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. 2020 Jun 2;15(8):579–590. doi: 10.2217/fmb-2019-0094

Synergistic action of substituted indole derivatives and clinically used antibiotics against drug-resistant bacteria

Danielle N Turner 1, Leslie Edwards 1, Alexander Kornienko 1,2, Liliya V Frolova 1, Snezna Rogelj 1,*
PMCID: PMC7546154  PMID: 32483987

Abstract

Aim:

The current report describes the discovery of indole derivatives that synergize with standard antibiotics.

Materials & methods:

The antibacterial activities were determined using an optimized time–kill method, while viability of mammalian cells was assessed using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.

Results:

The synergy is observed with methicillin- and vancomycin-resistant Staphylococcus aureus bacterial strains, against which the standard antibiotics show no activities of their own. Our indole derivatives in combination with antibiotics lack toxicity toward mammalian cells, do not promote the evolution of resistance of S. aureus in comparison to clinically established antibiotics, and likely work by permeabilizing bacterial cell membranes.

Conclusion:

The above-mentioned findings demonstrate the potential clinical applications of our indole derivatives.

Keywords: : antibiotics, Gram-positive bacteria, infectious disease therapeutics, MRSA, multidrug resistance

Graphical abstract

graphic file with name fmb-15-579-GA.jpg

Over the past 50 years antibiotics have been used as miracle drugs in the treatment of bacterial infections. However, the buildup of antibiotics in the environment [1,2], as a result of their wide-ranging use in healthcare [3] and agriculture [4], has put a selective evolutionary pressure on bacteria leading to appearance and spread of resistance [5]. Mutations that allow a microbe to survive the treatment with an antibiotic quickly spread in the microbial population [6]. Despite the social awareness campaigns [7] aimed at the inappropriate use of antibiotics, the resistance is rapidly growing and there is an urgent need for unconventional, therapeutic approaches to combat the resistant strains.

While the need for new strategies to fight resistant bacterial strains is urgent in general, Staphylococcus aureus is an especially dangerous and versatile opportunistic pathogen causing conditions, ranging from skin infections (resulting from cuts, abrasions, turf burns, etc.) to severe invasive diseases [8]. Originally responsive to penicillin, a number of its strains are now resistant to macrolides, beta-lactams and even vancomycin, the ‘drug of last resort’. Methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus represent a very important health threat and constitute a major cause of mortality from bacterial infections [9].

Another important Gram-positive pathogen causing hospital-acquired infections is Enterococcus faecalis [10]. About 5% of E. faecalis are resistant to ampicillin, while vancomycin resistance is present in 12.5% of these pathogens [11]. Vancomycin-resistant Enterococci (VRE) infections are responsible for severe crisis in hospitals as they are associated with high mortality and secondary infections, such as infective endocarditis [12].

Thus, there is an unmet clinical need to explore novel approaches to combat MRSA, VRE and other resistant bacterial strains. Compounds incorporating indole as the basic structural unit have been studied as potential antibiotics [13,14] and recently investigated by researchers in an attempt to develop antibacterial agents capable of overcoming bacterial resistance. For example, 3-(1H-imidazol-2-yl)-indoles were reported to be active against MRSA and VRE strains [15]. 2-Arylindole derivatives were shown to inhibit multidrug resistance mechanisms (MDR) in Gram-positive pathogens, including MRSA [16]. 3-Azoindole derivatives, compounds more relevant to those described in the current study, showed activity against a number of bacterial strains, but they were not evaluated against MDR bacteria [17]. Our own studies in this area led to new indole-based compounds that possessed activity against MRSA and a number of other bacterial strains [18]. While these advances are significant, the rapid emergence of bacterial resistance leads to examination of strategies that go beyond the ‘one drug, one site of action’ model characteristic of traditional antibiotics. Efforts aimed at the discovery of synergistic antibiotic combinations are becoming an important area of research [19–21]. Driven by these considerations, we reexamined our previously reported indole-based compounds and discovered a promising potentiation of the effects of known antibiotics against drug-resistant strains. The present report describes indole derivatives that synergize with antibiotics, such as norfloxacin, oxacillin and vancomycin, against drug-resistant Gram-positive organisms MRSA and VRE.

