli, P. aeruginosa, K. pneumoniae, and A. baumannii (Table 3, Figures S13–S15).
The MIC values of the compounds administered in combination generally decreased 2–4-fold compared to the compounds administered individually (Table 3). The calculated fractional inhibitory concentration (FIC) indices revealed an additive effect for all the combinations of 6 with the three ACIs in all the bacteria (Table 4). No significant synergistic effect was observed in any of the assayed combinations. FIC is a measure used to assess the interaction between two antimicrobial agents. An FIC index below 0.5 signifies synergistic interaction between the two tested antibacterials, values of the index between 0.5 and 4 are considered as an additive effect or indifference, and an index value above 4 signifies an antagonistic interaction between the two tested drugs.

2.4. Inhibition of A. baumannii Biofilm Formation and Elimination of Mature Biofilms

Compounds 6 and 7 showed a dose-dependent reduction in the A. baumannii biofilms, achieving 65% and 75% inhibition at the MIC concentration, respectively (Figure 3A). Compound 6 applied to 24 h old biofilms slightly stimulated the biofilm growth, while 7 had no significant effect on mature biofilms at concentrations of up to 4 × MIC (Figure 3B).
Since PAβN significantly lowered the MIC of the NBTIs, we investigated its effect on biofilm formation (Figure 4A,B) and mature biofilm disruption (Figure 4C,D) when used in combination with compounds 6 and 7 at different concentrations.
PAβN alone did not inhibit the biofilm formation at any of the concentrations tested (Figure S16). However, when PAβN was used in combination with compounds 6 and 7, a significant increase in the inhibition of biofilm formation was observed. In the case of compound 6, additional inhibition of biofilm formation occurred in combination with PAβN in all the combinations except at concentrations of compound 6 equal to MIC/4 and MIC/2 and 1 µg/mL PAβN, where the effect was similar to that of compound 6 alone (Figure 4A). All the combinations of compound 7 with PAβN resulted in additional inhibition of biofilm formation compared to compound 7 alone (Figure 4B). Approximately an 80% reduction in biofilm formation was achieved when the bacteria were exposed to the concentration of compounds 6 and 7 at 2 × MIC or when the compounds were combined with 100 µg/mL PAβN.
In combination with PAβN, a slight dose-dependent mature biofilm disintegration was observed for both compounds (Figure 4C,D). In combination with 100 µg/mL PAβN, a reduction in the biofilm mass of approximately 20 to 25% was achieved at each concentration of the compounds. PAβN alone showed no significant effect against mature biofilms (Figure S16).

3. Discussion

This study focused on discovering novel ways of enhancing the antimicrobial activity of a series of NBTIs against Gram-negative bacteria by assessing various different approaches, including combining them with different EPIs and ACIs. The difference in the intrinsic antibacterial activity of the NBTIs between E. coli D22 and the wild-type E. coli showed that our compounds struggle with cell permeability, especially compound 7, whose MIC for E. coli D22 is much lower compared to the MIC for the wild-type E. coli. The MIC values of the NBTIs against the E. coli N43 strains were significantly lower for all three tested compounds, indicating that the compounds are excellent substrates for E. coli efflux pumps; therefore, combining them with efflux pump inhibitors would be a sensible strategy for improving the antibacterial activity of these compounds. To verify that the tested EPIs were not acting as growth inhibitors, the MIC values for 1–5 were determined. Only 3 displayed weak–moderate antibacterial activity in E. coli, K. pneumoniae, and P. aeruginosa.
The EPIs used in this paper were selected because we wished to explore a range of differently acting commercially available EPIs that have been reported in the literature. PAβN was the only EPI where there was a significant improvement in the antibacterial activity of the NBTIs. As indicated in Table 2, an increase in the concentration of PAβN progressively lowers the MIC of the antibacterials. The MPC8 values for 6 and 7 in E. coli and K. pneumoniae were achieved at the PAβN concentrations of 4–8 µg/mL and 16 µg/mL in P. aeruginosa (Table 2). The exact values for 8 could not be determined due to the weak antibacterial activity of 8 alone (MIC > 128 µg/mL); however, at least an 8-fold reduction in MIC was observed with the addition of 32 µg/mL PAβN in P. aeruginosa and K. pneumoniae. NMP showed no significant effects in combination with the NBTIs in any bacteria (Figures S4–S6). The other tested EPIs, CCCP, reserpine, and berberine, in combination with 6 and 7, did not show any significant improvement in antibacterial activity in E. coli, P. aeruginosa, and K. pneumoniae (Figures S7–S12). This was expected due to a different mechanism of efflux pump inhibition by 3, and the fact that reserpine and berberine target the efflux pumps of Gram-positive bacteria. The efflux pump inhibitors were not used against Gram-positive bacteria since the Gram-positive bacteria (e.g., S. aureus and MRSA) were found to be more susceptible to the NBTIs tested (with MICs as low as 0.004 µg/mL) [37,39,40]. In addition to their potentiation of the antibacterial activity of NBTIs, an important aspect of the investigated EPIs is their toxicity. Toxicity can be measured in vivo and expressed as a minimum lethal dose (MLD), i.e., the minimum dose that results in lethality in ≥66% of the animals tested [45]. As mentioned in the Introduction, PAβN exhibits a moderate toxic effect in a mouse model with an MLD < 25 mg/kg [45]. In our case, a PAβN concentration of 16 µg/mL resulted in an 8–32-fold improvement in MIC for the tested NBTIs, suggesting that safety issues might arise at such PAβN concentrations in in vivo studies (Table 2). However, there are analogues of PAβN with better safety profiles, so we plan to continue our future studies by using them in combination with selected NBTIs. The combination approach, as described in this paper, shows improved efficacy regarding the antibacterials and could potentially be utilized for future treatment of Gram-negative bacterial infections. As mentioned in the Introduction, however, the clinical validation of such an approach is still needed. The discovery and development of novel less-toxic EPIs might pave the way for such an approach to become reality.
To explore whether the coadministration of NBTIs and ACIs is a valid strategy for improving the antibacterial potency of the two drug classes, the activity of compound 6 against various Gram-negative bacteria in the presence of different concentrations of 9, 10, and 11 was investigated. No synergy was observed in any combination, only additive effects (Table 4), meaning that the drugs show a combined effect similar to what would be expected if they were acting independently. The combination is neither particularly beneficial nor harmful. The coadministration of the two topoisomerase inhibitor classes with different modes of action could have resulted in synergism by increasing the overall target inhibition. An additive effect might have occurred rather than synergistic action because both classes of compounds act on the same target enzyme instead of inhibiting multiple targets at the same time. It would be interesting to combine NBTIs with antibacterials that disrupt other vital cellular processes to see if this results in synergy. Both synergy and additive effects are welcome outcomes. A synergistic effect is beneficial for the treatment of difficult-to-treat bacterial infections, whereas an additive effect may be applied to broadening antimicrobial coverage and avoiding toxicity issues by lowering the administered concentrations of both antibacterials.
Moreover, 6 and 7 alone inhibit A. baumannii biofilm formation in a dose-dependent manner; 6 achieved 65% inhibition and 7 achieved 75% inhibition at the concentration of the compounds equal to the MIC. Combining them with PAβN potentiates biofilm inhibition as compared to NBTIs alone, making them useful for the prevention of biofilms. This inhibition is especially relevant for A. baumannii, a pathogen known to cause persistent and difficult-to-treat infections due to its biofilm-forming ability. NBTIs alone or in combination with PAβN, however, are not able to significantly disrupt mature biofilms. While PAβN slightly improved mature biofilm disruption, the effect was relatively modest (around 20–25% biofilm reduction), and the compounds remained largely ineffective against mature biofilms and would therefore be ineffective for the treatment of chronic biofilm-associated infections.

