Berberine hydrochloride reduces staphyloxanthin synthesis by inhibiting fni genes in methicillin-resistant Staphylococcus aureus.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) poses a great health threat to humans. Looking for compounds that could reduce the resistance of S. aureus towards methicillin is an effective way to alleviate the antimicrobial resistance crisis. METHODS AND RESULTS: Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), Time-killing growth curve, staphyloxanthin and penicillin-binding protein 2a (PBP2a) were detected. A quantitative polymerase chain reaction was used to measure the effect of BBH on the gene transcription profiles of MRSA. The MIC of MRSA-ST59-t437 towards oxacillin was 8 µg/ml, and MBC was 128 µg/ml. After adding a sub-inhibitory concentration of BBH, the MIC and MBC of MRSA-ST59-t478 towards oxacillin went down to 0.125 and 32 µg/ml respectively. The amount of PBP2a and staphyloxanthin were reduced after treatment with BBH. Moreover, the transcription levels of sarA, mecA and fni genes were downregulated. CONCLUSIONS: It is for the first time reported that BBH could inhibit staphyloxanthin synthesis by inhibiting fni gene. Moreover, fni might be the target gene of sarA, and there might be another regulatory pathway to inhibit staphyloxanthin biosynthesis. BBH could effectively reduce the methicillin resistance of MRSA-ST59-t437 by downregulating fni, sarA and mecA genes.

One pot fabrication of diamino naphthalene -AuNPs decorated graphene nanoplatform for the MRSA detection in the biological sample.

Early monitoring of MRSA can effectively mitigate the disease risk by using Penicillin-binding protein 2a (PbP2a) biomarker. Diamino naphthalene-AuNPs decorated graphene (AuNPsGO-DN) nanocomposite was synthesized for a rapid and sensitive immunosensor detecting PbP2a. The synthesized AuNPsGO-DN nanocomposites were characterized by field emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (SEM-EDX), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, and X-ray diffraction spectroscopy (XRD). Electrochemical characterization done with cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrical impedance spectroscopy (EIS) techniques. Anti-PbP2a monoclonal antibodies immobilized at AuNPsGO-DN/GCE via covalent bonding. AuNPs enhanced the electrode surface area and the antibodies' loading. Mercaptopropionic acid (MPA) was a linker between the AuNPs and antibodies, orientated the antibodies as opposite to the PbP2a antigen, and improved the sensitivity and specificity. The antiPbP2a/MPA/AuNPsGO-DN/GCE electrode displayed sensitive and selective detection towards the PbP2a antigen in phosphate buffer saline (PBS pH 7.4). The broad linear range from 0.01 to 8000 pg/mL was obtained with LOD of 0.154 pg/mL and 0.0239 pg/mL, respectively. A label-free, simple, and sensitive immunosensor was developed with a 98-106 % recovery rate in spiked biological samples. It shows the potential applicability of the developed immunoelectrode.

Sensitive and Extraction-Free Detection of Methicillin-Resistant Staphylococcus aureus through Ag+ Aptamer-Based Color Reaction.

Refractory infections, such as hospital-acquired pneumonia, can be better diagnosed with the assistance of precise methicillin-resistant Staphylococcus aureus (MRSA) testing. However, traditional methods necessitate high-tech tools, rigorous temperature cycling, and the extraction of genetic material from MRSA cells. Herein, we propose a sensitive, specific, and extraction-free strategy for MRSA detection by integrating allosteric probe-based target recognition and exonuclease-III (Exo-III)-enhanced color reaction. The penicillin-binding protein 2a (PBP2a) aptamer in the allosteric probe binds with MRSA to convert protein signals to nucleic acid signals. This is followed by the DNA polymerase-assisted target recycle and the production of numerous single-strand DNA (ssDNA) chains which bind with silver ion (Ag+) aptamer to form a blunt terminus that can be identified by Exo-III. As a result, the Ag+ aptamer pre-coupled to magnetic nanoparticles is digested. After magnetic separation, the Ag+ in liquid supernatant catalyzes 3,3',5,5'-tetramethylbenzidine (TMB) for a color reaction. In addition, a concentration of 54 cfu/mL is predicted to be the lowest detectable value. Based on this, our assay has a wide linear detection range, covering 5 orders of magnitude and demonstrating a high specificity, which allows it to accurately distinguish the target MRSA from other microorganisms.