Materials & methods

Compounds

All compounds used in the work were dissolved in sterile DMSO (VWR International, PA, USA) and maintained at -20°C. A maximum of 0.1% DMSO as a vehicle was used for all synergy experiments.

Bacterial culture & synergistic studies against MRSA

Bacterial isolates were grown from a glycerol stock onto a Mueller–Hinton agar (MHA) plate overnight at 37°C. A bacterial broth suspension was grown in Mueller–Hinton broth (MHB) at 37°C on a rotary shaking incubator (100 r.p.m.). Bacterial cell concentration was adjusted to approximately 5 × 105 CFU/ml using optical density at 595 nm. For synergy studies, appropriate concentrations of vancomycin, norfloxacin and oxacillin were added to a broth suspension of MRSA ATCC BAA-44 at a cell concentration of 5 × 105 CFU/ml and vortexed to mix. The samples were incubated at 37°C on a rotary shaking incubator (100 r.p.m.) for 30 min, prior to the addition of 8 and 30 μM IM4, 1.4, 4 and 5 μM IM5, 8 μM IM7, 0.6 μM IM17 and 0.4 μM IM171. Samples were incubated for 18–24 h at 37°C on a rotary shaking incubator (100 r.p.m.). Following the incubation, samples were diluted tenfold and drip streaked (10 μl of each dilution) on to an MHA plate and incubated at 37°C overnight. Colony counts were performed and CFU/ml was calculated.

Bacterial culture & synergistic studies against VRE

Two hundred microgram per milliliter vancomycin was added to a broth suspension of VRE ATCC BAA-2573 that had been adjusted to a cell concentration of approximately 5 × 105 CFU/ml. The samples were vortexed to mix and incubated at 37°C on a rotary shaking incubator (100 r.p.m.) for 30 min, prior to the addition of 18 μM IM4, 2 μM IM5, 3 μM IM5, 5 μM IM7, 5 μM IM17 and 2.5 μM IM171. Samples were incubated for 18–24 h at 37°C on a rotary shaking incubator (250 r.p.m.). Following the incubation, samples were diluted tenfold and drip streaked (10 μl of each dilution) on to an MHA plate and incubated at 37°C overnight. Colony counts were performed and CFU/ml was calculated.

Cell culture viability assay

A HeLa (ATCC CCL-2) cell line was obtained from American Culture-Type Collection (ATCC, VA, USA), and was grown in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum in a humidified 5% CO2 incubator at 37°C. HeLa cells were seeded in a 96-well plate (4000 cells/well) and allowed to adhere overnight. The following day, cells were treated in fresh Dulbecco’s modified Eagle medium with the following concentrations as previously determined by synergistic combinations in MRSA ATCC BAA-44: 8 μM IM4 + 100 μg/ml oxacillin, 3 μM IM4 + 25 μg/ml norfloxacin, 8 μM IM4 + 1 μg/ml vancomycin, 1.4 μM IM5 + 100 μg/ml oxacillin, 5 μM IM5 + 25 μg/ml norfloxacin, 4 μM IM5 + 1 μg/ml vancomycin, 8 μM IM7 + 100 μg/ml oxacillin, 8 μM IM7 + 25 μg/ml norfloxacin, 8 μM IM7 + 1 μg/ml vancomycin, 0.3 μM IM17 + 100 μg/ml oxacillin, 0.3 μM IM17 + 25 μg/ml norfloxacin, 0.3 μM IM17 + 1 μg/ml vancomycin, 0.6 μM IM171 + 100 μg/ml oxacillin, 0.6 μM IM171 + 25 μg/ml norfloxacin and 0.6 μM IM171 + 1 μg/ml vancomycin for 48 h. Phenylarsine oxide (Sigma-Aldrich, MO, USA) at a final concentration of 100 μM served as a positive control, 1% DMSO (Sigma-Aldrich) served as a vehicle control and untreated cells served as a negative control. After 48-h incubation, 20 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma-Aldrich) solution (5 mg/ml stock in 1× phosphate-buffered saline) was added to each well. Cells were subsequently incubated at 37°C for 2 h. Following a 2-h incubation, the supernatant was aspirated and 100 μl of 100% DMSO was added to each well. Absorbance was measured at 595 nm using a microplate reader.

