Efflux pump inhibitor combined with ofloxacin decreases MRSA biofilm formation by regulating the gene expression of NorA and quorum sensing

Carbonyl cyanide p-nitrophenylhydrazone (2e) displayed a lone or synergistic efficacy against MRSA (RSC Adv., 2020, 10, 17854). In this work, the synergistic mechanism of 2e with ofloxacin was studied. MRSA2858 had potential for biofilm formation, and the value of MBEC of 2e alone was 0.78–1.56 μM, while that of 2e + ofloxacin was 0.39–0.78 μM. 2e combined with ofloxacin showed a synergistic anti-biofilm effect against MRSA. Efflux pump inhibitor 2e can better bind to NorA protein. After MRSA2858 was treated with 2e of 1/2MIC (0.78 μM) and ofloxacin of 1/8MIC (0.097 μM), the transcript levels of efflux genes (norA) and quorum-sensing (QS) regulatory genes (agrA, sarA, icaA, hla) were substantially down-regulated, and alpha-hemolysin (Hla) was inhibited by 99.15%. 2e combined with ofloxacin was more effective than 2e alone in reducing bacterial load in vivo. All in all, efflux pump inhibitor 2e enhanced the bactericidal activities of antibiotics through regulating the gene expression of NorA and QS system.


Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) has become a major global health threat. 1 Biolm-associated MRSA are difficult to eradicate, which can lead to acute to chronic infections, including diabetic foot infections, cystic brosis, and so on. [2][3][4][5] Therefore, it is urgently needed to nd unique therapeutic strategies or new chemical scaffolds against MRSA to counter this health challenge.
Carbonyl cyanide chlorophenylhydrazone (CCCP), an efflux pump inhibitor, is combined with antibiotics to enhance antimicrobial activity. CCCP reduces ATP production and increases membrane permeability in bacteria [6][7][8][9] by interfering with the transmembrane electrochemical gradient and proton motive force. 6,10 CCCP blocks the efflux of antibiotics outside the bacterial cells to decrease the biolm-forming capacity of bacteria. 11 However, it is toxic and has poor antibacterial activity when used alone. 12 The efflux pumps also play a role in biolm formation. 13 The inhibition of efflux pumps helps to enhance the therapeutic efficacy of traditional antibiotics by affecting QS genes and virulence factors to control biolm production. 11 Recently, CCCP analog 2e was found to display alone or synergistic efficacy against MRSA. 13 2e could inhibit biolm formation, effectively eradicated preformed biolm, and showed low toxicity for human hepatic L02 cells. 14 Studying the anti-biolm activity of 2e and 2e combined with ooxacin will help to understand the role and relationship of efflux pump and biolm formation in MRSA.
Quorum sensing (QS) is one of the signaling mechanisms which directly aids in biolm formation, and inhibiting QS can increase susceptibility to antibiotics. Formation and maintenance of biolms are controlled by complex regulatory circuits such as QS and SaeRS two-component system (TCS). 15,16 TCS is at the core of the QS system, which play an important role in bacterial survival, adaptation and pathogenesis, biolm formation and virulence factor production. 17,18 It is interesting how efflux pump inhibitors affect biolm formation by QS and TCS.
When 2e was combined with ooxacin, a synergistic anti-MRSA activity was observed (FICI = 0.28). In this work, we would further explore this synergistic antibacterial mechanism of efflux pump inhibitors on the biolm formation by downregulating QS system. The multi-drug resistant MRSA2858 strain was isolated and identied from clinical MRSA strains, which showed the potential of biolm formation. 2e combined with ooxacin inhibited biolm formation of MRSA2858 by down-regulating the transcript levels of efflux gene (norA) and QS regulatory genes (agrA, sarA, icaA, hla), and decreasing alphahemolysin (Hla). This study provided a novel strategy for MRSA treatment.

Strains and growth media
In this work, 16 clinical multi-resistant isolates of MRSA strains capable of establishing biolms were collected. All of MRSA strains were resistant to three or more antimicrobial agents. S. aureus reference strain was obtained from American Type Culture Collection (ATCC 6538). The ability of the 16 strains to establish biolms was tested using crystal violet (CV) assay (Table 2). MRSA2858 was the strongest biolm-forming strain isolated from the patient's pus. Inoculums for the antimicrobial testing and the biolm assays were prepared overnight in Mueller-Hinton (MH) broth and incubated at 37°C.

