Tobramycin-Linked Efflux Pump Inhibitor Conjugates Synergize Fluoroquinolones, Rifampicin and Fosfomycin against Multidrug-Resistant Pseudomonas aeruginosa

In this study, we examined the in vitro effect of tobramycin-efflux pump inhibitor (TOB-EPI) conjugates in combinations with fluoroquinolones, rifampicin and fosfomycin on the growth of multi-drug resistant (MDR) and extremely-drug resistant (XDR) Pseudomonas aeruginosa. The TOB-EPI conjugates include tobramycin covalently linked to 1-(1-naphthylmethyl)-piperazine (NMP) (1), paroxetine (PAR) (2) and a dibasic peptide analogue of MC-04,124 (DBP) (3). Potent synergism was found for combinations of TOB-NMP (1), TOB-PAR (2) or TOB-DBP (3) with either fluoroquinolones (moxifloxacin, ciprofloxacin), rifampicin or fosfomycin against a panel of multidrug-resistant/extensively drug-resistant (MDR/XDR) P. aeruginosa clinical isolates. In the presence of ≤8 mg/L (6.1–7.2 µM) (≤¼ × MICadjuvant) concentration of the three conjugates, the MIC80 of moxifloxacin, ciprofloxacin, rifampicin and fosfomycin were dramatically reduced. Furthermore, the MIC80 of rifampicin (0.25–0.5 mg/L) and fosfomycin (8–16 mg/L) were reduced below their interpretative susceptibility breakpoints. Our data confirm the ability of TOB-NMP (1), TOB-PAR (2) and TOB-DBP (3) conjugates to strongly synergize with moxifloxacin, ciprofloxacin, rifampicin and fosfomycin against MDR/XDR P. aeruginosa. These synergistic combinations warrant further studies as there is an urgent need to develop new strategies to treat drug-resistant P. aeruginosa infections.


Introduction
The opportunistic Pseudomonas aeruginosa is the leading cause of nosocomial and chronic lung infections in immunocompromised (e.g., cystic fibrosis) patients [1,2]. The World Health Organization (WHO) has listed carbapenem-resistant P. aeruginosa as one of the most critical (priority 1) pathogens that pose a serious threat to human health [3]. Among Gram-negative pathogens, infections caused by P. aeruginosa are particularly difficult to treat as the organism is both intrinsically resistant and capable of acquiring resistance (through mobile genetic elements) to most antibiotics [4]. The intrinsic resistance of P. aeruginosa is mostly due to its low outer membrane permeability, which is 12-100 times lower than that of Escherichia coli, presumably as a result of their relatively selective porins [4]. Overexpressed multidrug efflux pumps that limit the intracellular concentration of antibiotics is another key contributor of intrinsic resistance. Several small molecules such as porins [3]. Overexpressed multidrug efflux pumps that limit the intracellular concentration of antibiotics is another key contributor of intrinsic resistance. Several small molecules such as 1-(1-naphthylmethyl)-piperazine (NMP) [4], paroxetine (PAR) [5,6] and DBP [7], the analogue of dibasic dipeptide D-Ala-D-hPhe-aminoquinoline (MC-04,124) (Figure 1), have been reported to inhibit efflux pumps in Gram-negative and/or Gram-positive bacteria, thereby restoring activity to legacy antibiotics. In a previous study, we discovered that linking a tobramycin (TOB) vector to the efflux pump inhibitors (EPIs) NMP, PAR, and DBP generated TOB-EPI conjugates ( Figure 1) capable of sensitizing multidrug-resistant/extensively drug-resistant (MDR/XDR) Gram-negative bacilli, especially P. aeruginosa, to tetracycline antibiotics [8]. Mechanistic studies revealed tobramycin with a twelve carbon aliphatic chain (C12) to be a core fragment needed for outer membrane perturbation that leads to a 'self-promoted' uptake mechanism [8][9][10]. We also found that TOB-EPI conjugates are able to depolarize the inner membrane of P. aeruginosa, disrupting the electrical component (ΔΨ) of bacterial proton motive force (PMF) that results in a compromised transmembrane chemical component (ΔpH) [8]. An increase in ΔpH would consequently facilitate the increased uptake of tetracyclines as the process of accumulation of tetracyclines is ΔpH-dependent [11]. Moreover, a compromised PMF affects PMF-dependent efflux systems that effectively negate the active efflux of susceptible antibiotics [8,9]. Herein, we describe the synergistic interactions of TOB-NMP (1), TOB-PAR (2) and TOB-DBP (3) with either fluoroquinolones (moxifloxacin and ciprofloxacin), rifampicin or fosfomycin against MDR/XDR P. aeruginosa clinical isolates. In a previous study, we discovered that linking a tobramycin (TOB) vector to the efflux pump inhibitors (EPIs) NMP, PAR, and DBP generated TOB-EPI conjugates ( Figure 1) capable of sensitizing multidrug-resistant/extensively drug-resistant (MDR/XDR) Gram-negative bacilli, especially P. aeruginosa, to tetracycline antibiotics [9]. Mechanistic studies revealed tobramycin with a twelve carbon aliphatic chain (C 12 ) to be a core fragment needed for outer membrane perturbation that leads to a 'self-promoted' uptake mechanism [9][10][11]. We also found that TOB-EPI conjugates are able to depolarize the inner membrane of P. aeruginosa, disrupting the electrical component (∆Ψ) of bacterial proton motive force (PMF) that results in a compromised transmembrane chemical component (∆pH) [9]. An increase in ∆pH would consequently facilitate the increased uptake of tetracyclines as the process of accumulation of tetracyclines is ∆pH-dependent [12]. Moreover, a compromised PMF affects PMF-dependent efflux systems that effectively negate the active efflux of susceptible antibiotics [9,10]. Herein, we describe the synergistic interactions of TOB-NMP (1), TOB-PAR (2) and TOB-DBP (3) with either fluoroquinolones (moxifloxacin and ciprofloxacin), rifampicin or fosfomycin against MDR/XDR P. aeruginosa clinical isolates.