Evolution of resistance

The MICs of antibiotics and IM compounds were determined against S. aureus (ATCC 29213). In a 96-well microtiter plate, 100 μl of cell suspension (about 5 × 105 CFU/ml in tryptic soy broth were twofold diluted in triplicate with an IM compound or antibiotic. The mixture was allowed to incubate at 37°C on a rotary shaking incubator for approximately 16 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent (5 mg/ml w/v) was added to assess the viability (at 10% v/v). To evaluate for the resistance of evolution, sub-inhibitory concentrations (0.5 MIC; 0.25 MIC) of each compound and antibiotic were incubated with cells in tryptic soy broth. Cells were then streaked on to a tryptic soy agar (TSA) plate and allowed to incubate. The procedure was repeated for 30 days.

Membrane depolarization

MRSA ATCC BAA-44 was grown from a glycerol stock by placing a small volume of cells onto an MHA. The MHA plate was incubated overnight at 37°C. A single colony was subsequently used to inoculate MHB to obtain a bacterial broth suspension. This was grown in at 37°C on a rotary shaking incubator (100 r.p.m.) for 12 h. The bacterial cell concentration was adjusted to about 1 × 108 CFU/ml in MHB using optical density at 595 nm. Four hundred microliters of the adjusted bacterial cell suspensions were placed into borosilicate glass tubes, where the cell suspensions were treated with 5 μM 3,3′-diethyloxacarbocyanine iodide for 20 min before being treated with 4 × MIC of each compound as follows: 100 μM IM4, 20 μM IM5, 100 μM IM7, 5 μM IM17, 2.5 μM IM171; 20 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP), 100 μM CCCP and 20 μM melittin as a positive controls, and non-treated cells as a negative control. The treated cells were incubated for 25 min at 37°C and then fixed for 60 min in 2.5% paraformaldehyde. The relative fluorescence units were read on a FACScan flow cytometer on FL2.

Membrane permeability

MRSA ATCC BAA-44 was grown from a glycerol stock by placing a small volume of cells onto an MHA. The MHA plate was incubated overnight at 37°C. A single colony was subsequently used to inoculate MHB to obtain a bacterial broth suspension. This was grown in at 37°C on a rotary shaking incubator (100 r.p.m.) for 12 h. The bacterial cell concentration was adjusted to about 1 × 107 CFU/ml in MHB using optical density at 595 nm. 100 μl of the adjusted bacterial cell suspensions were placed into a 96-well plate, where the cell suspensions were treated with 4× MIC of each compound as follows: 100 μM IM4, 20 μM IM5, 100 μM IM7, 5 μM IM17, 2.5 μM IM171; 10 μM melittin as a positive control, and non-treated cells as a negative control. The treated cells were incubated for 15 min at 37°C. Eighteen micromolar propidium iodide (PI) was added to the cells and the cells were stained for 30 min at 37°C. The relative fluorescence units were read on a Tecan plate reader at 636 nm.

Results

The indole derivatives in question (Figure 1) contain the following set of substituents: 3-arylazo moiety (IM4, IM5 and IM7), 2-aryl moiety (IM4, IM171 and IM17) and 3-arylether group (IM171 and IM17). The syntheses of these 2,3-disubstituted and 3-monosubstituted indoles have been reported previously [18].

Figure 1. . Indole derivatives.

Figure 1. 

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3-monosubstituted (IM5 and IM7) and 2,3-disubstituted (IM4, IM17 and IM171) indole.

We have found that at specific dose ratios, compounds IM4, IM5, IM7, IM17 and IM171 synergize with clinically used antibiotics in their killing effect toward the multidrug-resistant MRSA strain ATCC BAA-44, which was derived from a sample obtained from a hospital in Lisbon, Portugal. Thus, when an IM compound and an antibiotic were combined at doses that show little antibacterial action on their own, normally a several-log reduction in bacterial count is observed for each of the combinations (Figure 2). For example, as shown in Figure 2C, oxacillin, vancomycin and norfloxacin utilized at 100, 0.01 and 25 μg/ml, respectively, produced no antibacterial action. Yet, a 4–5-log reduction in bacterial count was observed when these were potentiated with 8 μM IM7 also having no effect on its own.