MIC assay
The minimum inhibitory concentration (MIC) of tested compounds was determined using MH broth micro-dilution assay established by the Clinical Laboratory Standards Institute (CLSI) in 96-well microtest plates. Bacterial strains were grown overnight in MH broth at a concentration of 10 8 CFU ml −1 in microtiter plates. Then, 100 ml of culture medium containing different concentrations of test compounds and reference antibiotics were added to the plates. The nal bacterial concentration in the plate was 10 5 CFU ml −1 . Aer incubating for 20 h at 37°C, the MIC was determined by observing the lowest concentration of tested compound which inhibited bacterial growth. All assays were performed in quadruplicate.

Minimal biolm eradication concentration (MBEC) determination
The antibiolm activity of 2e was assessed in vitro against a mature biolm formed in a at-bottomed plate by an MRSA strain. For the assay, wells were lled with 200 ml of 1 : 100 dilution of a bacterial suspension cultured overnight in MH broth. Aer 24 h of incubation at 37°C in static conditions, the medium was discarded, and planktonic cells were removed by thorough washing of the biolm with sterile PBS. Then, 200 ml of MH broth supplemented with 2-fold dilutions of the compound (in the range 100-0.045 mM) were added in each well, and the plate was incubated further for 24 h at 37°C. 19 As an antibiotic control, dilutions of vancomycin, ooxacin, rifampin and erythromicin in the range 100-0.045 mM were included. Biolm formation was determined by crystal violet staining. To dissolve the crystal violet, 150 ml methanol per well was added to allow for minimal biolm eradication concentration determination (MBEC) analysis. 20 The optical density (OD) of each well was measured at 630 nm using an automated spectrophotometer (Bio-Tek Epoch-2, USA). Bio-lm growth inhibition (%) = 100 − (OD assay /OD positive ) × 100. Each experiment was repeated three times, with at least eight wells of each sample.

Molecular docking and toxicity prediction
A structure based in silico procedure was applied to discover the binding modes of the active compounds to NorA protein active site. The CDOCKER of Discovery Studio Client v18.1.0 (DS) was conducted to explain SAR of series compounds and further guide the design of more effective and concrete NorA protein inhibitors. The ligand binding to the crystal structure of NorA protein with PDB ID: 3WDO was selected as template. The target enzyme was prepared with Prepare Protein of DS to ensure the integrity of target. The ligands (CCCP and 2e) were processed by full minimization of the small molecular in DS. Then title compounds were docked into the active site of protein using CDOCKER. The view results of molecular docking were extracted aer the program running end, each docking result was analyzed for interaction and their different pose. Those poses with the lowest -CDOCKER_INTERACTION_ENERGY values were regarded as the most stable and picked to analysis binding interactions with target enzyme visualized.

Molecular dynamics simulations
The molecular dynamics (MD) simulations were performed in Yinfo Cloud Computing Platform using AmberTools 20 package with AMBER ff19SB and GAFF force elds for NorA and compound 2e, respectively. 21, 22 The system was solvated by a truncated octahedron (or cubic) water box using OPC water model with a margin of 10Å, the net charge was neutralized by (0.15 M of NaCl) sodium  Table 1 qRT-PCR primers in this study 23,24 Primer Sequence Reference   ions. Periodic boundary condition (PBC) was used and the net charge neutralized by Na + (or Cl − ) ions (or 0.15 M of NaCl). Nonbonded van der Waals interactions were calculated using the Lennard-Jones 12-6 potentials with a 10Å cutoff, while long-range electrostatics were treated using the Particle Mesh Ewald (PME) algorithm. The SHAKE algorithm was applied to constrain bonds involving hydrogen atoms. To removed improper atom contacts, the system was rst minimized by (1) the 5000 steps of steepest descent and the 5000 steps of the conjugate gradient, under a harmonic constraint of 10.0 kcal (molÅ 2 ) −1 on heavy atoms; (2) relaxing the entire system by steepest descent and conjugate gradient each in 20 000 steps. And then the system was gradually heated up to 300 K by a 20 ps NVT simulation. The equilibration phase was carried out in two steps: (1) a 200 ps NPT simulation with constraints on heavy atoms followed by (2) a 500 ps NVT simulation without restraint. The temperature and pressure were maintained at 300 K and 1 atm using the Berendsen thermostat and Monte Carlo barostat, with coupling constant and relaxation time of 1 ps. Finally, the system was subjected to a 20 ns NPT simulation with a time step of 1 fs. The root-mean-square deviation (RMSD), root-mean-square uctuation (RMSF), and hydrogen bonds were analyzed by the Cpptraj module. MM/PB(GB)SA calculations. The binding free energies were calculated using the Molecular Mechanics Poisson-Boltzmann Surface Area (MM/PBSA) method implemented in AmberTools 20 for 200 snapshots from the MD trajectory. For each snapshot, the free energy was calculated for protein NorA, compound 2e, and the complex NorA-2e using a single trajectory approach. The total binding free energy was calculated according to the following equation: where DE MM denotes the gas-phase interaction energy between the receptor and the ligand (including van der Waals energy contribution (DE vdw ) and electrostatic energy contribution (DE ele )); DG PB and DG SA are the polar and nonpolar components of the de-solvation free energy, respectively; TDS represents the conformational entropy contribution at temperature T. Here, DG PB was determined by the Poisson-Boltzmann approximation model, while DG SA was estimated based on the solvent accessible surface area model by the method: DG SA = g × SASA + b, where the values of the constants g and b were 0.00542 kcal A −2 and 0.92 kcal mol −1 , respectively. The solvent probe radius and ionic strength were set to be 1.4Å and 0.15 mM, respectively. The interior and exterior dielectric constant of MM/PBSA calculation systems was 1.0 and 80.0.

