Effect of new carbonyl cyanide aromatic hydrazones on biofilm inhibition against methicillin resistant Staphylococcus aureus

Carbonyl cyanide m-chlorophenylhydrazone (CCCP), as a protonophore, in combination with antibiotics exhibited potentiating antibacterial activity. To improve CCCP's potency and toxicity, a series of aromatic hydrazones were synthesized and their antimicrobial activity was evaluated; amongst them, compounds 2e and 2j with a strong para-electron-withdrawing substituent (–NO2 and –CF3) at the phenyl ring had the lowest MICs against both S. aureus and methicillin resistant Staphylococcus aureus (1.56 and 1.56 μM, respectively). Some compounds in combination with antibiotics exhibited potentiate Gram-positive antibacterial activity; compound 2e was found to display unaided or synergistic efficacy against MRSA. In particular, when compound 2e is combined with ofloxacin, it has a good synergistic effect against MRSA. Moreover, electron microscopy revealed that compound 2e inhibits biofilm formation and effectively eradicates preformed biofilm. MTT assay showed that compound 2e displays as low toxicity as CCCP. Overall, our data showed that the aromatic hydrazone is a promising scaffold for anti-staphylococcal drug development.


Introduction
Antimicrobial resistance (AMR) is an increasingly serious threat to global public health that requires a collaborative global approach across sectors. AMR largely reduces the antibiotic efficacies and increases health care costs, and the situation is getting worse due to the emergence of multidrug-resistant (MDR) bacterial pathogens, such as extended spectrum betalactamase Enterobacteriaceae, methicillin resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). 1,2 Therefore, there is now an urgent need to develop new antibacterial agents with novel targets and new approaches, which could be addressed by developing new antibacterial agents with unique chemical scaffolds. [3][4][5] Mitochondria are well-known for their role as biosynthetic and bioenergetic organelles, which play a critical role in the innate immune response against viral and bacterial infections. 6,7 As a chemical inhibitor of oxidative phosphorylation in the mitochondria, carbonyl cyanide m-chlorophenylhydrazone (CCCP) affects mitochondrial protein synthesis, causes an uncoupling of the proton gradient, acts essentially as an ionophore and reduces the ability of ATP synthase to function optimally. CCCP causes the gradual destruction of living cells and death of the organism by affecting the respiration and respiration-dependent phosphorylation. 8,9 Antibiotic accumulation in Gram-negative bacteria is one of the major causes of AMR. CCCP was widely used to study cellular accumulation in Gram-negative bacteria for many small molecules [10][11][12] due to its ability to collapse the proton motive force. 13 As AMR spreads, a promising approach is to restore the effectiveness of existing drugs via co-administration with adjuvants that inhibit the growth of drug-sensitive pathogens. 4,14,15 CCCP in combination with small molecules showed synergistic effect against most of the MDR pathogenic bacterial strains. [16][17][18][19][20] CCCP in combination with antibiotics could potentiate antibacterial activity.
Since CCCP acts as a protonophore which disperses the membrane proton motive force by modifying the transmembrane electrochemical potential, it simultaneously causes toxicity to the cell of the host. 21,22 Moreover, the concentration of synergistic antibacterial effect of CCCP is so high (at least 50 mM) that the effective dose may disrupt mitochondrial function to lead to toxicity. [23][24][25] Therefore, in this work, a series of aromatic hydrazones were synthesized and evaluated for their antibacterial activity to try and improve antibacterial potency and reduce toxicity. The preliminary screening showed that aromatic hydrazones exhibited potential Gram-positive antibacterial activities. New compounds alone or in combination with antibiotics exhibited potentiate Gram-positive antibacterial activities.
Therefore, the aromatic residue is a promising scaffold for further antibacterial modications. Further, a plausible antibacterial mechanism was proposed and investigated via scanning electron microscopy (SEM) and transmission electron microscopy (TEM) (Fig. 1).