Figure 2. . Synergy of IM compounds with antibiotics.

Figure 2. 

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(A) IM4, (B) IM5, (C) IM7, (D) IM17 and (E) IM171 with antibiotics oxacillin, norfloxacin and vancomycin against methicillin-resistant Staphylococcus aureus (ATCC BAA-44). The antibacterial activities were determined using an optimized time–kill method, as described in ‘Materials & methods’. The error bars represent technical triplicates in a representative biological replicate carried out at least three times.

CFU: Colony-forming unit; NT: No treatment.

We have evaluated the potential synergy of the IM compounds with vancomycin against VRE ATCC BAA-2573 strain at selected doses (Figure 3). Similar to the effects of this combination on MRSA, as described above, 1.2–4.5-log reduction in bacterial count was observed. It should be noted that vancomycin, which is generally effective at single digit μg/ml doses against susceptible strains, showed no VRE killing at doses as high as 200 μg/ml, but its activity was restored with the IM compounds. Particularly noteworthy is its 4.5-log potentiation of activity when 200 μg/ml vancomycin was used in combination with 3 μM IM5 (Figure 3B).

Figure 3. . Synergy of IM compounds with vancomycin.

Figure 3. 

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(A) IM4, (B) IM5, (C) IM7, (D) IM17 and (E) IM171 with vancomycin against vancomycin-resistant Enterococci (ATCC 2573). The antibacterial activities were determined using an optimized time–kill method, as described in Materials & methods. The error bars represent technical triplicates in a representative biological replicate carried out at least three times.

CFU: Colony-forming unit; NT: No treatment.

Importantly, we found that the IM compounds in combination with antibiotics show no toxicity to mammalian cells at doses that manifest synergistic antibacterial activities (Figure 4). For example, the combination treatment of MRSA involving 100 μg/ml oxacillin and 8 μM IM7 (see Figure 2C) shows 5.0-log reduction relative to individual doses of each ingredient. Yet, this combination treatment exhibited no toxicity to HeLa cervical cancer cells as compared with the untreated cells or cells treated with 1% DMSO vehicle control (Figure 4C).

Figure 4. . Toxicity of synergistic combinations toward human HeLa cervical carcinoma cells.

Figure 4. 

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(A) Synergistic combinations of IM4 with oxacillin, norfloxacin and vancomycin. (B) Synergistic combinations of IM5 with oxacillin, norfloxacin and vancomycin. (C) Synergistic combinations of IM7 with oxacillin, norfloxacin and vancomycin. (D) Synergistic combinations of IM17 with oxacillin, norfloxacin and vancomycin. (E) Synergistic combinations of IM171 with oxacillin, norfloxacin and vancomycin. One percent DMSO vehicle control was a negative control, phenylarsine oxide served as a positive control. Determined by the MTT method. The error bars represent technical triplicates.

MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NT: No treatment; PAO: Phenylarsine oxide.om

Another aspect of the present work involves the demonstration that the four out of five described indole derivatives do not promote the evolution of resistance in S. aureus. Indeed, serial passaging at fixed sub-inhibitory concentrations over extended periods, as often occurs in clinic, did not result in the evolution of resistance in the case of IM4, IM5, IM7 and IM17 [22]. In contrast, a similar experiment with the clinical antibiotics oxacillin and norfloxacin resulted in a dramatic increase of their MIC values at the end of the treatment period indicating the generation of a large resistant pathogen population (Table 1).

Table 1.  . Failure to promote the evolution of resistance by compounds IM4, IM5, IM7 and IM17, relative to the standard antibiotics.

Compound Initial MIC day 0 Final MIC day 32
IM4 1–2 μM 1–2 μM
IM5 4 μM 4 μM
IM7 8 μM 8 μM
IM17 2 μM 1 μM
IM171 0.25–5 μM >32 μM
Oxacillin 0.6 μM >500 μM
Norfloxacin 6.2 μM >310 μM
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The antibacterial activities were determined using a standard resistance study, as described in ‘Materials & methods’.

