Efflux pump inhibitory potential of indole derivatives as an arsenal against norA over-expressing Staphylococcus aureus

Abstract

NorA, an extensively studied efflux pump in Staphylococcus aureus, has been connected to fluoroquinolone, antiseptic, and disinfection resistance. Several studies have also emphasized how efflux pumps, including NorA, function as the first line of defense of S. aureus against antibiotics. In this study, we have screened some chemically synthesized indole derivatives for their activity as efflux pump inhibitors (EPIs). The derivative SMJ-5 was found to be a potent NorA efflux pump inhibitor among the screened indole derivatives, owing to increased ethidium bromide and norfloxacin accumulation in norA over-expressing S. aureus. The combination of SMJ-5 and ciprofloxacin demonstrated the eradication of S. aureus biofilm and prolonged the post-antibiotic effect more than ciprofloxacin alone. SMJ-5 was able to inhibit staphyloxanthin virulence. In in vitro time-kill trials and in vivo efficacy investigations, the combination enhanced the bactericidal activity of ciprofloxacin against S. aureus. Additionally, reverse transcription PCR results revealed that SMJ-5 also inhibits the NorA efflux pump indirectly at the transcriptional level. IMPORTANCE The NorA efflux pump is the most effective resistance mechanism in S. aureus. The clinical importance of NorA efflux pumps is demonstrated by the expression of pump genes in S. aureus strains in response to fluoroquinolones and biocides. Along with the repercussions of decreased fluoroquinolone sensitivity, increasing expression of efflux pump genes by their substrate necessitates the importance of efflux pump inhibitors. Reserpine and verapamil are clinically used to treat ailments and have proven NorA inhibitors, but, unfortunately, the concentration needed for these drugs to inhibit the pump is not safe in clinical settings. In the current study, we have screened some indole derivatives, and among them, SMJ-5 was reported to potentiate norfloxacin and ciprofloxacin at their sub-inhibitory concentration by inhibiting the norA gene transcriptionally. Here we highlight the promising points of this study, which could serve as a model to design a therapeutic EPI candidate against norA over-expressing S. aureus.

Keywords: Staphylococcus aureus; efflux pump inhibitors (EPIs); major facilitator superfamily (MFS); multidrug resistance (MDR); norA efflux pump.

FIG 1

(A) EtBr accumulation assay, RFF value of indole derivatives at 1/4×, 1/8×, and 1/16× MIC in the presence of EtBr on S. aureus SA-1199B (norA over-expression) and K-1758 (norA knockout) strain. (B) Norfloxacin accumulation assay on S. aureus SA-1199B strain. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. P value calculated using 95% class interval. Both experiments were done in biological duplicates, and this is the representation of one, comprising three and six technical replicates, respectively; panel A corresponds to average ± SD, and panel B is the representation of individual readings ± SD. ns, non-significant; SD, standard deviation.

FIG 2

EtBr efflux inhibition assay of indole derivatives at the sub-inhibitory concentration (1/4× MIC) in EtBr presence at 4 µg/mL concentration in the presence and absence of glucose. Reserpine was used as the positive control. The results correspond to the mean of two biological repeats performed in triplicates.

FIG 3

(A) Membrane depolarization was detected using fluorescence of DiSC₃(5) showing high fluorescence of positive control valinomycin; drug-free control was used as the negative control. (B) Membrane permeability was measured using nucleic acid binding dye propidium iodide; paenibacillin was used as the positive control, and drug-free control was used as the negative control. (C) ATP luciferin-luciferase Bioluminescence detection assay; CCCP, the positive control, quenches ATP compared to control. (D) Mammalian Ca²⁺ channel assay on human embryonic kidney cells. All the experiments represent biological duplicates performed in technical triplicates, and results correspond to average ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. P value calculated using 95% class interval. CCCP, carbonyl cyanide m-chlorophenyl hydrazone.