Effect of 2e on gene expression of MRSA strains
Total RNA from MRSA cultures treated with 2e alone or in combination with ooxacin for 24 h was extracted using the TRIzol method for RNA extraction and converted to cDNA using SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen, USA). The PCRs were performed in a 20 ml volume and contained Roche SYBR Green PCR Master Mix. The fold change in gene expression was calculated by the 2 −DDct method with gyrB as the housekeeping gene. Specic primers are listed in Table 1. Amplication was performed in a gradient thermal cycler (Bio-Rad, Hercules, USA). All samples were analyzed in triplicate.

Hemolytic activity
Hemolytic activity can be assessed by a rabbit red blood cell lysis technique, which consists of bringing the red blood cells into contact with the MRSA2858 or 2e, and quantifying the hemolytic activity of alpha-hemolysin (Hla) by spectrophotometric determination. Samples (100 ml) of MRSA2858 culture (1 × 10 9 CFU ml −1 ) were ltered and were added to 900 ml hemolysin buffer (0.145 mol per l NaCl, 0.02 mol per l CaCl 2 ) and 25 ml of debrinated rabbit blood. Rabbit blood was washed three times with 0.9% NaCl and resuspended to a concentration of 0.5 × 10 8 cells per ml, as determined by manual cell count. The solution was incubated for 15 min at 37°C. Unlysed blood cells were pelleted by centrifugation (5500g, 1 min). The hemolytic activity of the supernatant was determined by measuring the optical density at OD 541. Sterile culture medium served as the standard for 0% hemolysis, and a bacterial culture supernatant devoid of any inhibitor (control) was designated as the standard for 100% hemolysis. The percentage of hemolysis inhibition was calculated by comparison with the control culture. All assays were performed in triplicate.
In vivo anti-bacterial activity SPF female BALB/c mice were selected to avoid the re-infection induced by the ghting and barbering of males. SPF female BALB/c mice (aged 6-8 weeks; 18-21 g) were purchased from Changzhou Cavens Experimental Animal Co., Ltd, Animal certicate number: SCXK (SU) 2016-0010; mice were maintained with SPF food and water for 1 week, 6 mice per group. The test strain was refreshed on MH broth at 37°C for 24 h. From the overnight culture preparation, a single colony was diluted into 5 ml of MH broth and incubated overnight at 37°C. A log-phase subculture was obtained by adding 150 ml of overnight subculture in 10 ml MH broth and incubated for a further 2-3 h. A full dilution of the bacterial cell suspension in saline was achieved by washing (3220 g, 10 min) and the OD 600 in saline determined. The suspension of 1.5 × 10 9 CFU ml −1 solution was diluted in MH broth into the nal bacterial concentration of 10 5 CFU ml −1 . Mice were infected by intraperitoneal administration of MRSA2858 in 0.2 ml of MH broth.
Aer bacterial challenge for 2 h, mice were randomized to receive subcutaneous injection of saline as a control, ooxacin (5 mg kg −1 ), 2e (5 mg kg −1 ), or 2e combined with ooxacin group (5 mg kg −1 + 5 mg kg −1 ). To assess bacterial clearance, six mice in each group were euthanized, and bacterial counts were determined in the kidney and blood from each animal aer bacterial challenge for 24 h. In the MRSA infection model, parts of kidney tissues were xed in 10% neutral buffered formalin for 24 h, and then their morphologies were observed using H&E staining.