Bacterial Strains
Clinically-relevant bacterial strains were collected from the Canadian National Intensive Care Unit (CAN-ICU) study [13] and Canadian Ward Surveillance (CANWARD) studies [14,15]. All isolates were transported to the reference laboratory (Health Sciences Centre, Winnipeg, MB, Canada) on Amies charcoal swabs, subcultured onto LB broth, and stocked in skim milk with 10% glycerol at −80 • C until antimicrobial susceptibility testing was carried out. The efflux pump deficient strains, P. aeruginosa PAO200 and P. aeruginosa PAO750, were provided by Dr. Ayush Kumar from University of Manitoba, Canada. All pathogens obtained from CAN-ICU and CANWARD studies have received ethics approval from the University of Manitoba Ethics Committee. In addition, participating Canadian health centers have obtained appropriate ethics approval to submit clinical specimens.

Antimicrobial Agents
Tobramycin sulfate, moxifloxacin hydrochloride, rifampicin, and ciprofloxacin hydrochloride were obtained from AK Scientific, Inc. (Union City, CA, USA). Fosfomycin sodium was obtained from Sigma-Aldrich (St. Louis, MO, USA). Glucose-6-phosphate (Sigma-Aldrich) was added to the medium at a final concentration of 25 mg/L for all evaluations of fosfomycin.

Antimicrobial Susceptibility Testing
The antimicrobial activity of the compounds against a panel of bacteria was evaluated by broth microdilution assay in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines [16]. The overnight bacterial culture was diluted in saline to 0.5 McFarland turbidity, and then 1:50 diluted in Mueller−Hinton broth (MHB) for inoculation. The minimum inhibitory concentrations (MICs) of the antimicrobial agents were determined using 96-well plates containing doubling antimicrobial dilutions with MHB and incubated with equal volumes of inoculum for 18 h at 37 • C. The lowest concentration that inhibited visible bacterial growth was taken as the MIC for each antimicrobial agent which was also confirmed using EMax Plus microplate reader (Molecular Devices, San Jose, CA, USA) at a wavelength of 590 nm. We used a stock concentration of 10.24 mg/mL in deionized water or DMSO depending on the solubility of the compounds.

Antimicrobial Combination Screening
The checkerboard method [17] was used to assess synergism in all tested combinations. The fractional inhibitory concentration index (FICI) of each combination was calculated as follows: FICI is the sum of the fractional inhibitory concentration of antibiotic (FIC antibiotic ) and fractional inhibitory concentration of adjuvant (FIC ADJ ); FIC antibiotic = MIC combo /MIC antibiotic alone ; FIC adjuvant = MIC combo /MIC adjuvant alone , where MIC combo is the lowest inhibitory concentration of drug in the presence of the adjuvant; the combination is considered synergistic when the FICI is ≤0.5, no interaction is considered when the FICI is 0.5 < FICI ≤ 4.0, and the combination is considered antagonistic when the FICI is >4.0 [18].