Chalcogen-Varied Imidazolone Derivatives as Antibiotic Resistance Breakers in Staphylococcus aureus Strains.

In this study, a search for new therapeutic agents that may improve the antibacterial activity of conventional antibiotics and help to successfully overcome methicillin-resistant Staphylococcus aureus (MRSA) infections has been conducted. The purpose of this work was to extend the scope of our preliminary studies and to evaluate the adjuvant potency of new derivatives in a set of S. aureus clinical isolates. The study confirmed the high efficacy of piperazine derivatives of 5-arylideneimidazol-4-one (7-9) tested previously, and it enabled the authors to identify even more efficient modulators of bacterial resistance among new analogs. The greatest capacity to enhance oxacillin activity was determined for 1-benzhydrylpiperazine 5-spirofluorenehydantoin derivative (13) which, at concentrations as low as 0.0625 mM, restores the effectiveness of ß-lactam antibiotics against MRSA strains. In silico studies showed that the probable mechanism of action of 13 is related to the binding of the molecule with the allosteric site of PBP2a. Interestingly, thiazole derivatives tested were shown to act as both oxacillin and erythromycin conjugators in S. aureus isolates, suggesting a complex mode of action (i.e., influence on the Msr(A) efflux pump). This high enhancer activity indicates the high potential of imidazolones to become commercially available antibiotic adjuvants.

A Review on Five and Six-Membered Heterocyclic Compounds Targeting the Penicillin-Binding Protein 2 (PBP2A) of Methicillin-Resistant Staphylococcus aureus (MRSA).

Staphylococcus aureus is a common human pathogen. Methicillin-resistant Staphylococcus aureus (MRSA) infections pose significant and challenging therapeutic difficulties. MRSA often acquires the non-native gene PBP2a, which results in reduced susceptibility to ß-lactam antibiotics, thus conferring resistance. PBP2a has a lower affinity for methicillin, allowing bacteria to maintain peptidoglycan biosynthesis, a core component of the bacterial cell wall. Consequently, even in the presence of methicillin or other antibiotics, bacteria can develop resistance. Due to genes responsible for resistance, S. aureus becomes MRSA. The fundamental premise of this resistance mechanism is well-understood. Given the therapeutic concerns posed by resistant microorganisms, there is a legitimate demand for novel antibiotics. This review primarily focuses on PBP2a scaffolds and the various screening approaches used to identify PBP2a inhibitors. The following classes of compounds and their biological activities are discussed: Penicillin, Cephalosporins, Pyrazole-Benzimidazole-based derivatives, Oxadiazole-containing derivatives, non-ß-lactam allosteric inhibitors, 4-(3H)-Quinazolinones, Pyrrolylated chalcone, Bis-2-Oxoazetidinyl macrocycles (ß-lactam antibiotics with 1,3-Bridges), Macrocycle-embedded ß-lactams as novel inhibitors, Pyridine-Coupled Pyrimidinones, novel Naphthalimide corbelled aminothiazoximes, non-covalent inhibitors, Investigational-ß-lactam antibiotics, Carbapenem, novel Benzoxazole derivatives, Pyrazolylpyridine analogues, and other miscellaneous classes of scaffolds for PBP2a. Additionally, we discuss the penicillin-binding protein, a crucial target in the MRSA cell wall. Various aspects of PBP2a, bacterial cell walls, peptidoglycans, different crystal structures of PBP2a, synthetic routes for PBP2a inhibitors, and future perspectives on MRSA inhibitors are also explored.

Molecular Determinants of ß-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus (MRSA): An Updated Review.

The development of antibiotic resistance in Staphylococcus aureus, particularly in methicillin-resistant S. aureus (MRSA), has become a significant health concern worldwide. The acquired mecA gene encodes penicillin-binding protein 2a (PBP2a), which takes over the activities of endogenous PBPs and, due to its low affinity for ß-lactam antibiotics, is the main determinant of MRSA. In addition to PBP2a, other genetic factors that regulate cell wall synthesis, cell signaling pathways, and metabolism are required to develop high-level ß-lactam resistance in MRSA. Although several genetic factors that modulate ß-lactam resistance have been identified, it remains unclear how they alter PBP2a expression and affect antibiotic resistance. This review describes the molecular determinants of ß-lactam resistance in MRSA, with a focus on recent developments in our understanding of the role of mecA-encoded PBP2a and on other genetic factors that modulate the level of ß-lactam resistance. Understanding the molecular determinants of ß-lactam resistance can aid in developing novel strategies to combat MRSA.

