Methicillin-resistant Staphylococcus Aureus Cell Research Articles

Catalyst-free regeneration of plasma-activated water via ultrasonic cavitation: Removing aggregation concealment of antibiotic-resistant bacteria with enhanced wastewater sustainability

Aggregation is a crucial factor in bacterial biofilm formation, and comprehending its properties is vital for managing waterborne antibiotic-resistant bacteria. In this study, we examined Methicillin-resistant Staphylococcus aureus (MRSA) cell aggregation under varying conditions and assessed the inactivation efficiency of a novel disinfection method, micro-nano bubbles plasma-activated water via ultrasonic stirring cavitation (MPAW-US), on aggregated MRSA cells. Aggregation efficiency increased over time and at low salt concentrations but diminished at higher concentrations. Elevated MRSA cell aggregation in actual water samples represented significant real-life biohazard risks. Unlike conventional disinfection, MPAW-US treatment exhibited minimal change in the inactivation rate constant despite protective outer layers. Enhanced inactivation efficiency results from the synergistic effects of increased intracellular oxidative stress damage and extracellular substance disruption, triggered by ultrasound-activated micro-nano bubbles that improve PAW reactivity and applicability. This approach neither induced MRSA cross-resistance to unfavorable conditions nor increased toxicity or regrowth potential of aggregative MRSA, utilizing ATP levels as potential regrowth capability indicators. Ultimately, this energy-efficient disinfection technology functions effectively across diverse temperature ranges, showcasing exceptional sterilization and nutritional bean sprout production after cyclic filtering, thereby promoting wastewater sustainability amidst carbon emission concerns.

Napthalimide-based nuclease inhibitor: A multifunctional therapeutic material to bolster MRSA uptake by macrophage-like cells and mitigate pathogen adhesion on orthopaedic implant

The healthcare burden rendered by methicillin-resistant Staphylococcus aureus (MRSA) warrants the development of therapeutics that offer a distinct benefit in the clinics as compared to conventional antibiotics. The present study describes the potential of napthalimide-based synthetic ligands (C1-C3) as inhibitors of the staphylococcal nuclease known as micrococcal nuclease (MNase), a key virulence factor of the pathogen. Amongst the ligands, the most potent MNase inhibitor C1 rendered non-competitive inhibition, reduced MNase turnover number (Kcat) and catalytic efficiency (Kcat/Km) with an IC50 value of ~950 nM. CD spectroscopy suggested distortion of MNase conformation in presence of C1. Flow cytometry and confocal microscopy indicated that C1 restored the ability of activated THP-1 cells to engulf DNA-entrapped MRSA cells. Interestingly, C1 could inhibit MRSA adhesion onto collagen. For potential application, C1-loaded pluronic F-127 micellar nanocarrier (C1-PMC) was generated, wherein the anti-adhesion activity of the pluronic carrier (PMC) and C1 was harnessed in tandem to deter MRSA cell adhesion onto collagen. MRSA biofilm formation was hindered on C1-PMC-coated titanium (Ti) wire, while eluates from C1-PMC-coated Ti wires were non-toxic to HEK 293, MG-63 and THP-1 cells. The multifunctional C1 provides a blueprint for designing therapeutic materials that hold translational potential for mitigation of MRSA infections.

Cobalt pyridyl complexes against drug-resistant bacteria: Synthesis, characterization, antibacterial properties, mode of action, and molecular docking studies

This study describes the synthesis and characterization of four cobalt complexes using diverse physicochemical techniques. Notably, the co-grinding method was predominantly employed, resulting in higher yields and increased purity of the products. Thermal studies provided insights into the stability of the cobalt complexes, with dichoro-bis-4-((4-nitrophenoxy)methyl)pyridine cobalt(II) (CoL4) demonstrating the highest stability based on transformations and high melting points. High resolution electrospray ionization mass spectrometry (HRESI-MS) analysis further confirmed the successful synthesis of the cobalt complexes, reinforcing the characterization results. The dichloro-bis(pyridin-4ylmethyl-4aminobenzoate) cobalt (II) (CoL3) exhibited a cis tetrahedral structure with a high-symmetry monoclinic crystal system and a primitive lattice centering of an I2/a space group. The cobalt complexes displayed enhanced activity against Gram-negative bacteria, particularly Klebsiella pneumoniae, with some exhibiting potent bacteriostatic effects against Multidrug resistant K. pneumoniae (MDR-K. pneumoniae). Additionally, dichoro-bis-(4-[(pyridin-4-yl)methoxy] aniline) cobalt(II) (CoL2) and CoL3 were found to disrupt the folate biosynthesis of K. pneumoniae and Methicillin resistant Staphylococcus aureus (MRSA), respectively. Moreover, 4-chloromethylpyridinium tetrachlorocobaltate (II) (Co4PY-H), dichloro-bis-4-picolyl cobalt (II) (Co4PY), CoL2, and CoL3 showed significant disruption of the cell membrane integrity of K. pneumoniae, with CoL3 also affecting MRSA's cell membrane. Lastly, molecular docking calculations provided compelling evidence supporting the experimental modes of action of these active complexes.