Results
We recently reported the preparation and biological evaluation of three TOB-EPI conjugates (Figure 1), namely TOB-NMP (1), TOB-PAR (2) and TOB-DBP (3) [9]. We found that the three conjugates were mostly inactive (MIC = 2->1024 mg/L) alone but significantly potentiated minocycline, in combination, against MDR/XDR P. aeruginosa clinical isolates [9]. Preliminary results indicated that the adjuvant properties of 1-3 against P. aeruginosa are not limited to tetracycline antibiotics and can also be extended to other antimicrobial classes [9]. Herein, we further expand our understanding on the adjuvant properties of the three TOB-EPI conjugates to other antibacterial classes including rifampicin, fluoroquinolones (ciprofloxacin and moxifloxacin) and fosfomycin.
We further assessed the potency of TOB-EPI conjugates as adjuvants by comparing the absolute MICs of the four antibiotics, in the presence of ≤8 mg/L (6.1-7.2 µM) (≤ 1 4 × MIC adjuvant ) conjugates, to established susceptibility breakpoints. According to the Clinical and Laboratory Standards Institute (CLSI) [21], the susceptibility breakpoint of ciprofloxacin for Pseudomonas aeruginosa is ≤1 mg/L. However, no established susceptibility breakpoint of moxifloxacin, rifampicin and fosfomycin exists for Pseudomonas spp., and therefore we used other breakpoints in other organisms for comparison. We interpreted susceptibility to moxifloxacin for Pseudomonas aeruginosa to be similar to the established one for ciprofloxacin, as both belong to the fluoroquinolone class of antibiotics. CLSI denotes susceptibility to rifampicin for Enterococcus spp. as ≤1 mg/L [21]. Conversely, susceptibility to fosfomycin was described to be ≤64 mg/L for Enterobacteriaceae [21].
Next, we studied whether the absolute MIC of the four antibiotics in the presence of the three TOB-EPI conjugates at ≤8 mg/L (6.1-7.2 µM) (≤ 1 4 × MIC adjuvant ) reaches the expected susceptibility breakpoint of ciprofloxacin and moxifloxacin. Our results ( Table 2) show that in 6/8 cases, the adjuvants cannot reach the expected susceptibility breakpoint of the two fluoroquinolone antibiotics. The two P. aeruginosa strains which reach the susceptibility breakpoint (91433 and 101243) do not contain DNA gyrase A mutation, indicating that fluoroquinolone resistance is mostly due to active efflux in these strains [11]. Out of the two fluoroquinolones, moxifloxacin seemed to be strongly potentiated by the conjugates relative to ciprofloxacin (Figure 2). In contrast, the MIC of rifampicin was reduced below the susceptibility breakpoint in all strains tested by conjugates 1 and 2 (Table 2). However, conjugate 3 was able to reduce the MIC of rifampicin below the susceptibility breakpoint for all strains except P. aeruginosa PA260-97103 (absolute MIC = 16 mg/L). All the three conjugates lowered the absolute MIC of fosfomycin in all strains tested except P. aeruginosa 100036. which all but one are ciprofloxacin-resistant. All the three conjugates were found to be synergistic with the four tested antibiotics ( Table 2). Both TOB-NMP (1) and TOB-PAR (2) [20], the susceptibility breakpoint of ciprofloxacin for Pseudomonas aeruginosa is ≤1 mg/L. However, no established susceptibility breakpoint of moxifloxacin, rifampicin and fosfomycin exists for Pseudomonas spp., and therefore we used other breakpoints in other organisms for comparison. We interpreted susceptibility to moxifloxacin for Pseudomonas aeruginosa to be similar to the established one for ciprofloxacin, as both belong to the fluoroquinolone class of antibiotics. CLSI denotes susceptibility to rifampicin for Enterococcus spp. as ≤1 mg/L [20]. Conversely, susceptibility to fosfomycin was described to be ≤64 mg/L for Enterobacteriaceae [20].