The depsidones from marine sponge-derived fungus Aspergillus unguis IB151 as an anti-MRSA agent: Molecular docking, pharmacokinetics analysis, and molecular dynamic simulation studies.

Methicillin-resistant Staphylococcus aureus (MRSA) is an emerging nosocomial pathogen among hospitalized patients, with high morbidity and mortality rates. The discovery of a novel antibacterial is urgently needed to address this resistance problem. The present study aims to explore the antibacterial potential of three depsidone compounds: 2-clorounguinol (1), unguinol (2), and nidulin (3), isolated from the marine sponge-derived fungus Aspergillus unguis IB1, both in vitro and in silico. The antibacterial activity of all compounds was evaluated by calculating the Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) against MRSA using agar diffusion and total plate count methods, respectively. Bacterial cell morphology changes were  studied for the first time using scanning electron microscopy (SEM). Molecular docking, pharmacokinetics analysis, and molecular dynamics simulation were performed to determine possible protein-ligand interactions and the stability of the targeting penicillin-binding protein 2a (PBP2a) against 2-clorounguinol (1). The research findings indicated that compounds 1 to 3 exhibited MIC and MBC values of 2 µg/mL and 16 µg/mL against MRSA, respectively. MRSA cells displayed a distinct shape after the addition of the depsidone compound, as observed in SEM. According to the in silico study, 2-chlorounguinol exhibited the highest binding-free energy (BFE) with PBP2a (-6.7 kcal/mol). For comparison, (E)-3-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4H)-yl) benzoic acid inhibits PBP2a with a BFE less than -6.6 kcal/mol. Based on the Lipinski's rule of 5, depsidone compounds constitute a class of compounds with good pharmacokinetic properties, being easily absorbed and permeable. These findings suggest that 2-chlorounguinol possesses potential antibacterial activity and could be developed as an antibiotic adjuvant to reduce antimicrobial resistance.

Novel Thiazolylketenyl Quinazolinones as Potential Anti-MRSA Agents and Allosteric Modulator for PBP2a.

Bacterial infections caused by methicillin-resistant Staphylococcus aureus have seriously threatened public health. There is an urgent need to propose an existing regimen to overcome multidrug resistance of MRSA. A unique class of novel anti-MRSA thiazolylketenyl quinazolinones (TQs) and their analogs were developed. Some synthesized compounds showed good bacteriostatic potency. Especially TQ 4 was found to exhibit excellent inhibition against MRSA with a low MIC of 0.5 µg/mL, which was 8-fold more effective than norfloxacin. The combination of TQ 4 with cefdinir showed stronger antibacterial potency. Further investigation revealed that TQ 4, with low hemolytic toxicity and low drug resistance, was not only able to inhibit biofilm formation but also could reduce MRSA metabolic activity and showed good drug-likeness. Mechanistic explorations revealed that TQ 4 could cause leakage of proteins by disrupting membrane integrity and block DNA replication by intercalated DNA. Furthermore, the synergistic antibacterial effect with cefdinir might be attributed to TQ 4 with the ability to induce PBP2a allosteric regulation of MRSA and further trigger the opening of the active site to promote the binding of cefdinir to the active site, thus inhibiting the expression of PBP2a, thereby overcoming MRSA resistance and significantly enhancing the anti-MRSA activity of cefdinir. A new strategy provided by these findings was that TQ 4, possessing both excellent anti-MRSA activity and allosteric effect of PBP2a, merited further development as a novel class of antibacterial agents to overcome increasingly severe MRSA infections.

Antibacterial sensitizers from natural plants: A powerful weapon against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a drug-resistant bacterium that can cause a range of infections with high morbidity and mortality, including pneumonia, etc. Therefore, development of new drugs or therapeutic strategies against MRSA is urgently needed. Increasing evidence has shown that combining antibiotics with "antibacterial sensitizers" which itself has no effect on MRSA, is highly effective against MRSA. Many studies showed the development of antibacterial sensitizers from natural plants may be a promising strategy against MRSA because of their low side effects, low toxicity and multi-acting target. In our paper, we first reviewed the resistance mechanisms of MRSA including "Resistance to Beta-Lactams", "Resistance to Glycopeptide antibiotics", "Resistance to Macrolides, Aminoglycosides, and Oxazolidinones" etc. Moreover, we summarized the possible targets for antibacterial sensitizers against MRSA. Furthermore, we reviewed the synergy effects of active monomeric compounds from natural plants combined with antibiotics against MRSA and their corresponding mechanisms over the last two decades. This review provides a novel approach to overcome antibiotic resistance in MRSA.