Chemistry
The synthetic route to compounds 2a-2q is illustrated in Scheme 1. The nitrosation of the aromatic amines (1) with nitrous acid (in situ from sodium nitrite and concentrated hydrochloric acid) led to aromatic diazonium salts, which can be used to next reaction without purication. The diazonium salt as the key intermediate underwent a condensation reaction with methylene of malononitrile to yield the title compound 2.

Antibacterial activity of compounds 2a-2q
In order to determine the antimicrobial potential of aromatic hydrazones, they were evaluated in either Mueller-Hinton (MH) broth or Sabouraud Dextrose Agar (SDA) using a micro-broth dilution method against a panel of bacteria and fungi, including two Gram-positive bacteria: Staphylococcus aureus ATCC 25923 (SA) and Methicillin Resistant Staphylococcus  (Table 1). Amongst them, compounds 2e and 2j showed better activity than CCCP (2a) against both S. aureus and MRSA (MICs ¼ 1.56 mM), which are even better than cefoxitin and linezolid, and similar with the MICs of ooxacin. The growth inhibition effects of compounds 2a, 2e and 2j were further investigated against both S. aureus and MRSA. The results conrmed that both compounds 2e and 2j were able to inhibit the growth of S. aureus and MRSA effectively at the MIC or higher concentrations. Once the concentration drops down to half or less of the MICs, they could only slow down the growing rate of S. aureus and MRSA during logarithmic period, while the growth could be recovered aer being incubated for longer time (Fig. 2).

Cytotoxicity assays
The human hepatic L02 cells were treated with different concentrations of tested compound (3.125, 6.25, 12.5, 25, 50  and 100 mM), and cell viability was measured aer 24 h using MTT method. As shown in Fig. 3, compounds 2a, 2e and 2j at the test concentrations (3.125-50 mM) had no obvious cytotoxicity against L02 cells, and the relative cell viabilities of treated cells were all more than 70%.

Checkerboard assay
To develop a feasible medical application, active compounds 2a, 2e and 2j were tested in combination with clinical antibiotics on SA and MRSA by checkerboard assay in order to evaluate their ability to improve the anti-bacterial activity. 20 Each checkerboard test generates many different combinations and, by convention, the FIC value of the most effective combination was used in calculating the fractional inhibitory concentration index (FICI). FICI was calculated by adding both FICs: Fig. 3 Cell viability assay of tested compounds to L02 cells. Data are presented as the mean AE standard error (n ¼ 3), one-way ANOVA (vs. control), *P < 0.05, **P < 0.01.   are concentrations of compounds A and B at the isoeffective combinations. The FICI was interpreted as synergistic when it was #0.5, additional effects when 0.5 < FICI # 1.0, indifferent when 1.0 < FICI # 2.0, and antagonistic when FICI > 2.0, and any value between was interpreted as indifferent.
As shown in Table 2, when compound 2a was used in combination with antibiotics, MIC was reduced 2-fold, but for compound 2e, MIC was reduced 4-fold. Moreover, compounds 2a, 2e and 2j could signicantly improve the performance of clinical antibiotics, for example, ooxacin, cefoxitin and linezolid lowered their MIC values from 1.25, 50.0 and 7.5 mM to 0.04, 1.56 and 0.47 mM, respectively. Calculations of FIC and FICI (always less than 1.0) obtained by checkerboard assays on SA and MRSA showed at least additive effects of active compounds (2a, 2e and 2j) with clinical antibiotics. When compound 2e was combined with ooxacin, FICI value of 0.28 suggested a synergistic effect. Therefore, worthy of note is the prophylactic purpose that low doses of clinical antibiotics plus a protonophore may be developed as an anti-MRSA therapy by inhibiting biolm.