Next, we studied whether the absolute MIC of the four antibiotics in the presence of the three TOB-EPI conjugates at ≤8 mg/L (6.1-7.2 µM) (≤¼ × MICadjuvant) reaches the expected susceptibility breakpoint of ciprofloxacin and moxifloxacin. Our results ( Table 2) show that in 6/8 cases, the adjuvants cannot reach the expected susceptibility breakpoint of the two fluoroquinolone antibiotics. The two P. aeruginosa strains which reach the susceptibility breakpoint (91433 and 101243) do not contain DNA gyrase A mutation, indicating that fluoroquinolone resistance is mostly due to active efflux in these strains [10]. Out of the two fluoroquinolones, moxifloxacin seemed to be strongly potentiated by the conjugates relative to ciprofloxacin (Figure 2). In contrast, the MIC of rifampicin was reduced below the susceptibility breakpoint in all strains tested by conjugates 1 and 2 (Table 2). However, conjugate 3 was able to reduce the MIC of rifampicin below the susceptibility breakpoint for all strains except P. aeruginosa PA260-97103 (absolute MIC = 16 mg/L). All the three conjugates lowered the absolute MIC of fosfomycin in all strains tested except P. aeruginosa 100036.   The MIC 80 of moxifloxacin, ciprofloxacin, rifampicin and fosfomycin in combination with ≤8 mg/L (6.1-7.2 µM) (≤ 1 4 × MIC adjuvant ) TOB-EPIs conjugates (1, 2, or 3) against the tested P. aeruginosa panel were significantly lower than the MIC 80 of the antibiotic alone (Table 3 and Figure 2). More importantly, the absolute MIC 80 of rifampicin and fosfomycin were below their respective susceptibility breakpoints. In the presence of ≤8 mg/L (7.  Considering the possible effect of tobramycin-efflux pump inhibitor conjugates on the active efflux of fluoroquinolones, we assessed the synergy of moxifloxacin and the three conjugates in efflux-deficient P. aeruginosa strains (Table 4). PAO200 is a MexAB−OprM deletion strain while PAO750 is an efflux-sensitive strain that lacks five different clinically relevant RND pumps (MexAB−OprM, MexCD−OprJ, MexEF−OprN, MexJK, and MexXY) and the OM protein OpmH [22]. These efflux pumps confer resistance on P. aeruginosa by expelling a wide variety of antibiotic substrates including quinolones, tetracyclines and others. As expected, a significant reduction in MIC of moxifloxacin was observed for PAO200 (MIC = 0.125 mg/L) and PAO750 (MIC = 0.008 mg/L) as active efflux contributes greatly to fluoroquinolone resistance. Interestingly, a 16-fold MIC reduction was observed for TOB-NMP (1) from wild-type P. aeruginosa PAO1 (MIC = 128 mg/L) to PAO750 (MIC = 8 mg/L) while only a 2-to 4-fold difference was observed for the MIC of TOB-PAR (2) and TOB-DBP (3) against PAO1, PAO200 and PAO750. The combination of conjugate 1 and moxifloxacin remained synergistic across the efflux-deficient strains, albeit weakly synergistic (FICI = 0.31) against P. aeruginosa PAO750. Both conjugates 2 (FICI = 0.19) and 3 (FICI = 0.25) were found to be synergistic with moxifloxacin against the MexAB-OprM-deficient PAO200 strain. However, no interaction was found between moxifloxacin and conjugates 2 (FICI = 0.63) or 3 (FICI = 0.63) against PAO750.