Sensitivity of the PBP2a SA Culture Colony Test on shortly incubated subcultures of methicillin-resistant staphylococci from positive blood cultures.

The sensitivity of an immunochromatographic assay for detecting methicillin resistance (PBP2a SA Culture Colony Test, Alere-Abbott) on shortly incubated subcultures of staphylococci in blood cultures was evaluated. The assay is highly sensitive for the detection of methicillin-resistant Staphylococcus aureus after 4 hour-subculture but requires 6 hour-incubation for methicillin-resistant coagulase-negative staphylococci.

Brazilian red propolis in combination with ß-lactams exerts an efficient antibacterial action over methicillin-resistant Staphylococcus aureus (MRSA) strains.

AIMS: The antibacterial activity of red propolis extract (RPE) and brown propolis extracts (BPE) and the synergistic effect of RPE with cefoxitin (CEFO), imipenem (IMI), and ertapenem (ERTA) was evaluated in vitro against methicillin-resistant Staphylococcus aureus (MRSA) strains. METHODS AND RESULTS: MRSA ATCC 33591, community-associated (CA-MRSA) USA300, and four clinical isolates were used. A broth microdilution assay was performed to obtain inhibitory and bactericidal concentrations of BPE, RPE, CEFO, IMI, and ERTA. RPE in combination with CEFO, IMI, and ERTA was evaluated on the formation or eradication of biofilm. The bacterial relative membrane conductivity of the strains was assessed after RPE and combinations exposition. Surface/binding computational analyzes between RPE compounds and penicillin binding protein 2a (PBP2a) were performed. BPE samples had no activity against MRSA (MICs 3.2-5 g l-1; MBCs 10-15 g l-1), so the subsequent assays were carried out only with RPE and antimicrobials. RPE exerted a bacteriostatic action (MICs 0.0156-0.125 g l-1; MBCs 0.5-2 g l-1) but the combinations with IMI and ERTA showed the highest inhibition, as observed in the time-kill curve. However, the FICI index showed synergism (≥0.5) only to RPE + IMI. This combination was the most effective in inhibiting the biofilm and showed the highest values of membrane conductivity. Computational predictions indicated that RPE constituents may interact with PBP2a. CONCLUSION: RPE and RPE + IMI exerted an antibacterial and antibiofilm activity on MRSA strains probably due to membrane/wall damage and interactions with PBP2a.

New sulphonamide-peptide hybrid molecules as potential PBP 2a ligands and methicillin resistant Staphylococcus aureus actives.

Penicillin binding protein 2a (PbP 2a) expression accounts for the insusceptibility of methicillin-resistant Staphylocuccus aureus (MRSA) to ß-lactam antibiotics. Here we employed computational strategies to challenge PbP 2a with series of fifty-five 'ala-ala' and 'ala-pro' sulphonamide-dipeptides. Binding stability of two compounds (labeled: 10i and 10n) with theoretical Ki in nM and µM ranges, for PbP 2a active and allosteric sites respectively, were investigated using molecular dynamics simulations. In addition, the results of the sensitivity of four strains of MRSA for compounds 10i and 10n obtained revealed the compounds at 10 µg/ml caused two isolates (S4 and S10) to revert to being susceptible. Finally, a reliable binding conformations of both compounds in the two binding sites of PbP 2a are described to provide rationale for structure-activity optimization of this series.Communicated by Ramaswamy H. Sarma.