FIG 4

(A) Hemolysis assay on 4% red blood cell (RBC) at 64-, 128-, 250-, and 500-µg/mL concentrations. (B) Cytotoxicity assay on human peripheral blood mononuclear cells and human embryonic kidney cells (HEK 293T) at 500- and 250-µg/mL concentrations. (C) Spectrometric quantification of staphyloxanthin intermediates, namely, 4,4-diapophytoene (286 nm), 4,4-diaponeurosporene (435 nm), 4,4-diaponeurosporenic acid (455 nm), and staphyloxanthin (462 nm) extracted from control and indole derivatives (1/4 × MIC) treated ATCC 29,213 cells. Results were considered significant (*) when P < 0.05 and highly significant (**) when P < 0.01 and (***) P < 0.001. P values were calculated using 95% class interval. Panels A through C represent two biological readings performed in triplicates. (D) Schematic representation of the staphyloxanthin biosynthesis pathway. The figure demonstrates SMJ-5 inhibits dehydrosqualene synthase (CrtM) while miconazole inhibits 4,4-diapophytoene desaturase (CrtN). CrtM is an enzyme that catalyzes the condensation of two farnesyl diphosphates to produce 4,4-diapophytoene, further converted to 4,4-diaponeurosporene by CrtN.

FIG 5

Confocal microscopic images of biofilm disruption in the presence of ciprofloxacin and SMJ-5 alone and in combination after 24 h of treatment on poly-L-lysine-coated glass coverslips. Confocal microscopy of biofilm in the sequence control, ciprofloxacin (4 µg/mL), SMJ-5 (32 µg/mL), ciprofloxacin (16 µg/mL), and ciprofloxacin (1 µg/mL) + SMJ-5 (32 µg/mL). Scale 50 µm. The image is the best representation of the biological triplicates.

FIG 6

Post-antibiotic effect of ciprofloxacin at 1×, 1/2×, and 1/4× MIC alone and in combination with SMJ-3, SMJ-5, SMJ-9, and SMJ-10 at 1×, 1/2×, and 1/4× MIC by turbiditory method on S. aureus SA-1199B strain in H; the results are the representation of two biological repeats performed in triplicates ± SD.

FIG 7

(A) Time-kill kinetic assay, the combination of ciprofloxacin (2 µg/mL) and SMJ-5 (32 µg/mL), demonstrated a bactericidal effect as compared to ciprofloxacin alone against S. aureus SA-1199B strain. (B) Persister-kill kinetics, ciprofloxacin at 20× MIC, forms persisters hereditarily for 3 days on S. aureus ATCC 29213; on the fourth day, combination of ciprofloxacin at 20× MIC and SMJ-5 at 1/4× MIC lowers the persister count by 2 log₁₀ CFU/mL. (C) RT-PCR of S.aureus SA-1199B norA in untreated, ciprofloxacin (1/4× MIC), SMJ-5 (1/4× MIC), and ciprofloxacin (1/32 MIC) + SMJ-5 (1/4× MIC), ciprofloxacin (1/32× MIC) + SMJ-5 (1/8× MIC) and ciprofloxacin (1/32× MIC) + SMJ-5 (1/16× MIC). (D) Macrophage invasion assay on S. aureus SA-1199B (norA over-expressing), SA-1199 (norA wild type), and K-1758 (norA knockout) strain in the presence of SMJ-5 at sub-inhibitory concentration; the results in B correspond to the mean of triplicate readings ± SD. Experiments A and D are representations of three biological repeats, and experiments B and C are the representations of two biological repeats. Results were considered significant (*) when P < 0.05 and highly significant (**) when P < 0.01 . P value calculated using 95% class interval.

FIG 8

(A) Histopathological analysis of six major organs of BALB/c mice after sub-cutaneous administration of 1,000- and 500-mg/kg SMJ-5 and control, respectively (n = 3); and ciprofloxacin-SMJ-5 combination is efficacious in two mouse models of infection. (B) Neutropenic mouse thigh infection model: single sub-cutaneous dose treatment (10-mg/kg ciprofloxacin and 10-mg/kg SMJ-5 alone and in combination, six mice per group, 4 h after infection). CFU was determined 24 h post-infection for drug-treated mice; for controls, CFU was calculated 4 and 24 h post-infection. (C) Mouse peritonitis survival model: three sub-cutaneous dose treatments (10-mg/kg ciprofloxacin and 10-mg/kg SMJ-5 alone and in combination, 10 mice per group; after 1, 4, and 6 h post-infection). *P < 0.05, **P < 0.01, ****P < 0.0001. P values were calculated using 95% class interval.

FIG 9

Protocol for the synthesis of 2-(2′-aminophenyl) indole [RP2] derivatives.