5-Methylindole kills various bacterial pathogens and potentiates aminoglycoside against methicillin-resistant Staphylococcus aureus

Strains, medium and reagents

Various Gram-positive (S. aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus pyogenes, Micrococcus luteus, Streptococcus iniae) and Gram-negative (E. coli, P. aeruginosa and Shigella flexneri) bacterial strains, as well as M. tuberculosis strain (H37Ra) were used in this study and their characteristics are described in Table S1. Briefly, each bacterial strain was cultured over-night and then diluted at 1:500 in Luria-Bertani (LB) medium (Note: the MRS medium was used for E. faecalis) to further agitated in a shaker (37 °C, 220 rpm) for 18–24 h to prepare stationary-phase cells. All chemical reagents are of analytical purity. Stock solutions of indole, 2-methylindole, 5-methylindole and 5-iodoindole were prepared with DMSO and placed in brown, opaque Eppendorf tubes to avoid photo-induced damage.

Cell survival assay

Briefly, bacterial cells in stationary-phase were mixed with indole, 2-methylindole, 5-methylindole at varying concentrations and agitated for 3 h. A total of 100 μL treated cell cultures were centrifuged (13,000 rpm, 1 min) in Eppendorf tubes and washed with PBS (0.27 g/L KH₂PO₄, 1.42 g/L Na₂HPO₄, 8 g/L NaCl, 0.2 g/L KCl, pH 7.4). Afterwards, 5 μL of tenfold serially diluted cell suspension were spot plated onto LB agar dishes for cell survival assay. In addition, stationary-phase and exponential-phase S. aureus cell cultures were mixed with aminoglycoside antibiotics in the presence of indole, 2-methylindole or 5-methylindole for varying length of time, and cells were washed with PBS before cell survival assay as described above. Independent experiments were repeated at least three times.

Minimum inhibitory concentration (MIC) assay

The MICs of 5-methylindole for S. aureus ATCC25923, E. faecalis ATCC 29212, S. iniae, E. coli BW25113, P. aeruginosa PAO1, S. flexneri 24T7T, K. pneumoniae KP-D367 were measured by incubating freshly inoculated cultures in LB for 24 h containing varying concentrations of indoles. Results were determined by observing visual turbidity and experiments were performed using at least two independent cultures.

Preparation and eradication of antibiotic-tolerant S. aureus persister cells

Nutrient shift-induced persisters were prepared as previously reported (Chen et al., 2019). In brief, over-night cultures of S. aureus were diluted at 1:100 into LB medium (37 °C, 220 rpm) and cultured to mid-exponential phase at a cell density of OD₆₀₀ = 0.5–0.6. Cells were centrifuged and washed with M9 medium twice before transferred to M9 medium plus 2 g/L fumarate and agitated for four hours before indole treatment. Starvation-induced persisters were prepared according to an earlier report (Eng et al., 1991). Briefly, S. aureus cells were diluted at 1:500 in Mueller-Hinton broth medium and cultured for 24 h (37 °C, 220 rpm) to a cell density of around 10⁹ CFU/mL. Cells were centrifuged, re-suspended in yeast nitrogen broth medium without amino acids, diluted to a cell density of around 10⁸ CFU/mL and agitated for 5 h before indole treatment.

Hemolysis effect assay

Mouse blood cells were obtained from mice discarded from other animal experiments and sodium citrate at a final concentration of 2.5% was used as anticoagulant. Blood cells were collected by centrifugation (1,000 rpm, 15 min) and washed twice with 0.9% NaCl containing 2.5% sodium citrate. The cell pellets were re-suspended in 0.9% NaCl solution containing 2.5% sodium citrate plus indole, 2-methylindole or 5-methylindole at increasing concentrations, incubated at 37 °C for 3 h and centrifuged, with the supernatant being subjected to absorbance measurement at 545 nm on a 96-well plate reader.

Ethics statement

Animal experiments were performed in accordance with the National Standards of China (GB/T 35892-2018 and GB/T 35823-2018) and after the approval issued by the Animal Ethical and Welfare Committee of Fujian Normal University (approval no. IACUC 20190006).

5-Methylindole kills various Gram-positive bacteria in stationary-phase

We evaluated the potential bactericidal activity of indole and its two derivatives, i.e., 5-methylindole and 2-methylindole, against typical Gram-positive bacteria, including S. aureus and Staphylococcus epidermidis. We prepared these bacteria in stationary-phase that are generally tolerant to bactericidal antibiotics (Keren et al., 2004) and widely used for antibiotic research (Allison, Brynildsen & Collins, 2011; Peng et al., 2015), and then subjected them to the treatment with indole derivatives at varying concentrations for 3 h. Cell survival assay revealed that (i) while indole hardly exhibited bactericidal activity, 5-methylindole was able to effectively eradicate these pathogens in a concentration-dependent manner (Figs. 1A–1E); (ii) 2-methylindole eradicated S. aureus, S. epidermidis and E. faecalis as effectively as did 5-methylindole but had no killing effect on S. pyogenes and M. luteus. We also examined the aquatic pathogen Streptococcus iniae and found that 5-methylindole could kill S. iniae in a concentration-dependent manner, with 2-methylindole and indole being less effective (Fig. 1F). Furthermore, we determined MICs of 5-methylindole to S. aureus, E. faecalis and S. iniae, which were 4, 16 and 16 mM, respectively (Table 1).