Statistic analysis
Statistical analysis was performed using SPSS 13.0 soware package, and one-way ANOVA followed was used to assess the signicance. A p-value # 0.05 was set as statistically signicant. Data were analyzed by Microso Excel 2003 spreadsheet soware, and expressed as the mean ± standard deviation.

Antibacterial activity of 2e
The results showed that 2e performed the best against S. aureus with MIC value of 1.56 mM, and against MRSA with MIC value of 1.56 mM, which was superior to the front-line antibiotics including cefoxitin and linazolid. 14 As shown in Table 2, 16 clinical MRSA were isolated from different specimens, and all were resistant to at least three antimicrobial agents of furantoin, penicillin, rifampicin, tetracycline, tegacyclin, cipro-oxacin, erythromycin, gentamicin, linezolid, benzacillin or clindamycin. Among them, MRSA2858 was a clinical isolate with multi-drug resistance and the strongest biolm formation ability. Although certain antibiotics have MIC values even more favorable than compound 2e in Table 2, levooxacin and ciprooxacin are alternative or complementary drugs and are not clinical rst-line anti-MRSA agents according to CLSI guidelines.
Studying the synergistic mechanism of 2e with ooxacin will help to enhance the therapeutic efficacy of traditional antibiotics against resistant bacteria. Data indicated that 2e strongly inhibited the bacterial growth of all clinical strains with the potency of MIC values less than 1.56 mM and the results had no statistically signicant differences for all strains, which validated 2e as a promising anti-MRSA agent.

Anti-biolm activity of 2e
To assess the anti-biolm potential of 2e, the MRSA clinical isolates were assayed in vitro in a preliminary set of experiments for their ability to form adherent biolm layers under static conditions. 19 Clinical strain MRSA2858 demonstrated high biolm capacity and planktonic cells that were susceptible to 2e ( Table 2). MRSA2858 biolms were initially grown for 24 h and then treated with dilutions of 2e and vancomycin, ooxacin, rifampin, erythromicin, as antibiotic control, for a further 24 h. Biolm production and eradication were quantitatively evaluated using crystal violet staining.
As shown in Fig. 2 and Table 3, 2e alone and combination with 1/8MIC ooxacin was active against MRSA biolms, the value of MBEC was 0.78-1.56 mM and 0.39-0.78 mM, similar to it MIC, superior to those of vancomycin, ooxacin, rifampin and erythromycin ( Fig. 2A). Although low level of b-lactam antibiotic was reported to induce biolm formation of S. aureus, 25,26 2e combined with subinhibitory concentration of ooxacin (1/8MIC) showed a synergistic anti-biolm effect against MRSA (Fig. 2B).

Molecular docking and toxicity prediction
In the molecular docking study, two ligands (CCCP and 2e) were docked into NorA protein, and they were located in the same activity site. 4-NO 2 of 2e formed hydrogen bond interaction with   (Fig. 3), which implied that 2e may be a new NorA protein inhibitor. In order to assess the feasibility of 2e in vivo application, we performed an in silico analysis of physicochemical, pharmacokinetic and toxicological properties using Discovery Studio2018 soware. Although 2e was predicted to be carcinogenic risk and mutagenic risk in humans, this cannot be considered a major problem in a drug discovery process, as many widely used drugs in the market are exist these risks. Outstandingly, the chronic oral lowest toxic dose of 2e (90.2 mg kg −1 ) is much higher than the actual dose (5 mg kg −1 ).