Specifically targeted antimicrobial peptides synergize with bacterial-entrapping peptide against systemic MRSA infections

IntroductionThe design of precision antimicrobials aims to personalize the treatment of drug-resistant bacterial infections and avoid host microbiota dysbiosis. ObjectivesThis study aimed to propose an efficient de novo design strategy to obtain specifically targeted antimicrobial peptides (STAMPs) against methicillin-resistant Staphylococcus aureus (MRSA). MethodsWe evaluated three strategies designed to increase the selectivity of antimicrobial peptides (AMPs) for MRSA and mainly adopted an advanced hybrid peptide strategy. First, we proposed a traversal design to generate sequences, and constructed machine learning models to predict the anti-S. aureus activity levels of unknown peptides. Subsequently, six peptides were predicted to have high activity, among which, a broad-spectrum AMP (P18) was selected. Finally, the two targeting peptides from phage display libraries or lysostaphin were used to confer specific anti-S. aureus activity to P18. STAMPs were further screened out from hybrid peptides based on their in vitro and in vivo activities. ResultsAn advanced hybrid peptide strategy can enhance the specific and targeted properties of broad-spectrum AMPs. Among 25 assessed peptides, 10 passed in vitro tests, and two peptides containing one bacterial-entrapping peptide (BEP) and one STAMP passed an in vivo test. The lead STAMP (P18E6) disrupted MRSA cell walls and membranes, eliminated established biofilms, and exhibited desirable biocompatibility, systemic distribution and efficacy, and immunomodulatory activity in vivo. Furthermore, a bacterial-entrapping peptide (BEP, SP5) modified from P18, self-assembled into nanonetworks and rapidly entrapped MRSA. SP5 synergized with P18E6 to enhance antibacterial activity in vitro and reduced systemic MRSA infections. ConclusionsThis strategy may aid in the design of STAMPs against drug-resistant strains, and BEPs can serve as powerful STAMP adjuvants.

Medicinal Remedies for the Treatment of Methicillin Resistant Staphylococcus Aureus

Antimicrobial resistance has led to the rise of formidable microbes like methicillin-resistant Staphylococcus aureus (MRSA). This infection can be acquired at the hospital and in a community, making the search for efficient treatment critical, given the rise in inefficacy of conventional treatments such as vancomycin. Phytocompounds can offer protection against these threats. They not only have the ability to kill MRSA but also enhance its susceptibility to previously ineffective antimicrobial agents. Plant-derived anti-MRSA agents can produce therapeutic effects comparable to some conventional treatments, and in some cases, they may even outperform an antibiotic. This article provided many examples of botanical compounds that have proven potent agents against MRSA in both in vitro and in vivo studies. These compounds achieve these effects by inhibiting the resistance mechanisms in MRSA, breaking down its defenses, making it vulnerable, and eventually killing it. Many of these compounds are inherently bactericidal; however, they may produce a synergistic response when combined with allopathic antibiotics. For example, plumbagin, from Plumbago zeylanica, not only disrupts MRSA cell wall morphology, eliciting cytoplasmic changes but also produces a synergistic effect with ciprofloxacin and piperacillin. Moreover, honokiol and magnolol glycosides, from Magnolia officinalis, reversed the quintessential resistance of MRSA by repressing genes, responsible for resistance development. The possibilities are indeed endless, but further research and financial investments are required for proper drug development.