4. Materials and Methods

4.1. Bacterial Strains

Reference strains of E. coli, P. aeruginosa, K. pneumoniae, and A. baumannii were obtained from different libraries, E. coli from ATCC (E. coli (ATCC 25922)), P. aeruginosa from DSM (P. aeruginosa RDK 184 (DSM 939)), K. pneumoniae from RDK 070A (ATCC 51503), and A. baumannii from 8C6 (GES-14).

4.2. Chemicals and Media

PAβN, NMP, CCCP, reserpine, and berberine were purchased from Sigma-Aldrich (Steinheim, Germany). NBTIs and ACIs were synthesized in house and have been previously characterized for purity [36,38,40,46,47,48].

4.3. Determination of MIC—Susceptibility Testing

The minimum inhibitory concentrations (MICs) were determined by broth micro-dilution method in 96-well plate format according to the guidelines of Clinical and Laboratory Standards Institute and recommendations of European Committee on Antimicrobial Susceptibility Testing [49,50]. Bacterial suspension of the specific bacterial strain (E. coli, P. aeruginosa, K. pneumoniae, and A. baumannii) equivalent to the 0.5 McFarland turbidity standard was diluted with cation-adapted Mueller–Hinton broth with TES (Thermo Fisher Scientific, Waltham, MA, USA) to obtain a final inoculum of 105 CFU/mL. The compounds (NBTIs, ACIs, and EPIs), dissolved in DMSO, and the inoculum were mixed together and incubated at 35 °C for 20 h. The assay concentrations of the tested compounds ranged from 256 to 0.004 µg/mL. After incubation, the MIC values were determined by visual inspection as the lowest dilution of the compounds showed no turbidity. Tetracycline was used as a positive control on each test plate.

4.4. Determination of MPCn

Minimum potentiation concentration (MPC) is defined as the lowest concentration of an EPI that causes a n-fold reduction in the minimum inhibitory concentration MIC of an antibiotic (expressed as MPCn for an n-fold reduction) [23]. To determine MPC8 values, a checkerboard assay was designed with single-agent concentrations of NBTI and EPI to determine the MIC and combinations of different concentrations of NBTI and EPI. We added serial dilutions of EPI to columns 1–10; the highest concentration was in column 1 and the lowest in column 10. No EPI was added to column 11. Serial dilutions of an NBTI were added to rows A–H; the highest concentration was in row A and the lowest in row G. No NBTI was added to row H. Column 12 contained dilutions of tetracycline as a control. We first determined MICNBTI in the presence of different concentrations of PAβN (Table 2) and then chose MICEPI required to reduce the MICNBTI at least 8-fold (MPC8). Indeed, 8-fold greater reduction in MICNBTI values after addition of EPI was considered significant. The assay concentrations of the EPIs used in the checkerboard assay ranged from 256 to 0.5 µg/mL for 2, from 128 to 0.25 µg/mL for 1, 3, and 4, and from 64 to 0.125 µg/mL in 5. The assay concentrations of NBTIs depended on the bacterial strains used in the assay. Assay concentrations of 6 ranged from 16 to 0.25 µg/mL in P. aeruginosa, from 4 to 0.063 µg/mL in K. pneumoniae, and from 1 to 0.016 µg/mL in E. coli or A. baumannii. Assay concentrations of 7 ranged from 32/16 to 0.5/0.25 µg/mL in P. aeruginosa, from 16/8 to 0.25/0.125 µg/mL in K. pneumoniae, from 2 to 0.031 µg/mL in E. coli, and from 2/1 to 0.031/0.016 µg/mL in A. baumannii. Assay concentrations of 8 ranged from 128 to 2 µg/mL in all tested bacterial strains.