Electron microscope
To elucidate the effects of compound 2e on MRSA, both the Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) were used to observe the bacteria aer being treated with either compound 2e alone or in combination with ooxacin. As shown in Fig. 4, SEM results revealed that untreated MRSA form biolms in normal growth condition, while the biolms were eradicated when treated with compound 2e at 1/2 MIC concentration or in its combination with 1/8 MIC of ooxacin. Furthermore, TEM results revealed that the regular cell conformation was destructed and the leakage of cellular substances under the treatment of compound 2e in combination with ooxacin.

Conclusion
In summary, a series of aromatic hydrazones were synthesized and evaluated for their antibacterial activities. Some compounds showed potential antimicrobial activity against Gram-positive bacteria, amongst them, compounds 2e and 2j had the lowest MICs against both S. aureus and MRSA (1.56 mM), and the growth inhibition assay conrmed the inhibition effects. SAR showed that (i) for aromatic hydrazones containing heterocycles, the antibacterial activity against Gram-positive bacteria was signicantly decreased (2a > 2b, 2c, 2k); (ii) the phenyl ring with strong electron-withdrawing substituent (-NO 2 and -CF 3 ) showed the moderate antibacterial activity (2d-2h, 2j and 2p); (iii) further, para-substituted group (-NO 2 and -CF 3 ) exhibited better activity than meta-substituted group, e.g. for S. aureus and MRSA (MIC values), 2e, 2h > 2f, 2g; 2j > 2d, 2p. Aromatic hydrazones in combination with clinical antibiotics exhibited better Gram-positive antibacterial activities, especially when compound 2e was used in combination with ooxacin, in which the synergistic effect was observed. MTT assay showed that the toxicity of compound 2e was low as that of CCCP. Further, electron microscopy showed that compound 2e possesses the capability to inhibit the formation of biolm and eradicate already existing biolm. In sum, compound 2e displayed antibacterial activity against MRSA through inhibiting biolm, especially improved the bactericidal effects of clinical antibiotics by synergistic effect. Therefore, the aromatic residue is a promising scaffold for further antibacterial modications.

Chemistry
All reagents were purchased from commercial sources and were used without further purication. Melting points (uncorrected) were determined on a XT4MP apparatus (Taike Corp., Beijing, China). 1 H NMR and 13 C NMR spectra were recorded on Bruker AV-600 or AV-400 MHz instruments in CDCl 3 . Chemical shis are reported in parts per million (d) downeld from the signal of tetramethylsilane (TMS) as internal standards. Coupling constants are reported in Hz. The multiplicity is dened by s (singlet), d (doublet), t (triplet), or m (multiplet). High resolution mass spectra (HRMS) were obtained on an Agilent 1260-6221 TOF mass spectrometry. Column and thin-layer chromatography (CC and TLC, resp.) were performed on silica gel (200-300 mesh) and silica gel GF 254 (Qingdao Marine Chemical Factory) respectively.

General procedures for synthesis of 2-(2-arylhydrazono) malononitriles 2a-2q
To a solution of the aromatic amine (15 mmol) and concentrated HCl (37%, 13.8 mL) in H 2 O (75 mL) was dropwise added NaNO 2 (15 mmol 1.04 g) in H 2 O (50 mL) for 1 h in an ice bath, and the mixture was stirred for 30 min. Then, the reaction solution was added to a solution of CH 2 (CN) 2 (20 mmol, 1.26 mL) and NaOAc (31 mmol, 38.1 g) in H 2 O (130 mL) under continuous stirring at 0 C. Aer 2 hours, the reaction mixture was ltrated, washed twice with water, and the residue was recrystallized from ethanol to give the title compounds 2a-2q.

Minimum inhibitory concentrations (MICs)
The MICs of tested compounds were determined using Mueller-Hinton (MH) broth micro-broth dilution assay established by the Clinical Laboratory Standards Institute (CLSI) in 96-well micro-test plates. The nal test concentration ranged from 0.39 to 200 mM and the bacterial inocula was 10 8 CFU mL À1 . Aer 18-20 hours of incubation at 37 C, the MICs were determined to be the lowest concentration of tested compound that inhibited the apparent increase in microorganisms. Each experiment was repeated at least 3 times to report the MIC value. 26

Inhibition of bacterial growth
The effect of concentrations ranging from 0.5 to 4 times MIC of the active compounds on the growth of S. aureus or MRSA was quantied aer incubation at 35 C for 0, 4, 8, 10, 18, 22 and 26 hours. At each time point, an aliquot (100 mL) was pipetted and measured for the A 450 nm. The experiment was performed in three biologically independent assays, each tested in triplicate.