Molecular dynamics simulation
MD simulation mainly relies on Newtonian mechanics to simulate the motion of a molecular system. This approach can not only obtain the trajectory of atoms but also enables the observation of various microscopic details during atomic motion. Compound 2e was docked into the NorA protein pocket and then subjected to MD simulation to examine the structural stability of simulation system. MD simulation of complexes was performed for 20 ns period. In this study, the kinetic energy of 2e started to increase by temperature and time until a steady state was reached. As shown in Fig. 4A, before 10 000 ps, the entire system of the complex NorA-2e was in a stable state. Aer 10 000 ps, an obvious upward trend was emerged, especially at 12 000 ps, aer which the overall system was relatively stable, and the wave range of RMSD was within 1Å. Subsequently, calculated by the MM-PB(GB)SA method (Fig. 4B), the total binding free energy was −43.76 kcal mol −1 for the complex of NorA-2e, and the van der Waals energy was −30.01 kcal mol −1 . Fig. 4C was the contributions of hot residues in the binding pocket of NorA protein.
Under normal circumstances, a residue with lower interaction energy than −1 kcal mol −1 was considered to be essential for ligand recognition and combination. As disclosed in docking studies, hydrogen bonding of 2e with Gln65 (−2.44 kcal mol −1 ) was signicant for binding to NorA protein.
Effect of 2e on norA gene expression 2e was used to examine the effect on the expression of norA gene in MRSA2858 using qRT-PCR (Fig. 5). 2e alone signicantly down-regulated the gene expression of norA by 41.8%. Combination of 2e with 1/8MIC ooxacin reduced the expression of norA by 65.9%. The results showed that 2e combined with low concentration of ooxacin showed a synergistic inhibitory effect against MRSA by regulating NorA efflux pump.

Effect on expression of QS regulatory genes
The QS system plays a role in the precise regulation of genes controlling virulence factors and biolm formation in MRSA. 2e  exerted dose-dependent inhibitory effects on virulence phenotypes (hla) regulated by QS in MRSA using qRT-PCR (Fig. 6). Moreover, the expression levels of QS regulatory genes, including agrA, sarA and icaA, were repressed aer 2e combined with 1/8MIC ooxacin treatment in a dose-dependent manner, and genes hla, agrA, sarA and icaA were down-regulated by 73.0%, 68.7%, 62.1% and 71.0%, respectively. These results conrmed the expression of QS regulatory genes (agrA, sarA, icaA, hla) were substantially down-regulated in treated with 1/ 2MIC 2e at 0.78 mM and 1/8MIC ooxacin at 0.097 mM.

Effect on MRSA2858 virulence
Alpha-hemolysin (Hla) was a major virulence factor in the pathogenesis of S. aureus infection, being active against a wide range of host cells. Hla created holes in the membranes of various host cells, such as immune system cells and erythrocytes. The amount of hemoglobin released from lysed red blood cells was taken as a measure of Hla production. 2e signicantly decreased the level of hemolysis in a dosedependent manner at 0.097-0.781 mM. When 2e was combined with 1/8MIC ooxacin, the inhibition rate of ahemolysin was 99.15%, while the inhibition rate of a-hemolysin for 2e alone was 93.75% (Table 4, Fig. 7). These results suggested that the synergistic antibacterial effect came along with the virulence inhibition of MRSA.

In vivo antibacterial activity
Effect of 2e or/and ooxacin treatment on bacterial load of blood and kidney. The antibacterial effect of 2e on MRSA was evaluated in the mouse abdominal infection model. MRSA2858 was used to observe the effect of 2e, ooxacin, and their combination on reducing the bacterial load in the blood and kidney of mice. 2e combined with ooxacin could signicantly reduce the bacterial load in the blood and kidney of mice, bacterial colonies counts were dropped to 1.18 and 0.97 log 10 CFU ml −1 , respectively (Fig. 8). The efficacy of 2e combined with ooxacin was superior to that of 2e or ooxacin, which suggested that 2e combined with ooxacin could effectively reduce the MRSA abdominal infection.   Effect of 2e or/and ooxacin treatment on hematological parameters in vivo. Hematological studies provided a good indication of the progress of bacterial infection as well as treatment. WBC, neutrophils and monocytes may drop abruptly due to massive consumption in a short period of time by acute bacterial infection. MRSA was known to contain hemolysin that can help in targeted killing of host cells. A reduction in Hb and RBC was noticed in untreated infected group. In treated group, Fig. 7 Inhibitory effect of 2e on rabbit blood hemolysis by MRSA2858. Fig. 8 Effect of treatment with 2e or/and ofloxacin on bacterial load of different tissues (comparison between each administration group and control group, **p < 0.05, ***p < 0.01). all compounds displayed improvement in haemalogical parameters. Aer treatment with 2e combined with ooxacin, the best adjustment of RBC, WBC and monocytes was observed, and HGB and neutrophil count were similar to negative control, showing their higher potency in controlling infection ( Table 5).
Effect of 2e or/and ooxacin on the pathological changes of kidney tissue. Firstly, kidney tissues were removed and xed in 10% formaldehyde. Secondly, aer dehydration in gradient concentration of alcohol, the tissues were embedded in paraffin and sliced. Finally, sections were stained by H&E, and imaged under an optical microscope. As shown in Fig. 9, in the blank control group, normal morphology of the glomeruli and proximal tubules were clearly shown in the kidneys of mice, while in abdominal infection mouse model, the kidneys were severely damaged, resulting in renal tubular epithelial injury, inammatory cell inltration, renal tubular cell swelling, and tubular dilatation. Therefore, the treatment of 2e combined with ooxacin signicantly improved the necrosis of kidney tissue and inammatory inltrating cells caused by MRSA in model mice.