4.5. Checkerboard Assay and the Determination of FIC Index

Fractional inhibitory concentration (FIC) is defined as the sum of the FICs of each drug tested ( FIC A ,   FIC B ), where the FIC for each drug is determined by dividing the MIC of each drug in combination ( C A ,   C B ) by the MIC of each drug when used alone ( MIC A ,   MIC B ) (Equation (1)):
FIC = FIC A + FIC B = C A MIC A + C B MIC B
Synergy is assumed when the value of the FIC index is <0.5 and antagonism when FIC > 4. In synergy, the combination of compounds increases the inhibitory activity (decrease in MIC) of one or both compounds compared to just the compounds alone, while in antagonism more compound would be required in order to produce the same effect as the compounds alone. When the combination of compounds results in an FIC value of 0.5–4, the combination has no increase in inhibitory activity or a slight increase in inhibitory activity from the additive effect of both compounds combined. A checkboard assay, similar to the MPC8 determination assay, was designed. NBTIs were added to columns 2–11; column 2 contained the lowest concentration, followed by a 2-fold increase in concertation in each consecutive column. No NBTI was added to the first column; therefore, intrinsic antibacterial activity of the ACIs could be determined. ACIs were added to rows A–G; row A contained the highest concentration of ACI, followed by a 2-fold decrease in concertation in each consecutive row. No ACI was added to row H; therefore, we could determine the intrinsic antibacterial activity of the NBTI. Column 12 contained 2-fold dilutions of tetracycline as a control. Assay concentrations ranged from 16 to 0.031 µg/mL for 6, from 64 to 1 µg/mL for 9 and 11, and from 16 to 0.031 µg/mL for 10 in all tested combinations.

4.6. Biofilm Formation Inhibition Assay

Biofilm quantification assays were performed in 96-well microtiter plates using a crystal violet (CV) method to stain adherent cells [51]. Overnight cultures of A. baumanii ATCC 19606 were diluted to 5 × 107 cells mL−1 in brain heart infusion media (BHI), and 100 µL was added to the wells without treatment (negative control) or in the presence of the compounds. Biofilms formed for 24 h at 37 °C were washed and adherent cells stained with 0.1% CV (v/v). Assays were performed in six wells and repeated two times.

4.7. Mature Biofilm Disruption Assay

Overnight cultures of A. baumannii were diluted to 5 × 107 cells mL−1 in BHI and 100 μL was added to the wells in 96-well microtiter plates and incubated for 24 h at 37 °C. After removal of the culture from the wells and two washing steps with PBS, adherent cells were exposed to different concentrations of the compounds for additional 24 h and the biofilm biomass was quantified using CV as described above. The wells without treatment served as a control. Assays were performed in six wells and repeated two times.

4.8. Potentiation of Compounds’ Antibiofilm Activity with Efflux Pump Inhibitors

To investigate whether PAβN can potentiate antibiofilm activity of the compounds, compounds 6 and 7 at concentrations MIC/4, MIC/2, MIC, and 2 × MIC were combined with increasing concentrations of PAβN (1, 10, and 100 μg/mL), and biofilm formation inhibition assay and mature biofilm disruption assay were performed as described above.

5. Conclusions

With the steady increase in antimicrobial resistance, multidrug-resistant bacteria are becoming increasingly difficult to fight. There are fewer and fewer effective antimicrobials, so new approaches are needed. In this study, we presented a few such approaches: coadministration of an antimicrobial agent (NBTI) and an efflux pump inhibitor (EPI), coadministration of two antibacterials with distinct modes of action (NBTI and ACI), as well as inhibition of biofilm formation and mature biofilm disruption. We investigated a combination of three NBTIs with different EPIs against Gram-negative bacteria belonging to ESKAPE pathogens, as well as a combination of an NBTI with three different ACIs. Among the EPIs tested, PAβN significantly improved the antibacterial activity of NBTIs against Gram-negative bacteria (more than 8-fold). In contrast, the other EPIs tested (e.g., NMP, CCCP, reserpine, and berberine) did not improve the antibacterial activity of the NBTIs against selected Gram-negative bacteria. The investigated combinations of NBTI with ACIs resulted in an additive effect or indifference but did not produce a synergistic antibacterial effect. NBTIs alone and in combination with PAβN inhibited biofilm formation but were less effective in inhibiting mature biofilms. Our results demonstrate for the first time that the combination of NBTIs with EPI could improve the effectiveness of NBTIs and enable their clinical use, especially when the NBTIs are known efflux pump substrates. However, it is vital to develop new EPIs with improved safety profiles in the future. Combining NBTIs with ACIs did not show a synergistic effect, yet the combination itself might prove to be beneficial, especially in the therapy of resistant strains. Inhibition of biofilm formation and mature biofilm disruption are strategies aimed at reducing the virulence of aggregated bacteria known to cause chronic infections. Although our compounds were not successful in disrupting mature biofilms, they strongly inhibited biofilm formation. Overall, the results of this study will undoubtedly help to pave the way to developing more effective treatments for Gram-negative bacterial infections in the future.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/antibiotics13111081/s1, Figure S1: Checkerboard assay of PAβN (1) and 6 in different Gram-negative bacterial strains; Figure S2: Checkerboard assay of PAβN (1) and 7 in different Gram-negative bacterial strains; Figure S3: Checkerboard assay of PAβN (1) and 8 in different Gram-negative bacterial strains; Figure S4: Checkerboard assay of NMP (2) and 6 in different Gram-negative bacterial strains; Figure S5: Checkerboard assay of NMP (2) and 7 in different Gram-negative bacterial strains; Figure S6: Checkerboard assay of NMP (2) and 8 in different Gram-negative bacterial strains; Figure S7: Checkerboard assay of CCCP (3) and 6 in different Gram-negative bacterial strains; Figure S8: Checkerboard assay of CCCP (3) and 7 in different Gram-negative bacterial strains; Figure S9: Checkerboard assay of reserpine (4) and 6 in different Gram-negative bacterial strains; Figure S10: Checkerboard assay of reserpine (4) and 7 in different Gram-negative bacterial strains; Figure S11: Checkerboard assay of barberine (5) and 6 in different Gram-negative bacterial strains; Figure S12: Checkerboard assay of barberine (5) and 7 in different Gram-negative bacterial strains; Figure S13: Checkerboard assay 6 and 9 in different Gram-negative bacterial strains; Figure S14: Checkerboard assay 6 and 10 in different Gram-negative bacterial strains; Figure S15: Checkerboard assay 6 and 11 in different Gram-negative bacterial strains; Figure S16: Inhibition of A. baumannii biofilm formation (A) and biofilm disintegration (B) with PAβN at different concentrations.