Checkerboard assays
The synergistic effect of the combination of clinical antibacterials with the tested compounds was determined by checkerboard microdilution assays. In brief, checkerboards were set up with double dilutions of compounds 2a (0-12.5 mM) or 2e (0-3.12 mM) or 2j (0-3.12 mM) in the horizontal wells and ooxacin (0-2.5 mM) or cefoxitin (0-100 mM) or linezolid (0-14 mM) in the vertical wells. Then 50 mL each was arranged on the rows and columns of the plate, and 100 mL of MRSA was added to the wells and bacteria inocula of 5 Â 10 8 CFU mL À1 . Aer incubation at 35 C for 20 hours in 96-well micro-test plates. Aromatic hydrazones were further tested to determine their nature of interaction (synergy, antagonism, additive or no interaction) with ooxacin, cefoxitin and linezolid and expressed as the fractional inhibitory concentration index (FICI) for each agent.

Cell viability
Cell viability was performed against L02 (normal human liver cell line) cells using the MTT assay. L02 cells were grown in DMEM containing 10% fetal calf serum, 100 units per mL penicillin and 100 mg mL À1 streptomycin at 37 C in a 5% CO 2 incubator. L02 cells were seeded at 1 Â 10 4 cells per well in 96well micro-test plates. Aer 24 h of culture, the cells were treated with different concentrations of tested compound. Aer 24 h, 20 mL of 0.5 mg mL À1 MTT reagent was added to the cells and incubated for 4 h. Aer 4 h, the liquid in the well was discarded, and then 150 mL of DMSO was added to dissolve the formazan. The absorbance value (OD 570 ) was measured at 570 nm. The cell percentage survival rate was calculated by setting the density of formazan formed in the blank group to 100% viability as a control. Cell viability (%) ¼ compound (OD 570 )/ blank (OD 570 ) Â 100%. Each compound was tested in triplicate.

Electron microscope
MRSA (ATCC 43300) was grown overnight at 37 C on Mueller-Hinton Agar. The bacteria were harvested and the OD of bacteria suspended in MHB was adjusted to $0.5 MacFarlane units so as to give 5 Â 10 7 CFU mL À1 . Bacteria were then aliquoted into 10 mL tubes and compound 2e dissolved in DMSO was added to give a nal concentration ranging from 0.5 to 4 mg L À1 (two fold serial dilutions). Aer incubation at 37 C for 24 h, the bacteria were harvested by centrifugation at 4000 rpm, and cell pellets were then re-suspended with 10 mM PBS, pH 7.2 and harvested at 4000 rpm. The bacteria were xed using 2.5% glutaraldehyde for 3 h, following by washing with 0.1 M PBS (pH 7.2) for three times. The washing buffer was then removed and the bacteria were post-xed in 1% OsO 4 for 2 h. The OsO 4 were then pipetted out into an osmium waste bottle and the bacteria were washed in PBS (pH 7.2) for three times. Fixed microbial pellets were processed in graded alcohols, propylene oxide, and araldite and cured for 48 h at 60 C. Sample were nally stained with uranyl acetate and lead citrate before examine with Hitachi TEM system at an accelerating voltage of 80 kV. The SEM model used is the Hitachi su8100 at 3.0 kV voltage. 27

Statistical analysis
All results were expressed as mean values AE standard deviation. One-way analysis of variance followed by Dunnett's post hoc test was used for all comparisons.

Conflicts of interest
There are no conicts of interest to declare.