WYBQ-4: a New Bactericidal Agent against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant pathogen that currently poses a serious threat to global health. Novel antimicrobial agents against MRSA are urgently being developed. In this study, we investigated WYBQ-4, which is an effective antibacterial agent with potent bactericidal activity and bactericidal efficiency against MRSA USA300 and clinical isolate strains. In addition, WYBQ-4 exhibited low cytotoxicity without hemolytic activity according to a safety evaluation. Importantly, WYBQ-4 showed potent in vivo efficacy in an MRSA-induced mouse pneumonia model, systemic infection model, and intramuscular infection model. The efficacy of this new cephalosporin against MRSA was associated with a high affinity for penicillin-binding proteins (PBP1, PBP2, PBP3, PBP4, PBP2a) evaluated in a competition assay using bocillin as a reporter. In conclusion, WYBQ-4 has a significant bactericidal effect in vitro and in vivo, indicating that it is a promising compound to control MRSA infection. IMPORTANCE Antibiotic resistance is spreading faster than the introduction of new compounds into clinical practice, causing a public health crisis. Novel antimicrobial agents against MRSA are urgently being developed. In this study, we investigated WYBQ-4, which is an effective antibacterial agent with potent bacteriostatic activity and bactericidal efficiency against MRSA USA300 and clinical isolate strains. WYBQ-4 showed potent in vivo efficacy in MRSA-induced mouse models. Subsequently, we further revealed its antibacterial mechanism. In conclusion, WYBQ-4 has a significant bactericidal effect in vitro and in vivo, indicating that it is a promising compound to control MRSA infection.

Synergistic Antibacterial Activity with Conventional Antibiotics and Mechanism of Action of Shikonin against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a troublesome pathogen that poses a global threat to public health. Shikonin (SKN) isolated from Lithospermum erythrorhizon (L. erythrorhizon) possesses a variety of biological activities. This study aims to explore the effect of the combined application of SKN and traditional antibiotics on the vitality of MRSA and the inherent antibacterial mechanism of SKN. The synergies between SKN and antibiotics against MRSA and its clinical strain have been demonstrated by the checkerboard assay and the time-kill assay. The effect of SKN on disrupting the integrity and permeability of bacterial cell membranes was verified by a nucleotide and protein leakage assay and a bacteriolysis assay. As determined by crystal violet staining, SKN inhibited the biofilm formation of clinical MRSA strains. The results of Western blot and qRT-PCR showed that SKN could inhibit the expression of proteins and genes related to drug resistance and S. aureus exotoxins. SKN inhibited the ability of RAW264.7 cells to release the pro-inflammatory cytokines TNF-α and IL-6, as measured by ELISA. Our findings suggest that SKN has the potential to be developed as a promising alternative for the treatment of MRSA infections.

Recombinant PBP2a/autolysin conjugate as PLGA-based nanovaccine induced humoral responses with opsonophagocytosis activity, and protection versus methicillin-resistant Staphylococcus aureus infection.

Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) reasons extreme infections, can resist various conventional antimicrobial agents, and cause morbidity and mortality worldwide. Vaccination seems to help modulate MRSA infections. Nanovaccine is considered a novel strategy in vaccine technology. The primary purpose of the present study was to develop a conjugate vaccine based on recombinant PBP2a and MRSA autolysin formulated in PLGA as a nanoparticle capable of enhancing protective responses against MRSA in the murine model. Materials and Methods: Recombinant PBP2a and autolysin have been expressed and purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity column and characterized by SDS-PAGE and western blot. PLGA was bound to recombinant proteins by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and adipic acid dihydrazide (ADH) as a linker and spacer, respectively. Conjugation of recombinant proteins to PLGA was confirmed by the AFM assay, zeta potential, and size distribution, and its efficacy was evaluated in mice. Total IgG, IgG1, IgG2a, IgG2b, and IgM titers were analyzed to assess immune responses. Lastly, the bioactivity of antibodies was tested by using the opsonophagocytosis assay. Results: Mice immunized with the r-PBP2a-r-autolysin-PLGA nanovaccine led to increased levels of opsonic antibodies and IgG1, IgG2a, IgG2b, and IgM when compared with other experimental groups. Our results confirmed that vaccination with nanovaccine could reduce the mortality rate against the sub-lethal dose of MRSA challenge. Furthermore, the nanovaccine could eliminate MRSA from the kidney of infected mice. Conclusion: This study may provide valuable insights into the protective power of the r-PBP2a-r-autolysin-PLGA conjugate vaccine against MRSA infection.

Prevalence, Mechanism, Genetic Diversity, and Cross-Resistance Patterns of Methicillin-Resistant Staphylococcus Isolated from Companion Animal Clinical Samples Submitted to a Veterinary Diagnostic Laboratory in the Midwestern United States.