Author Contributions

M.Z., M.H.R., N.M., M.A. and M.K. contributed to conception and design of the study. M.Z., M.K., M.H.R. and I.Z. were involved in antimicrobial susceptibility testing. L.S. and M.M. performed the biofilm inhibition and mature biofilm disruption assays. N.Z., A.E.C., M.D. and L.P.M. kindly provided some of the compounds necessary for this study. Data analysis and interpretation were performed by M.Z., M.K. and M.H.R., as well as L.S. and M.M. All authors have read and agreed to the published version of the manuscript.

Funding

The financial support of this work from the Slovenian Research Agency (Grants J3-50123, P1-0017, and P1-0208, the young researcher’s program number 39013) is gratefully acknowledged.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Antimicrobial Resistance Collaborators. Global Burden of Bacterial Antimicrobial Resistance in 2019: A Systematic Analysis. Lancet 2022, 399, 629–655. [Google Scholar] [CrossRef] [PubMed]
  2. De Oliveira, D.M.P.; Forde, B.M.; Kidd, T.J.; Harris, P.N.A.; Schembri, M.A.; Beatson, S.A.; Paterson, D.L.; Walker, M.J. Antimicrobial Resistance in ESKAPE Pathogens. Clin. Microbiol. Rev. 2020, 33, 10–1128. [Google Scholar] [CrossRef] [PubMed]
  3. WHO Publishes List of Bacteria for Which New Antibiotics Are Urgently Needed. Available online: https://www.who.int/news/item/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed (accessed on 21 August 2023).
  4. Blair, J.M.A.; Webber, M.A.; Baylay, A.J.; Ogbolu, D.O.; Piddock, L.J.V. Molecular Mechanisms of Antibiotic Resistance. Nat. Rev. Microbiol. 2014, 13, 42–51. [Google Scholar] [CrossRef]
  5. Reygaert, W.C. An Overview of the Antimicrobial Resistance Mechanisms of Bacteria. AIMS Microbiol. 2018, 4, 482. [Google Scholar] [CrossRef]
  6. Høiby, N.; Bjarnsholt, T.; Moser, C.; Bassi, G.L.; Coenye, T.; Donelli, G.; Hall-Stoodley, L.; Holá, V.; Imbert, C.; Kirketerp-Møller, K.; et al. ESCMID Guideline for the Diagnosis and Treatment of Biofilm Infections 2014. Clin. Microbiol. Infect. 2015, 21, S1–S25. [Google Scholar] [CrossRef]
  7. Lewis, K. Multidrug Tolerance of Biofilms and Persister Cells. Curr. Top. Microbiol. Immunol. 2008, 322, 107–131. [Google Scholar] [CrossRef] [PubMed]
  8. Santajit, S.; Indrawattana, N. Mechanisms of Antimicrobial Resistance in ESKAPE Pathogens. Biomed Res. Int. 2016, 2016, 2475067. [Google Scholar] [CrossRef] [PubMed]
  9. Masi, M.; Réfregiers, M.; Pos, K.M.; Pagès, J.M. Mechanisms of Envelope Permeability and Antibiotic Influx and Efflux in Gram-Negative Bacteria. Nat. Microbiol. 2017, 2, 17001. [Google Scholar] [CrossRef]
  10. Nikaido, H. Molecular Basis of Bacterial Outer Membrane Permeability Revisited. Microbiol. Mol. Biol. Rev. 2003, 67, 593–656. [Google Scholar] [CrossRef]
  11. Stewart, P.S.; Costerton, J.W. Antibiotic Resistance of Bacteria in Biofilms. Lancet 2001, 358, 135–138. [Google Scholar] [CrossRef]
  12. Pao, S.S.; Paulsen, I.T.; Saier, M.H., Jr. Major Facilitator Superfamily. Microbiol. Mol. Biol. Rev. 1998, 62, 1. [Google Scholar] [CrossRef]
  13. Tseng, T.T.; Gratwick, K.S.; Kollman, J.; Park, D.; Nies, D.H.; Goffeau, A.; Saier, M.H. The RND Permease Superfamily: An Ancient, Ubiquitous and Diverse Family That Includes Human Disease and Development Proteins. J. Mol. Microbiol. Biotechnol. 1999, 1, 107–125. [Google Scholar]
  14. Li, X.Z.; Nikaido, H. Efflux-Mediated Drug Resistance in Bacteria. Drugs 2004, 64, 159–204. [Google Scholar] [CrossRef]
  15. Poole, K. Efflux-Mediated Antimicrobial Resistance. J. Antimicrob. Chemother. 2005, 56, 20–51. [Google Scholar] [CrossRef]
  16. Piddock, L.J.V. Clinically Relevant Chromosomally Encoded Multidrug Resistance Efflux Pumps in Bacteria. Clin. Microbiol. Rev. 2006, 19, 382. [Google Scholar] [CrossRef]
  17. Stavri, M.; Piddock, L.J.V.; Gibbons, S. Bacterial Efflux Pump Inhibitors from Natural Sources. J. Antimicrob. Chemother. 2007, 59, 1247–1260. [Google Scholar] [CrossRef]
  18. Compagne, N.; Vieira Da Cruz, A.; Müller, R.T.; Hartkoorn, R.C.; Flipo, M.; Pos, K.M. Update on the Discovery of Efflux Pump Inhibitors against Critical Priority Gram-Negative Bacteria. Antibiotics 2023, 12, 180. [Google Scholar] [CrossRef]
  19. Pagès, J.M.; Masi, M.; Barbe, J. Inhibitors of Efflux Pumps in Gram-Negative Bacteria. Trends Mol. Med. 2005, 11, 382–389. [Google Scholar] [CrossRef]
  20. Marquez, B. Bacterial Efflux Systems and Efflux Pumps Inhibitors. Biochimie 2005, 87, 1137–1147. [Google Scholar] [CrossRef]
  21. Mahamoud, A.; Chevalier, J.; Alibert-Franco, S.; Kern, W.V.; Pagès, J.M. Antibiotic Efflux Pumps in Gram-Negative Bacteria: The Inhibitor Response Strategy. J. Antimicrob. Chemother. 2007, 59, 1223–1229. [Google Scholar] [CrossRef]