COMBATTING METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) IN THE FOOD INDUSTRY BY HARNESSING THE POWER OF NATURE: A SYSTEMATIC REVIEW

Antibiotic resistance is a critical global health concern, with Methicillin-resistant Staphylococcus aureus (MRSA) posing a significant challenge due to its resistance to commonly used antibiotics. Recent research has revealed the potential of natural compounds and microorganisms in combatting MRSA and other antibiotic-resistant bacteria. In this systematic review, we studied the effect of essential oils, bacteriophages, bacteriocins, and probiotics on S. aureus, including MRSA in particular, in the food industry. Essential oils (EOs) have gained significant attention because of their antimicrobial properties, inhibiting MRSA growth by damaging bacterial cells and inhibiting essential enzymes and compounds. Cinnamon oil liposomes caused the most significant decrease in MRSA populations among our reviewed essential oils. Bacteriophages can lyse the bacterial host. They encode peptidoglycan hydrolases called endolysins that target the bacterial cell wall. In our study, S. aureus phage (containing CHAPLysGH15 and LysGH15), and phage SA11 endolysin (LysSA11) were the most effective against S. aureus. Bacteriocins, antimicrobial peptides produced by bacteria, also show potential in combatting MRSA, mainly by generating organic acids that interfere with bacterial metabolism. According to our review, the most effective bacteriocins against S. aureus were Enterocin AS-48 with phenolic compounds or with 2NPOH, Bacteriocin isolated from Lactobacillus pentosus - Pentocin JL-1, and bacteriocin produced by S. pasteuri RSP-1, respectively. Probiotics can compete with pathogens by producing antimicrobial compounds that disrupt MRSA cell production and ultimately lead to bacterial death. In our review, the most effective probiotics were Streptomyces griseus, Pediococcus acidilactici strains A11 and C12, Lactococcus lactis, and Lactobionic acid respectively. A multi-hurdle approach combining these natural agents has shown promising results in targeting and eliminating MRSA cells. By harnessing the power of nature, we can potentially overcome the challenges posed by MRSA and other antibiotic-resistant bacteria.

Smartphone-triggered targeted inactivation of MRSA under SERS monitoring

Silver nanoparticles-based multifunctional agents show tremendous potential for simultaneous imaging and against the methicillin-resistant Staphylococcus aureus (MRSA)-induced superficial infection. However, it is still a challenge to achieve a balance between high precision, high antibacterial efficiency, and biocompatibility. Herein, we present a 4-mercaptophenylboronic acid (4-MPBA)-modified Ag2CO3/Ag2O nano-heterojunctions (AgH@M) that can recognize single MRSA cell by surface-enhanced Raman scattering (SERS) and effectively eradicate MRSA under smartphone irradiation. Moreover, the 4-MPBA molecule acts as triple identities for MRSA targeted binding, SERS tag, and inhibition of excessive release of toxic silver ions for the Raman Imaging guided bacterial treatment. AgH@M simultaneously targeted binding on the surface of bacteria and made full use of the released limited Ag+ into the MRSA, thereby reducing the toxicity to mammalian cells. In addition, the potential antibacterial mechanism involves ROS production from photocatalytic behavior, interference with functional genes of quorum sensing, virulence, biofilm, and ATP-binding cassette transporter. Also, the AgH@M can monitor the residual bacteria in MRSA-infected wounds and keratitis animal models in real-time for at least 7 days by SERS and accomplishes a satisfactory therapeutic effect. This work may provide promising visualized heterojunctions for the targeted eradication of MRSA with a portable light source.

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 β-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-β-lactam allosteric inhibitors, 4-(3H)-Quinazolinones, Pyrrolylated chalcone, Bis-2-Oxoazetidinyl macrocycles (β-lactam antibiotics with 1,3-Bridges), Macrocycle-embedded β-lactams as novel inhibitors, Pyridine-Coupled Pyrimidinones, novel Naphthalimide corbelled aminothiazoximes, non-covalent inhibitors, Investigational-β-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.

Quinoxaline-based membrane-targeting therapeutic material: Implications in rejuvenating antibiotic and curb MRSA invasion in an in vitro bone cell infection model.