Discussion
The low permeability of the outer membrane and overexpressed multidrug efflux pumps in Gram-negative bacteria, especially in P. aeruginosa, limits effective antibiotics for treatment [23]. The compounding effect of the restrictive lipid bilayer and active efflux prevents the intracellular accumulation of antibiotics to concentrations needed to achieve biological effect. The problem is further exacerbated in drug-resistant organisms as they express genetically encoded resistance mechanism that may actively incapacitate antibiotics. Unfortunately, no new antibiotics with a novel mode of action for Gram-negative bacteria have been introduced in the clinic for more than five decades. There is a definite need to develop new strategies which are able to overcome resistance in Gram-negative pathogens, for which the combination therapy of existing antibiotics with adjuvants is a promising option [24].
We recently described the preparation of TOB-EPI conjugates (1, 2 or 3) that synergize tetracycline antibiotics [9]. Moreover, we also demonstrated their ability to permeabilize the outer membrane of P. aeruginosa in a dose-dependent manner [9]. Herein, TOB-EPI conjugates (1, 2 or 3) were found to significantly potentiate the outer membrane impermeable rifampicin (32-128 fold) against a panel of MDR/XDR P. aeruginosa clinical isolates. At ≤8 mg/L (6.1-7.2 µM) (≤ 1 4 × MIC adjuvant ) concentration of either the three conjugates, the absolute MIC 80 of rifampicin was significantly reduced below susceptibility breakpoint. This suggest that conjugates 1, 2 and 3 are good candidates for future adjuvant therapy development in combination with rifampicin. As rifampicin is a poor substrate for P. aeruginosa RND efflux pumps [9,10], membrane permeabilization may be responsible for the observed synergism with TOB-EPI conjugates. The P. aeruginosa inactive efflux pump inhibitors NMP and PAR were found to exhibit no interactions with rifampicin. In contrast, the P. aeruginosa active DBP was found to be synergistic with rifampicin against wild-type P. aeruginosa PAO1. A previous report of DBP analog PAβN revealed its ability to permeabilize bacterial membranes in a concentration-dependent manner [25], therefore this may have contributed to the observed rifampicin potentiation.
Out of the two fluoroquinolones tested, combinations of the three TOB-EPI conjugates with moxifloxacin yielded stronger potentiation relative to ciprofloxacin ( Figure 2). However, the conjugates were not able to bring down the absolute MIC 80 of both fluoroquinolones below their susceptibility breakpoint. It should be noted that the MICs of both fluoroquinolones were reduced below the susceptibility breakpoint in only two isolates (91433 and 101243 isolates which lack T 83 to I 83 mutation). This suggests that the conjugates enhance the intracellular concentration of fluoroquinolones. However, this effect cannot compensate acquired resistance caused by genetic mutations of the target enzyme.
The synergy of the conjugates with fluoroquinolones may not only be attributed to adjuvant-induced enhanced membrane permeability but may also be due to a compromised activity of PMF-dependent efflux pumps. We recently demonstrated that the TOB-EPI conjugates strongly reduce motility at sub-MIC concentration and disrupt the electrical component (∆Ψ) of the PMF [9]. This action in turn may affect efflux systems that are dependent to PMF, leading to reduced efflux of fluoroquinolones. Our data revealed that the three conjugates were poor substrates of the MexAB-OprM RND efflux pump (Table 4). However, TOB-NMP (1) may be a substrate of other efflux systems in P. aeruginosa since a 16-fold MIC reduction was observed from wild-type PAO1 to the multiple efflux pump-deficient PAO750. We found that the synergism between moxifloxacin and TOB-EPI conjugates was independent of the MexAB-OprM RND efflux pump. Yet, there was a clear effect on the tested combinations of moxifloxacin and TOB-EPI conjugates against PAO750. The potent synergistic interaction with moxifloxacin found against wild-type PAO1 were drastically reduced to either weakly synergistic (for conjugate 1) or no interaction (for conjugates 2 and 3) against PAO750. Therefore, we assume that either MexCD-OprJ, MexEF-OprN, MexXY, or MexJK efflux pumps is affected by the TOB-EPI conjugates action on PMF. Certainly, moxifloxacin is a good substrate of many efflux pumps in P. aeruginosa.
Fosfomycin is a bactericidal antibiotic that inhibits cell wall biosynthesis [26]. Specifically, fosfomycin inactivates the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) that catalyzes the formation of peptidoglycan precursor UDP N-acetylmuramic acid (UDP-MurNAc) [26,27]. The three TOB-EPI conjugates strongly potentiated the activity of fosfomycin (2-64 fold) against wild-type and MDR/XDR P. aeruginosa clinical isolates susceptible or resistant to fosfomycin. In the presence of a ≤8 mg/L (6.1-7.2 µM) (≤ 1 4 × MIC adjuvant ) concentration of the conjugates, the absolute MIC for 7/8 isolates was ≤16 mg/L, which is 4-fold lower than the expected susceptibility breakpoint of fosfomycin (≤64 mg/L). Fosfomycin is known to be a poor substrate of the multidrug efflux system in P. aeruginosa [28] and it is understood that its cellular entry occurs through porins [29]. We hypothesize that the observed synergy of fosfomycin with TOB-EPI adjuvants reflects the enhanced cellular permeation of fosfomycin via the self-promoted uptake of TOB-EPI adjuvants.

Conclusions
In conclusion, we demonstrate promising synergistic combinations of TOB-EPI conjugates with either fluoroquinolones, rifampicin or fosfomycin against MDR/XDR P. aeruginosa. More importantly, the conjugates TOB-NMP (1), TOB-PAR (2) and TOB-DBP (3) significantly reduced the MIC 80 of rifampicin and fosfomycin below their respective susceptibility breakpoints. These findings show that the adjuvant potency of TOB-EPI conjugates is not limited to tetracyclines [9] but can be expanded to other legacy antibiotics.
Author Contributions: F.S. supervised the studies. X.Y., Y.L., G.G.Z. and F.S. designed, performed, or supervised the biochemical assays. X.Y. wrote the original draft. R.D. and F.S. were involved in the writing-review and editing.