  22. Lomovskaya, O.; Bostian, K.A. Practical Applications and Feasibility of Efflux Pump Inhibitors in the Clinic—A Vision for Applied Use. Biochem. Pharmacol. 2006, 71, 910–918. [Google Scholar] [CrossRef] [PubMed]
  23. Renau, T.E.; Léger, R.; Flamme, E.M.; Sangalang, J.; She, M.W.; Yen, R.; Gannon, C.L.; Griffith, D.; Chamberland, S.; Lomovskaya, O.; et al. Inhibitors of Efflux Pumps in Pseudomonas Aeruginosa Potentiate the Activity of the Fluoroquinolone Antibacterial Levofloxacin. J. Med. Chem. 1999, 42, 4928–4931. [Google Scholar] [CrossRef]
  24. Lomovskaya, O.; Warren, M.S.; Lee, A.; Galazzo, J.; Fronko, R.; Lee, M.; Blais, J.; Cho, D.; Chamberland, S.; Renau, T.; et al. Identification and Characterization of Inhibitors of Multidrug Resistance Efflux Pumps in Pseudomonas Aeruginosa: Novel Agents for Combination Therapy. Antimicrob. Agents Chemother. 2001, 45, 105. [Google Scholar] [CrossRef] [PubMed]
  25. Coban, A.Y.; Ekinci, B.; Durupinar, B. A Multidrug Efflux Pump Inhibitor Reduces Fluoroquinolone Resistance in Pseudomonas Aeruginosa Isolates. Chemotherapy 2004, 50, 22–26. [Google Scholar] [CrossRef]
  26. Samosorn, S.; Bremner, J.B.; Ball, A.; Lewis, K. Synthesis of Functionalized 2-Aryl-5-Nitro-1H-Indoles and Their Activity as Bacterial NorA Efflux Pump Inhibitors. Bioorg. Med. Chem. 2006, 14, 857–865. [Google Scholar] [CrossRef] [PubMed]
  27. Lamut, A.; Peterlin Mašič, L.; Kikelj, D.; Tomašič, T. Efflux Pump Inhibitors of Clinically Relevant Multidrug Resistant Bacteria. Med. Res. Rev. 2019, 39, 2460–2504. [Google Scholar] [CrossRef]
  28. Reza, A.; Mark Sutton, J.; Rahman, K.M. Effectiveness of Efflux Pump Inhibitors as Biofilm Disruptors and Resistance Breakers in Gram-Negative (ESKAPEE) Bacteria. Antibiotics 2019, 8, 229. [Google Scholar] [CrossRef] [PubMed]
  29. Zechini, B.; Versace, I. Inhibitors of Multidrug Resistant Efflux Systems in Bacteria. Recent Pat. Anti-Infect. Drug Discov. 2009, 4, 37–50. [Google Scholar] [CrossRef]
  30. Donlan, R.M. Biofilms: Microbial Life on Surfaces. Emerg. Infect. Dis. 2002, 8, 881. [Google Scholar] [CrossRef]
  31. Roy, R.; Tiwari, M.; Donelli, G.; Tiwari, V. Strategies for Combating Bacterial Biofilms: A Focus on Anti-Biofilm Agents and Their Mechanisms of Action. Virulence 2018, 9, 522. [Google Scholar] [CrossRef]
  32. Desai, J.; Sachchidanand, S.; Kumar, S.; Sharma, R. Novel Bacterial Topoisomerase Inhibitors (NBTIs)—A Comprehensive Review. Eur. J. Med. Chem. Rep. 2021, 3, 100017. [Google Scholar] [CrossRef]
  33. Charrier, C.; Salisbury, A.M.; Savage, V.J.; Duffy, T.; Moyo, E.; Chaffer-Malam, N.; Ooi, N.; Newman, R.; Cheung, J.; Metzger, R.; et al. Novel Bacterial Topoisomerase Inhibitors with Potent Broad-Spectrum Activity against Drug-Resistant Bacteria. Antimicrob. Agents Chemother. 2017, 61. [Google Scholar] [CrossRef]
  34. Aldred, K.J.; Kerns, R.J.; Osheroff, N. Mechanism of Quinolone Action and Resistance. Biochemistry 2014, 53, 1565–1574. [Google Scholar] [CrossRef] [PubMed]
  35. Khan, T.; Sankhe, K.; Suvarna, V.; Sherje, A.; Patel, K.; Dravyakar, B. DNA Gyrase Inhibitors: Progress and Synthesis of Potent Compounds as Antibacterial Agents. Biomed. Pharmacother. 2018, 103, 923–938. [Google Scholar] [CrossRef]
  36. Durcik, M.; Nyerges, Á.; Skok, Ž.; Skledar, D.G.; Trontelj, J.; Zidar, N.; Ilaš, J.; Zega, A.; Cruz, C.D.; Tammela, P.; et al. New Dual ATP-Competitive Inhibitors of Bacterial DNA Gyrase and Topoisomerase IV Active against ESKAPE Pathogens. Eur. J. Med. Chem. 2021, 213, 113200. [Google Scholar] [CrossRef]
  37. Kokot, M.; Weiss, M.; Zdovc, I.; Hrast, M.; Anderluh, M.; Minovski, N. Structurally Optimized Potent Dual-Targeting NBTI Antibacterials with an Enhanced Bifurcated Halogen-Bonding Propensity. ACS Med. Chem. Lett. 2021, 12, 1478–1485. [Google Scholar] [CrossRef]
  38. Kokot, M.; Weiss, M.; Zdovc, I.; Senerovic, L.; Radakovic, N.; Anderluh, M.; Minovski, N.; Hrast, M. Amide Containing NBTI Antibacterials with Reduced HERG Inhibition, Retained Antimicrobial Activity against Gram-Positive Bacteria and in Vivo Efficacy. Eur. J. Med. Chem. 2023, 250, 115160. [Google Scholar] [CrossRef] [PubMed]
  39. Kolarič, A.; Kokot, M.; Hrast, M.; Weiss, M.; Zdovc, I.; Trontelj, J.; Žakelj, S.; Anderluh, M.; Minovski, N. A Fine-Tuned Lipophilicity/Hydrophilicity Ratio Governs Antibacterial Potency and Selectivity of Bifurcated Halogen Bond-Forming Nbtis. Antibiotics 2021, 10, 862. [Google Scholar] [CrossRef]