Our mechanistic studies point to the membrane-permeabilizing effect of the IM compounds. Thus, to assess whether the IM compounds cause membrane depolarization in MRSA, we used membrane potential probe 3,3′-diethyloxacarbocyanine iodide (DiOC2[3]) [20]. This dye aggregates in membranes with intact membrane potential emitting red fluorescence, and thus, the decrease of red fluorescence is indicative of the dissipation of the membrane potential. The positive controls included melittin, a membrane pore-forming basic peptide [23], and CCCP, a proton translocator [20]. Figure 5 shows that in a manner similar to both positive controls, the IM compounds reduce the magnitude of red fluorescence, providing evidence for the loss of membrane potential in the treated MRSA cells.

Figure 5. . The IM compounds induce membrane depolarization in methicillin-resistant Staphylococcus aureus.

Figure 5. 

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Determined using the decrease in fluorescence of membrane potential probe 3,3′-diethyloxacarbocyanine iodide. Compounds were used at 4× MIC. Melittin and CCCP were used as positive controls. The error bars represent technical triplicates in a representative biological replicate carried out at least three times.

DiOC2(3): 3,3′-diethyloxacarbocyanine iodide.

To obtain further evidence of membrane-permeabilizing effect of the IM compounds, we utilized a DNA intercalating dye PI. Cells with intact membranes exclude PI, but if the membrane integrity is compromised, PI inters the cells and binds to DNA, resulting in a large increase of its fluorescence [24]. As can be seen in Figure 6, the IM compounds induce an increase of PI fluorescence in a manner similar to melittin after MRSA cells have been treated at 4 × MIC. The choice of this concentration is based on established literature precedent for these types of assays [23].

Figure 6. . The IM compounds induce membrane permeabilization in methicillin-resistant Staphylococcus aureus.

Figure 6. 

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Determined using the increase in fluorescence of DNA-intercalating dye propidium iodide. Compounds were used at 4× MIC. Melittin was used as a positive control. The error bars represent technical triplicates in a representative biological replicate carried out at least three times.

PI: Propidium iodide

Discussion

In this work, we utilized the multidrug-resistant MRSA strain ATCC BAA-44 from a sample obtained in a hospital in Portugal. In addition to -lactams, this strain is resistant to antibiotics differing in their structures and modes of action, such as erythromycin, ciprofloxacin, tetracycline and tobramycin [21,25]. It was found that when an IM compound and an antibiotic were combined at doses that show little antibacterial action on their own, normally a several-log reduction in bacterial count is observed for each of the combinations (Figure 2). This finding is important, especially when one takes into account that the antibiotics selected for this study work through dissimilar mechanisms: fluoroquinolone norfloxacin is a bacterial DNA gyrase inhibitor, beta-lactam oxacillin a cell-wall biosynthesis inhibitor and glycopeptide vancomycin, also a cell-wall inhibitor, albeit utilizing a different intracellular target than oxacillin.

In an effort to address the increasing global resistance to antibiotics, on 27 February 2017 the WHO released a list of bacteria that present the greatest danger to human health generating the need for new effective antibiotics [26]. The list of bacteria was prioritized on the basis of the reported levels of resistance, the occurrence of infection, mortality they cause and the burden they place on healthcare systems. In addition to MRSA, another Gram-positive organism on the list is vancomycin-resistant E. faecalis, known as VRE [27]. As mentioned in the introduction, this bacterial strain has acquired resistance to vancomycin, in the past the drug of choice for this infection. Again, it was found that the activity of vancomycin absent at doses as high as 200 μg/ml was restored with the IM compounds (Figure 3).

Further findings in this work include the lack of mammalian cell toxicity of the synergistic mixtures of the IM compounds with antibiotics (Figure 4) and the demonstration of the absence of the evolution of resistance in S. aureus (Table 1). The latter finding is in contrast to the clinical antibiotics oxacillin and norfloxacin that gave rise to large resistant pathogen populations (Table 1). This is an important and unexpected discovery that bodes well for the potential clinical applications of these compounds.