5-Methylindole kills various Gram-negative bacteria in stationary-phase

We then examined the potential bactericidal activity of 5-methylindole against typical Gram-negative bacteria in stationary-phase, including E. coli, P. aeruginosa and Shigella flexneri. The cell survival assay showed that 5-methylindole was able to effectively kill all these pathogens in a concentration-dependent manner (Figs. 2A–2C). Similarly, 2-methylindole and indole were also able to kill these pathogens in a concentration-dependent manner, albeit at lower efficacy than 5-methylindole. Notably, Gram-negative bacteria overall seemed more sensitive to 5-methylindole than Gram-positive bacteria (Fig. 2 vs Fig. 1). In addition, we determined MICs of 5-methylindole to E. coli, P. aeruginosa and Shigella flexneri, which were 8, 16 and 2 mM, respectively (Table 1).

5-Methylindole kills antibiotic-resistant/tolerant bacteria and Mycobacterium tuberculosis

Considerable bactericidal activity of 5-methylindole against both Gram-positive and Gram-negative bacterial pathogens, as described in Figs. 1 and 2, prompted us to further examine whether it could eradicate those antibiotic-resistant/tolerant pathogens that give rise to the failure of antibiotic therapy. For this purpose, we first examined methicillin-resistant Staphylococcus aureus (MRSA) that has been designated on the WHO priority pathogens list (WHO, 2017), as well as multidrug-resistant pathogen Klebsiella pneumoniae that has been reported as one of the most critical antibiotic-resistant pathogens in China (China Antimicrobial Resistance Surveillance System, 2020). The cell survival assay showed that 5-methylindole was able to eradicate these two antibiotic-resistant pathogens in a concentration-dependent manner (Figs. 3A and 3B) while 2-methylindole was less effective. Notably, we found that 5-methylindole and 2-methylindole were also able to kill Mycobacterium tuberculosis H37Ra (an avirulent strain) in a concentration-dependent manner (Fig. 3C). In addition, MIC of 5-methylindole to K. pneumoniae was determined to be 4 mM (Table 1).

We next examined the effect of 5-methylindole on bacterial persisters, which are transiently tolerant to the attack of bactericidal antibiotics and play an important role in chronic infections and the evolution of antibiotic resistance (Lewis, 2010; Levin-Reisman et al., 2017; Liu et al., 2020). To this end, nutrient shift-induced S. aureus persisters were prepared by switching the carbon source of S. aureus cells in exponential-phase to fumarate (Radzikowski et al., 2016), and starvation-induced S. aureus persisters were made by transferring S. aureus cells in stationary-phase to medium without any nutrients (Chen et al., 2019). These nutrient shift-induced and starvation-induced S. aureus persister cells were kept in non-replicating status and highly tolerant to the attack by tobramycin under conventional treatment condition (Fig. S1; or refer to our earlier reports (Sun et al., 2020; Chen et al., 2019)). However, both of them were killed by 5-methylindole and 2-methylindole in a concentration-dependent manner, with indole being much less effective (Figs. 3D and 3E).

5-Methylindole potentiates aminoglycosides killing of conventional S. aureus and MRSA cells in stationary-phase but not the cells in exponential-phase

We previously reported that 5-methylindole could potentiate aminoglycoside antibiotics against conventional S. aureus and MRSA cells under hypoionic conditions (i.e., in ion-free solutions) and that such potentiation could be achieved within a couple of minutes (Sun et al., 2020). Here we examined whether 5-methylindole could potentiate aminoglycosides under conventional treatment conditions (i.e., agitation in LB medium for a couple of hours). The cell survival assay showed that 5-methylindole could significantly enhance the bactericidal actions of tobramycin against conventional S. aureus cells in stationary-phase, in both time-dependent (Fig. 4A) and concentration-dependent manners (Fig. 4B). In addition, tobramycin alone at 100 μg/mL only exhibited marginal bactericidal activity, but eradicated S. aureus cells by more than four orders of magnitude when combined with 5-methylindole (Fig. 4C). More importantly, we found that both 5-methylindole and 2-methylindole significantly increased the killing efficacy of streptomycin against MRSA cells, with streptomycin alone hardly killing the bacteria (Fig. 4D). We also observed that 5-methylindole, 2-methylindole and indole could dramatically potentiate gentamicin, streptomycin and kanamycin against S. aureus cells in stationary-phase (Figs. S2B–S2D). However, they had little effects on fluoroquinolone antibiotics ofloxacin and ciprofloxacin (Figs. S2E and S2F), in contrast to earlier reports showing that ofloxacin was potentiated by ascorbic acid against Staphylococcus aureus (Dey & Bishayi, 2018) and certain oils could reduce the MIC of ofloxacin against E. coli and S. aureus (Pereira et al., 2020). In addition, we found that none of 5-methylindole, 2-methylindole and indole could potentiate tobramycin and other types of aminoglycosides against S. aureus cells in exponential-phase (Figs. S3). Together, these results suggest that 5-methylindole could potentiate aminoglycoside antibiotics against S. aureus cells in stationary-phase.

5-Methylindole at sub-toxic concentrations potentiates aminoglycosides killing of S. aureus in stationary-phase

Because 5-methylindole alone could kill bacteria, we thus evaluated its cytotoxicity towards mammalian cells by examining its hemolysis effect. Cell lysis assay revealed that 5-methylindole could disrupt mouse blood cells in a concentration-dependent manner (Fig. 5A, Fig. S4), with the hemolysis effect of 1 mM 5-methylindole being minimal. In comparison, the hemolysis effects of 2-methylindole and indole were less potent than that of 5-methylindole. Based on this, we then evaluated the potentiation effects of 5-methylindole, 2-methylindole and indole at sub-toxic concentrations (1, 2 and 4 mM, respectively) on tobramycin. The cell survival assay revealed that all of them could effectively enhance the killing efficacy of tobramycin against S. aureus cells in stationary-phase after agitation for 3 h (Fig. 5B) or 5 h (Fig. 5C).