  40. Kokot, M.; Weiss, M.; Zdovc, I.; Anderluh, M.; Hrast, M.; Minovski, N. Diminishing HERG Inhibitory Activity of Aminopiperidine-Naphthyridine Linked NBTI Antibacterials by Structural and Physicochemical Optimizations. Bioorg. Chem. 2022, 128, 106087. [Google Scholar] [CrossRef]
  41. Kim, L. In Search of Natural Substrates and Inhibitors of MDR Pumps. J. Mol. Microbiol. Biotechnol. 2001, 3, 247–254. [Google Scholar]
  42. Bambeke, F.; Pages, J.-M.; Lee, V. Inhibitors of Bacterial Efflux Pumps as Adjuvants in Antibiotic Treatments and Diagnostic Tools for Detection of Resistance by Efflux. Recent Pat. Antiinfect. Drug Discov. 2006, 1, 157–175. [Google Scholar] [CrossRef] [PubMed]
  43. Bush, K. Synergistic Antibiotic Combinations. Top. Med. Chem. 2018, 25, 69–88. [Google Scholar] [CrossRef]
  44. Acar, J.F. Antibiotic Synergy and Antagonism. Med. Clin. N. Am. 2000, 84, 1391–1406. [Google Scholar] [CrossRef]
  45. Renau, T.E.; Léger, R.; Filonova, L.; Flamme, E.M.; Wang, M.; Yen, R.; Madsen, D.; Griffith, D.; Chamberland, S.; Dudley, M.N.; et al. Conformationally-Restricted Analogues of Efflux Pump Inhibitors That Potentiate the Activity of Levofloxacin in Pseudomonas Aeruginosa. Bioorg. Med. Chem. Lett. 2003, 13, 2755–2758. [Google Scholar] [CrossRef]
  46. Cotman, A.E.; Durcik, M.; Benedetto Tiz, D.; Fulgheri, F.; Secci, D.; Sterle, M.; Možina, Š.; Skok, Ž.; Zidar, N.; Zega, A.; et al. Discovery and Hit-to-Lead Optimization of Benzothiazole Scaffold-Based DNA Gyrase Inhibitors with Potent Activity against Acinetobacter Baumannii and Pseudomonas Aeruginosa. J. Med. Chem. 2023, 66, 1380–1425. [Google Scholar] [CrossRef]
  47. Cotman, A.E.; Fulgheri, F.; Piga, M.; Peršolja, P.; Benedetto Tiz, D.; Skok, Ž.; Durcik, M.; Sterle, M.; Dernovšek, J.; Cruz, C.D.; et al. New N-Phenylpyrrolamide Inhibitors of DNA Gyrase with Improved Antibacterial Activity. RSC Adv. 2024, 14, 28423–28454. [Google Scholar] [CrossRef] [PubMed]
  48. Durcik, M.; Cotman, A.E.; Toplak, Ž.; Možina, Š.; Skok, Ž.; Szili, P.E.; Czikkely, M.; Maharramov, E.; Vu, T.H.; Piras, M.V.; et al. New Dual Inhibitors of Bacterial Topoisomerases with Broad-Spectrum Antibacterial Activity and In Vivo Efficacy against Vancomycin-Intermediate Staphylococcus Aureus. J. Med. Chem. 2023, 66, 3968–3994. [Google Scholar] [CrossRef]
  49. The European Committee on Antimicrobial Susceptibility Testing. Breakpoint Tables for Interpretation of MICs and Zone Diameters. Version 14.0. 2024. Available online: https://www.eucast.org/ (accessed on 10 July 2024).
  50. Clinical Laboratory Standards Institute. M07 Ed12|Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically, 12th Edition. Available online: https://clsi.org/standards/products/microbiology/documents/m07/ (accessed on 10 July 2024).
  51. Merritt, J.H.; Kadouri, D.E.; O’Toole, G.A. Growing and Analyzing Static Biofilms. Curr. Protoc. Microbiol. 2005, 22, 1B-1. [Google Scholar] [CrossRef]
Antibiotics 13 01081 g001
Figure 1. Structural representations of the investigated efflux pump inhibitors.
Figure 1. Structural representations of the investigated efflux pump inhibitors.
Antibiotics 13 01081 g001
Antibiotics 13 01081 g002
Figure 2. Structural representations of the investigated novel bacterial topoisomerase inhibitors (6 (N-(4-bromo-3,5-difluorobenzyl)-1-(2-(6-methoxy-1,5-naphthyridin-4-yl)ethyl)piperidin-4-amine), 7 (2-(4-((4-bromo-3,5-difluorobenzyl)amino)cyclohexyl)-1-(6-methoxy-1,5-naphthyridin-4-yl)ethan-1-ol), and 8 (4-bromo-3,5-difluoro-N-(6-(((6-methoxy-1,5-naphthyridin-4-yl)oxy)methyl)tetrahydro-2H-pyran-3-yl)benzamide)) and ATP-competitive inhibitors of bacterial type II topoisomerases (9 (1-(2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-5-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl)piperidin-3-aminium chloride), 10 (2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylic acid), and 11 (2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-morpholinobenzo[d]thiazole-6-carboxylic acid)).
Figure 2. Structural representations of the investigated novel bacterial topoisomerase inhibitors (6 (N-(4-bromo-3,5-difluorobenzyl)-1-(2-(6-methoxy-1,5-naphthyridin-4-yl)ethyl)piperidin-4-amine), 7 (2-(4-((4-bromo-3,5-difluorobenzyl)amino)cyclohexyl)-1-(6-methoxy-1,5-naphthyridin-4-yl)ethan-1-ol), and 8 (4-bromo-3,5-difluoro-N-(6-(((6-methoxy-1,5-naphthyridin-4-yl)oxy)methyl)tetrahydro-2H-pyran-3-yl)benzamide)) and ATP-competitive inhibitors of bacterial type II topoisomerases (9 (1-(2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-5-(5-oxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)phenyl)piperidin-3-aminium chloride), 10 (2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-(1-phenylethoxy)benzo[d]thiazole-6-carboxylic acid), and 11 (2-(3,4-dichloro-5-methyl-1H-pyrrole-2-carboxamido)-4-morpholinobenzo[d]thiazole-6-carboxylic acid)).