Finally, we began studies aimed at elucidation of the mechanism underlying the synergy between the IM compounds and clinically used antibiotics. Since the indole derivatives synergize with structurally and mechanistically diverse antibiotics, we hypothesized that the IM compounds act as membrane permeabilizers, explaining such a general effect. To test this hypothesis, we performed membrane depolarization and permeabilization assays, whose results are consistent with this mechanistic hypothesis and provide strong evidence for membrane permeabilization as the mode of action of our indole derivatives.

Conclusion

The rapidly growing antibiotic resistance among the strains of pathogenic bacteria may be bringing to an end ‘the antibiotic era’, a period extending over the past 50 years. New antibacterial strategies are urgently needed to combat the emergence of superbugs, organisms resistant to most, if not all, currently available antibiotics. Because genetic elements encoding the resistant components are transferred easily throughout a microbial population or even between species, new compounds that potentiate the effects of many different families of antibiotics against the resistant organisms are urgently needed. The clinical effectiveness of combination antibiotic therapies has already been established for a number of bacterial infections for which individual therapy was ineffective. These include, but not limited to, aztreonam-amikacin against Pseudomonas aeruginosa [28], azithromycin-ceftazidime against Burkholderia cepacian [29] and rifampicin-meropenem-colistin against multidrug-resistant Klebsiella pneumoniae [30]. The current work describes compounds that potentiate the activities of the standard antibiotics that are ineffective on their own against the MRSA and VRE strains and these synergistic mixtures show no toxicity to mammalian cells. Further, these compounds do not promote the evolution of resistance of S. aureus and, based on our preliminary mechanistic studies, act as membrane permeabilizers. All in all, our new indole derivatives warrant further development as promising antibacterial agents that restore the activity of clinically used antibiotics against MDR bacterial strains.

Future perspective

The ease of transfer of genes throughout a microbial population will result in resistant strains that will require new treatment strategies. New compounds that potentiate the effects of many different families of antibiotics against the resistant bacterial strains will be an important focus of future research.

Summary points.

  • The article addresses the need for new antibacterial strategies to combat the emergence of superbugs, organisms resistant to most, if not all, currently available antibiotics.

  • The clinical effectiveness of combination antibiotic therapies has already been established for a number of bacterial infections for which individual therapy was ineffective.

  • We have found that at specific dose ratios, compounds IM4, IM5, IM7, IM17 and IM171 synergize with clinically used antibiotics in their killing effect toward the methicillin-resistant Staphylococcus aureus strain ATCC BAA-44.

  • The synergy was found for antibiotics working through dissimilar mechanisms: fluoroquinolone norfloxacin, a bacterial DNA gyrase inhibitor, beta-lactam oxacillin, a cell-wall biosynthesis inhibitor and glycopeptide vancomycin, also a cell-wall inhibitor, albeit utilizing a different intracellular target than oxacillin.

  • When an IM compound and an antibiotic were combined at doses that show little antibacterial action against methicillin-resistant S. aureus on their own, normally a several-log reduction in bacterial count is observed for each of the combinations.

  • The synergy was also found for IM4, IM5, IM7, IM17 and IM171 with vancomycin against vancomycin-resistant Enterococci ATCC BAA-2573 strain at selected doses.

  • When an IM compound and vancomycin were combined at doses that show little antibacterial action against vancomycin-resistant Enterococci on their own, normally a several-log reduction in bacterial count is observed for each of the combinations.

  • We found that the IM compounds in combination with antibiotics show no toxicity to mammalian cells at doses that manifest synergistic antibacterial activities.

  • We demonstrated that the four out of five described indoles do not promote the evolution of resistance in S. aureus.

  • Our mechanistic studies identify membrane permeabilization with IM compounds as their likely mode of action.

Acknowledgments

S Rogelj and LV Frolova acknowledge their NMT Presidential Research Support.

Footnotes

Author contributions

DN Turner, L Edwards and LV Frolova performed the experiments. A Kornienko designed the study and wrote the manuscript. S Rogelj designed the study and oversaw the laboratory work.

Financial & competing interests disclosure

This project was supported by a grant from the National Institute of General Medical Sciences (P20GM103451). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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