Manifestation of resistance in methicillin-resistant Staphylococcus aureus (MRSA) against multiple antibiotics demands an effective strategy to counter the menace of the pathogen. To address this challenge, the current study explores quinoxaline-based synthetic ligands as an adjuvant material to target MRSA in a combination therapy regimen. Amongst the tested ligands (C1-C4), only C2 was bactericidal against the MRSA strain S. aureus 4s, with a minimum inhibitory concentration (MIC) of 32μM. C2 displayed a membrane-directed activity and could effectively hinder MRSA biofilm formation. A quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that C2 downregulated expression of the regulator gene agrC and reduced the fold change in the expression of adhesin genes fnbA and cnbA in MRSA in a dose-dependent manner. C2 enabled a 4-fold reduction in the MIC of ciprofloxacin (CPX) and in presence of 10μM C2 and 8.0μM CPX, growth of MRSA was arrested. Furthermore, a combination of 10μM C2 and 12μM CPX could strongly inhibit MRSA biofilm formation and reduce biofilm metabolic activity. The minimum biofilm inhibitory concentration (MBIC) of CPX against S. aureus 4s biofilm was reduced and a synergy resulted between C2 and CPX. In a combinatorial treatment regimen, C2 could prevent emergence of CPX resistance and arrest growth of MRSA till 360 generations. C2 could also be leveraged in combination treatment (12μM CPX and 10μM C2) to target MRSA in an in vitro bone cell infection model, wherein MRSA cell adhesion and invasion onto cultured MG-63 cells was only ~17% and~0.37%, respectively. The combinatorial treatment regimen was also biocompatible as the viability of MG-63 cells was high (~ 91%). Thus, C2 is a promising adjuvant material to counter antibiotic-refractory therapy and mitigate MRSA-mediated bone cell infection.

Evaluation of Antibacterial Activity of Thiourea Derivative TD4 against Methicillin-Resistant Staphylococcus aureus via Destroying the NAD+/NADH Homeostasis

To develop effective agents to combat bacterial infections, a series of thiourea derivatives (TDs) were prepared and their antibacterial activities were evaluated. Our results showed that TD4 exerted the most potent antibacterial activity against a number of Staphylococcus aureus (S. aureus), including the methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis and Enterococcus faecalis strains, with the minimum inhibitory concentration (MIC) at 2-16 µg/mL. It inhibited the MRSA growth curve in a dose-dependent manner and reduced the colony formation unit in 4× MIC within 4 h. Under the transmission electron microscope, TD4 disrupted the integrity of MRSA cell wall. Additionally, it reduced the infective lesion size and the bacterial number in the MRSA-induced infection tissue of mice and possessed a good drug likeness according to the Lipinski rules. Our results indicate that TD4 is a potential lead compound for the development of novel antibacterial agent against the MRSA infection.

The Synergistic Antimicrobial Effect and Mechanism of Nisin and Oxacillin against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for skin and soft tissue infections with multi-resistance to many antibiotics. It is thus imperative to explore alternative antimicrobial treatments to ensure future treatment options. Nisin (NIS), an antibacterial peptide produced by Lactococcus lactis, was selected to combine with Oxacillin (OX), to evaluate the antimicrobial effect and potential mechanism against MRSA. The synergistic antimicrobial effect of OX and NIS was verified by Minimal Inhibitory Concentration (MIC) assays, checkerboard analysis, time-kill curve, biofilm producing ability, and mice skin infection model in vivo. For the potential synergistic antimicrobial mechanism, the microstructure and integrity change of MRSA cells were determined by Scanning and Transmission Electron Microscope (SEM and TEM), intracellular alkaline phosphatase activity and propidium iodide staining were assayed; And transcription of mecA, main gene of MRSA resistant to OX, were detected by qRT-PCR. The results showed NIS could restore the sensitivity of MRSA to OX and inhibit biofilm production; OX + NIS can make MRSA cell deform; NIS may recover OX sensitivity by inhibiting the transcription of mecA. In vivo, mice skin infection models indicate that OX + NIS can substantially alleviate MRSA infections. As a safe commercially available biological compound, NIS and the combination of antibiotics are worth developing as new anti-MRSA biomaterials.

