Overcoming Multidrug‐Resistant MRSA Using Conventional Aminoglycoside Antibiotics

Abstract Global multidrug‐resistant (MDR) bacteria are spreading rapidly and causing a great threat to human health due to the abuse of antibiotics. Determining how to resensitize MDR bacteria to conventional inefficient antibiotics is of extreme urgency. Here, a low‐temperature photothermal treatment (PTT, 45 °C) is utilized with red phosphorus nanoparticles to resensitize methicillin‐resistant Staphylococcus aureus (MRSA) to conventional aminoglycoside antibiotics. The antibacterial mechanism is studied by the proteomic technique and molecular dynamics (MD) simulation, which proves that the aminoglycoside antibiotics against MRSA can be selectively potentiated by low‐temperature PTT. The catalytic activity of 2‐aminoglycoside phosphotransferase (APH (2″))—a modifying enzyme—is demonstrated to be obviously inhibited via detecting the consumption of adenosine triphosphate (ATP) in the catalytic reaction. It is also found that the active site of aspartic acid (ASP) residues in APH (2″) is thermally unstable from the results of molecular dynamics simulation. Its catalytic ability is inhibited by preventing the deprotonating procedure for the target —OH of gentamycin. The combined therapy also exhibits great biocompatibility and successfully treats MRSA infections in vivo. This low‐temperature PTT strategy has the potential to be an exogenous‐modifying enzyme inhibitor for the treatment of MDR bacterial infection.

4×MIC, 16×MIC and 32×MIC were used to evaluate the growth inhibition when combined with photothermal treatment. The experiment groups could be divided as control (no treatment), RPNPs (200 g/mL), Gen and RPNPs-Gen (200 g/mL, 1×MIC). 150 L of bacterial suspension (5×10 6 CFU/mL) was added to each group and subsequently irradiated with 808 nm laser (0.5 W cm -2 , Hi-Tech Optoelectronics 7. Bacterial membrane permeability assay. ANS was used to investigate the membrane permeability change after treatments. [4] The experiment groups could be divided as control (no treatment), 1% TritonX-100, RPNPs (200 g/mL), Gen (4×MIC) and RPNPs-Gen (200 g/mL, 4×MIC). The MRSA was washed with PBS and resuspended in PBS solution (10 8 CFU/mL). ANS (10 M) was added to the washed MRSA and equilibrated for 30 min. After 30 min treatments as above, the fluorescence emission of samples was detected under excitation at 380 nm through microplate reader. Then, the leakage of protein from MRSA (10 7 CFU/mL) of each group was also measured by a BCA kit (Beyotime, China). After treatments, the MRSA solution was centrifuged at 5000 rpm/min for 10 min at 3 °C. The supernatant (40 L) was taken out and added to BCA working solution (200 L). After 30 min incubation at 37 °C, the optical density (OD) value of each group was measured at 562 nm.
8. Proteomics evaluation. The MRSA treated by Gen and RPNPs-Gen were first lysed with SDT solution (4% Sodium dodecyl sulfate, 100 mM Tris-HCl, 1 mM dithiothreitol, pH 7.6) and heated with boiling water for 15 min. after centrifugation at 14000 g for 30 min, the supernatant were obtained and then quantified by BCA kit.
The protein enzymolysis process was operated FASP (Filter-aided sample preparation) method. [5] The peptides of samples were labeled with TMT reagent (Thermo Scientific). The TMT-labeled peptides were fractionated using a fractionation kit (Thermo Scientific). Then, the samples were fractionated by HPLC (high performance liquid chromatography) system (Easy nLC, Thermo Scientific) in Shanghai Applied Protein Technology Co., Ltd. After that, the samples were analyzed by a mass spectrometry (Q Exactive, Thermo Scientific). For GO annotation, the protein sequences were compared with Staphylococcus aureus subsp. aureus NCTC 8325 database. For KEGG pathway annotation, the KEGG Automatic Annotation Server software was used to perform KEGG annotation. The differential proteins were compared with the online KEGG database (http://www.kegg.jp/) to extract the corresponding KEGG pathway. The enrichment analysis of GO and KEGG annotation were performed using Fisher's Exact Test. The PPI information of differential proteins were obtained from IntAct molecular interaction database (http://www.ebi.ac.uk/intact/) using their corresponding gene symbols. The PPI network was created by Cytoscape software.
Subsequently, the samples were centrifuged at 12000 g for 5 min and the ATP in the supernatant was determined by ATP assay kit. The fluorescence intensity of ATP was recorded by microplate reader. The consumption of ATP was calculated from the standard curve of ATP. The generation of Gen-P was calculated from the consumption of ATP over time.
10. Molecular dynamics simulation. The MD simulation was carried out by GROMACS 5.1.4 to analyze the conformation change of APH (2") and examine the interaction between APH (2") and Gen at 37 °C and 48 °C (100 ns), respectively. The system was optimized by steepest descent method to realize energy minimization. The protein was solvated into water using SPC model and placed into a cubic box. Na + and Cl − were added to neutralize the system. The protein modeling was performed with Modeller 9.20 and we chose high similarity protein template (4ORK, 4OCJ, 3TDV, 3N4V, 3HAM, 3ATT, 2QG7, 2QG7 and 1ZYL) through blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to model. 20 L) and RPNPs-Gen (4 mg/mL, 20 L; 10 mg/mL, 20 L). Before surgery, the mice were anaesthetized through intraperitoneal injection using 3% pentobarbital. The back of mice were shaved and cut using a biopsy punch. Subsequently, 2.0×10 7 CFU MRSA was injected into the wound of each mouse. These wounds were then covered by bandage. After 2 days infection, the samples were added and following by carrying out 808 nm laser treatment (0.3 W cm -2 ) for 30 min at 45 °C. The wound tissues were cut to harvest MRSA and the CFU of the containing MRSA were calculated through spread plate method on day 3. On day 4, the wound tissues were cut, fixed by formalin, dehydrated, paraffined and sectioned. The Hematoxylin-Eosin (H&E) and Gram staining was used to analyze the inflammatory reaction and residue bacteria of the wounds. On Day 10, the condition of wound healing was investigated by Hematoxylin-Eosin (H&E) and Masson staining. The wounds were observed and photographed on day 0, 2, 4, 8 and 10, respectively. The histological analysis of heart, liver, spleen, lung and kidney were also performed via H&E staining after 10 days.
The collagen content from Masson staining and wounds areas (%) were calculated by Image J software.
14. Statistical analysis. All experiments were shown as mean values ± standard deviation of at least three tests. A one-way analysis of variance (ANOVA) program combined with a student t-test was used to evaluate the statistical significance of the variance and a difference of *P < 0.05, **P < 0.01 and ***P < 0.001 were considered significant.