Results and discussion
MRSA infection is a serious threat to human health. The ability of biolm formation in MRSA can lead to resistance to most currently used antibiotics. [27][28][29] Novel antibacterial agents can be developed to inhibit their function, to help reduce the virulence of bacteria and avoid drug resistance. There's different methods for evaluating biolm formation, and the most common ones are CV staining for biomass and XTT (tetrazolium salt reduction) assay for viability. 30 Because CV staining is oen used for quantication of S. aureus biolm formation, 31 the CV method is more suitable to evaluate the inhibition of 2e for biolm formation and biomass production.
This study showed that 2e could inhibit the biolm formation of MRSA in vitro. The transcription proling demonstrated that the NorA efflux pathway was obviously inhibited by 2e treatment. As one of the most important efflux pump genes of S. aureus, norA gene was over expressed in 43% of strains. 32,33 NorA was capable of extruding multiple structurally dissimilar substrates such as hydrophilic uoroquinolones. 34 The use of efflux pump inhibitors (EPIs) could decrease the MIC of antibacterial agents, increase susceptibility restoration in clinical resistant strains, and inhibit biolm formation. NorA EPIs were capable of restoring drug susceptibility in resistant strains have been found, such as reserpine, verapamil, omeprazole, chlorpromazine, etc. 35 The efflux pump can expel metabolites, toxic substances and antibacterial drugs out of the membrane of bacteria, which helps to maintain the vitality and virulence of starving bacteria in the biolm. Therefore, the expression level of efflux pump was positively correlated with the formation of biolm. 34 We found that 2e markedly inhibited NorA pump activity.
The norA pump is regulated by the QS system. QS system has been known to be essential for robust biolm production and development in numerous bacteria, including clinical multidrug resistant isolates. 36 Consistent with these observations, 2e treatment could down-regulate the expression of QS regulatory genes (agrA, sarA, icaA, hla) in a dose-dependent manner, decrease the virulence by blocking the NorA efflux pathway, and contribute to the biolm inhibitory activity. Although biolm development in MRSA is ica independent, 37 we observed that the regulatory gene of MRSA2858 was ica dependent.
One of the key features of S. aureus infection was the production of a series of virulence factors, including secreted enzymes and toxins. 38 In this study, the pore-forming toxin alphahemolysin (Hla) was a S. aureus secreted factor which participated in the activation of the NorA efflux pathway. This study showed that the anti-virulence activity of compound 2e combined with ooxacin was superior to that of 2e alone against MRSA.
QS-controlled biolm formation and virulence factor secretion by MRSA in clinical settings has remained controversial due to emerging drug resistance. Therefore, it is important to nd diverse molecules for anti-biolm or anti-QS activities.

Conclusion
It was urgent for the discovery of new anti-resistant agents with anti-biolm and anti-efflux pump activity, especially therapeutic alternatives for multi-drug resistant MRSA. Carbonyl cyanide p-nitrophenylhydrazone 2e alone or combined with ooxacin was found to have potent anti-MRSA activity. 2e combined with ooxacin was exhibited anti-resistant activity through inhibiting NorA efflux pump, as well as decreasing biolm formation, decreasing Hla and regulating QS systems of MRSA. Therefore, our results are signicant and relevant to nd novel therapeutic methods against MRSA to reduce the use of antibiotics.

Ethical statement
In this study, MRSA strains were isolated and identied from diverse sources including blood, peritoneal uid, and purulent leukorrhea of clinical infected patients, and the samples were collected by technically trained persons. These samples were evaluated and permitted by the Institutional Ethical Committee (IEC), Anhui No. 2 Provincial People's Hospital, China (IEC No. 2019814). The procedures for maintenance and treatment of laboratory animals were approved by the Animal Care and Use Committee of the Anhui No. 2 Provincial People's Hospital. All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.