Antibiotics 13 01081 g002
Antibiotics 13 01081 g003
Figure 3. Inhibition of A. baumannii biofilm formation (A) and mature (24 h old) A. baumannii biofilm disintegration (B) with compounds 6 and 7 at different concentrations. Treatment with DMSO (w/o (without the compound), grey) was used as negative control.
Figure 3. Inhibition of A. baumannii biofilm formation (A) and mature (24 h old) A. baumannii biofilm disintegration (B) with compounds 6 and 7 at different concentrations. Treatment with DMSO (w/o (without the compound), grey) was used as negative control.
Antibiotics 13 01081 g003
Antibiotics 13 01081 g004
Figure 4. Inhibition of biofilm formation (A,B) and biofilm disintegration (C,D) with compounds 6 and 7 at concentrations equal to MIC/4, MIC/2, MIC, and 2MIC (MIC1 = 0.125 µg/mL; MIC2 = 1 µg/mL), alone (w/o), and with increasing concentrations of PAβN (1 µg/mL, 10 µg/mL, and 100 µg/mL).
Figure 4. Inhibition of biofilm formation (A,B) and biofilm disintegration (C,D) with compounds 6 and 7 at concentrations equal to MIC/4, MIC/2, MIC, and 2MIC (MIC1 = 0.125 µg/mL; MIC2 = 1 µg/mL), alone (w/o), and with increasing concentrations of PAβN (1 µg/mL, 10 µg/mL, and 100 µg/mL).
Antibiotics 13 01081 g004
Table 1. Antibacterial activity of NBTIs and EPIs against various Gram-negative “ESKAPE” pathogens.
Table 1. Antibacterial activity of NBTIs and EPIs against various Gram-negative “ESKAPE” pathogens.
Bacterial StrainMIC (µg/mL)
EPINBTIACI
1234567891011
E. coli (ATCC 25922)>128>25632>128>640.5–12128818
E. coli D22 aNDNDNDNDND0.250.016128NDNDND
E. coli N43 (CGSC# 5583) bNDNDNDNDND0.008<0.0080.031NDNDND
P. aeruginosa RDK 184>128>256128>128>641616>1281618
K. pneumoniae RDK 070A>128>25664>128>6428>12816–32216
A. baumannii 8C6 (GES-14)>128>256NDNDND0.125–0.50.5–1>1288–1614
a With a mutation in the lpxC gene that increases membrane permeability. b With AcrA knock-out (cell membrane efflux pump). ND: not determined.
Table 2. Antibacterial activity of NBTIs in combination with efflux pump inhibitor PAβN.
Table 2. Antibacterial activity of NBTIs in combination with efflux pump inhibitor PAβN.
Bacterial StrainCmpdMIC of NBTI in the Presence of PAβN (µg/mL)MPC8 b (µg/mL)
PAβN a Concentration (µg/mL)
00.51248163264128
E. coli610.50.50.250.1250.0630.0310.0160.0160.0164
722210.50.250.0630.0310.0310.0318
P. aeruginosa6168888810.250.0630.03116
7168888820.50.250.12516
8>128>128>128>128>128>128>12882216–32
K. pneumoniae6222210.1250.0630.0310.0310.0168
78888210.250.1250.0630.0318
8>128>128>128>128>128>128>12822216–32
A. baumannii60.250.0630.0630.0310.0310.0310.0160.0160.0160.0162
70.50.50.50.50.250.250.1250.0630.0310.03132
MIC values of the antibacterials alone are shown in the third column, where PAβN concentration equals 0. All MPCs were determined by comparing the MICs of the antibacterials alone with the MICs of the antibacterials in combination with PAβN. a PAβN: efflux pump inhibitor Phe-Arg-naphthylamide. b MPC8: minimum concentration of efflux pump inhibitor in µg/mL required to reduce the MIC of NBTI at least 8-fold.
Table 3. MIC values of individual compounds or in combination.
Table 3. MIC values of individual compounds or in combination.
Bacterial StrainMIC (µg/mL)
NBTIACI
691011
I aC b, 9C b, 10C b, 11I aC b, 6I aC b, 6I aC b, 6
E. coli0.50.250.250.1258410.584
P. aeruginosa1688816210.582
K. pneumoniae2–422116–32821168
A. baumannii0.1250.0630.0630.06316410.2544
a Intrinsic antibacterial activity, i.e., MIC when the compound is administered individually (I). b MIC when the compound is administered in combination (C). 9 MIC in combination with 9. 10 MIC in combination with 10. 11 MIC in combination with 11. 6 MIC in combination with compound 6.
Table 4. FIC index values of compound 6 in combination with various ACIs.
Table 4. FIC index values of compound 6 in combination with various ACIs.
Bacterial StrainFIC
C b, 9C b, 10C b, 11
E. coli1.0001.0000.750
P. aeruginosa0.6251.0000.750
K. pneumoniae0.7501.0000.750
A. baumannii0.7500.7501.500
b MIC when the compound is administered in combination (C). 9 MIC in combination with 9. 10 MIC in combination with 10. 11 MIC in combination with 11.
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Zorman, M.; Kokot, M.; Zdovc, I.; Senerovic, L.; Mandic, M.; Zidar, N.; Cotman, A.E.; Durcik, M.; Peterlin Mašič, L.; Minovski, N.; et al. Enhancing Antibacterial Efficacy: Combining Novel Bacterial Topoisomerase Inhibitors with Efflux Pump Inhibitors and Other Agents Against Gram-Negative Bacteria. Antibiotics 2024, 13, 1081. https://doi.org/10.3390/antibiotics13111081