Methicillin-resistant Staphylococcus (MRS) is a leading cause of skin and soft tissue infections in companion animals, with limited treatment options available due to the frequent cross-resistance of MRS to other antibiotics. In this study, we report the prevalence, species distribution, genetic diversity, resistance mechanism and cross-resistance patterns of MRS isolated from companion animal (mostly dog and cat) clinical cases submitted to Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) between 2012 and 2019. The majority of isolates were identified as Staphylococcus pseudintermedius (68.3%; 2379/3482) and coagulase-negative Staphylococcus (CoNS) (24.6%; 857/3482), of which 23.9% and 40.5% were phenotypically resistant to methicillin, respectively. Cross resistance to other ß-lactams (and to a lesser extent to non-ß-lactams) was common in both methicillin-resistant S. pseudintermedius (MRSP) and CoNS (MRCoNS), especially when oxacillin MIC was ≥4 µg/mL (vs. ≥0.5−<4 µg/mL). The PBP2a protein was detected by agglutination in 94.6% (521/551) MRSP and 64.3% (146/227) MRCoNS. A further analysis of 31 PBP2a-negative MRS isolates (all but one MRCoNS) indicated that 11 were mecA gene-positive while 20 were negative for mecA and other mec genes by PCR. The resistance to last-resort anti-staphylococcal human drugs (e.g., tigecycline, linezolid, vancomycin) among the MRS tested was none to very low. Even though genotyping indicated an overall high level of genetic diversity (87 unique PFGE patterns and 20 MLST types) among a subset of MRSP isolates tested (n = 106), certain genotypes were detected from epidemiologically connected cases at the same or different time points, suggesting persistence and/or nosocomial transmission. These results indicate a relatively high prevalence of MRS from companion animals in the Midwestern US; therefore, it is important to perform routine susceptibility testing of Staphylococcus in veterinary clinical settings for the selection of appropriate antimicrobial therapy.

The NaHCO3-Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus (MRSA) Is Influenced by mecA Genotype.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a leading cause of many invasive clinical syndromes, and pose treatment difficulties due to their in vitro resistance to most ß-lactams on standard laboratory testing. A novel phenotype frequently identified in MRSA strains, termed 'NaHCO3-responsiveness', is a property whereby strains are susceptible in vitro to many ß-lactams in the presence of NaHCO3. Specific mecA genotypes, repression of mecA/PBP2a expression and perturbed maturation of PBP2a by NaHCO3 have all been associated with this phenotype. The aim of this study was to define the relationship between specific mecA genotypes and PBP2a substitutions, on the one hand, with NaHCO3-responsiveness in vitro. Mutations were made in the mecA ribosomal binding site (RBS -7) and at amino acid position 246 of its coding region in parental strains MW2 (NaHCO3-responsive) and C36 (NaHCO3- nonresponsive) to generate 'swap' variants, each harboring the other's mecA-RBS/coding region genotypes. Successful swaps were confirmed by both sequencing, as well as predicted swap of in vitro penicillin-clavulanate susceptibility phenotypes. MW2 swap variants harboring the nonresponsive mecA genotypes became NaHCO3-nonresponsive (resistant to the ß-lactam, oxacillin [OXA]), in the presence of NaHCO3. Moreover, these swap variants had lost NaHCO3-mediated repression of mecA/PBP2a expression. In contrast, C36 swap variants harboring the NaHCO3-responsive mecA genotypes remained NaHCO3-nonresponsive phenotypically, and still exhibited nonrepressible mecA/PBP2a expression. These data demonstrate that in addition to the mecA genotype, NaHCO3-responsiveness may also depend on strain-specific genetic backgrounds.

Pyridine coupled pyrazole analogues as lethal weapon against MRSA: An in-vitro and in-silico approach.

The treatment of Methicillin-resistant staphylococcus aureus (MRSA) infections has become challenging due to the growth of multidrug resistance in the bacteria. Here we report the synthesis of pyridine-coupled pyrazoles as an antimicrobial agent against MRSA. A series of pyridine coupled pyrazoles were synthesized and synthesized compounds were characterized using FT-IR, 1H NMR, and Mass spectroscopy. The ADMET results of all the 14 active compounds are interpreted. To identify the potent compound the synthesized compounds screened for minimum inhibitory concentrations against MRSA and compared with standard drug vancomycin. Among the synthesized compounds 6d exhibited good antibacterial activity with MIC value 21 µg/mL, bacterial cell membrane damage study was studied potassium efflux, and cellular content leakage assay. Anticoagulant study for the potent compound also studied and validated by molecular docking and molecular dynamics simulation studies. The docking study of the synthesized compound was carried out and the study depicted that the pyridine ring of all the analogues binds with the various amino acids in the binding pocket of the active site of the Staphylocoagulase and PBP2a protein of MRSA.