AMA Style

Zorman M, Kokot M, Zdovc I, Senerovic L, Mandic M, Zidar N, Cotman AE, Durcik M, Peterlin Mašič L, Minovski N, et al. Enhancing Antibacterial Efficacy: Combining Novel Bacterial Topoisomerase Inhibitors with Efflux Pump Inhibitors and Other Agents Against Gram-Negative Bacteria. Antibiotics. 2024; 13(11):1081. https://doi.org/10.3390/antibiotics13111081

Chicago/Turabian Style

Zorman, Maša, Maja Kokot, Irena Zdovc, Lidija Senerovic, Mina Mandic, Nace Zidar, Andrej Emanuel Cotman, Martina Durcik, Lucija Peterlin Mašič, Nikola Minovski, and et al. 2024. "Enhancing Antibacterial Efficacy: Combining Novel Bacterial Topoisomerase Inhibitors with Efflux Pump Inhibitors and Other Agents Against Gram-Negative Bacteria" Antibiotics 13, no. 11: 1081. https://doi.org/10.3390/antibiotics13111081

APA Style

Zorman, M., Kokot, M., Zdovc, I., Senerovic, L., Mandic, M., Zidar, N., Cotman, A. E., Durcik, M., Peterlin Mašič, L., Minovski, N., Anderluh, M., & Hrast Rambaher, M. (2024). Enhancing Antibacterial Efficacy: Combining Novel Bacterial Topoisomerase Inhibitors with Efflux Pump Inhibitors and Other Agents Against Gram-Negative Bacteria. Antibiotics, 13(11), 1081. https://doi.org/10.3390/antibiotics13111081

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