Structural and Antibacterial Characterization of a New Benzamide FtsZ Inhibitor with Superior Bactericidal Activity and In Vivo Efficacy Against Multidrug-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant (MDR) bacterial pathogen of acute clinical significance. Resistance to current standard-of-care antibiotics, such as vancomycin and linezolid, among nosocomial and community-acquired MRSA clinical isolates is on the rise. This threat to global public health highlights the need to develop new antibiotics for the treatment of MRSA infections. Here, we describe a new benzamide FtsZ inhibitor (TXH9179) with superior antistaphylococcal activity relative to earlier-generation benzamides like PC190723 and TXA707. TXH9179 was found to be 4-fold more potent than TXA707 against a library of 55 methicillin-sensitive S. aureus (MSSA) and MRSA clinical isolates, including MRSA isolates resistant to vancomycin and linezolid. TXH9179 was also associated with a lower frequency of resistance relative to TXA707 in all but one of the MSSA and MRSA isolates examined, with the observed resistance being due to mutations in the ftsZ gene. TXH9179 induced changes in MRSA cell morphology, cell division, and FtsZ localization are fully consistent with its actions as a FtsZ inhibitor. Crystallographic studies demonstrate the direct interaction of TXH9179 with S. aureus FtsZ (SaFtsZ), while delineating the key molecular contacts that drive complex formation. TXH9179 was not associated with any mammalian cytotoxicity, even at a concentration 10-fold greater than that producing antistaphylococcal activity. In serum, the carboxamide prodrug of TXH9179 (TXH1033) is rapidly hydrolyzed to TXH9179 by serum acetylcholinesterases. Significantly, both intravenously and orally administered TXH1033 exhibited enhanced in vivo efficacy relative to the carboxamide prodrug of TXA707 (TXA709) in treating a mouse model of systemic (peritonitis) MRSA infection. Viewed as a whole, our results highlight TXH9179 as a promising new benzamide FtsZ inhibitor worthy of further development.

Novel copper-containing ferrite nanoparticles exert lethality to MRSA by disrupting MRSA cell membrane permeability, depleting intracellular iron ions, and upregulating ROS levels.

The widespread use of antibiotics has inevitably led to the emergence of multidrug-resistant bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA), making treatment of this infection a serious challenge. This study aimed to explore new treatment strategies for MRSA infection. The structure of Fe3O4 NPs with limited antibacterial activity was optimized, and the Fe2+ ↔ Fe3+ electronic coupling was eliminated by replacing 1/2 Fe2+ with Cu2+. A new type of copper-containing ferrite nanoparticles (hereinafter referred to as Cu@Fe NPs) that fully retained oxidation-reduction activity was synthesized. First, the ultrastructure of Cu@Fe NPs was examined. Then, antibacterial activity was determined by testing the minimum inhibitory concentration (MIC) and safety for use as an antibiotic agent. Next, the mechanisms underlying the antibacterial effects of Cu@Fe NPs were investigated. Finally, mice models of systemic and localized MRSA infections was established for in vivo validation. It was found that Cu@Fe NPs exhibited excellent antibacterial activity against MRSA with MIC of 1 μg/mL. It effectively inhibited the development of MRSA resistance and disrupted the bacterial biofilms. More importantly, the cell membranes of MRSA exposed to Cu@Fe NPs underwent significant rupture and leakage of the cell contents. Cu@Fe NPs also significantly reduced the iron ions required for bacterial growth and contributed to excessive intracellular accumulation of exogenous reactive oxygen species (ROS). Therefore, these findings may important for its antibacterial effect. Furthermore, Cu@Fe NPs treatment led to a significant reduction in colony forming units within intra-abdominal organs, such as the liver, spleen, kidney, and lung, in mice with systemic MRSA infection, but not for damaged skin in those with localized MRSA infection. The synthesized nanoparticles has an excellent drug safety profile, confers high resistant to MRSA, and can effectively inhibit the progression of drug resistance. It also has the potential to exert anti-MRSA infection effects systemically in vivo. In addition, our study revealed a unique multifaceted antibacterial mode of Cu@Fe NPs: (1) an increase in cell membrane permeability, (2) depletion of Fe ions in cells, (3) generation of ROS in cells. Overall, Cu@Fe NPs may be potential therapeutic agents for MRSA infections.

Antibacterial activity and synergistic antibiotic mechanism of trialdehyde phloroglucinol against methicillin-resistant Staphylococcus aureus.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) has become a critical global concern. Identifying new anti-S. aureus agents or therapeutic strategies are urgently needed to treat S. aureus infection. The present study investigated the antibacterial activity of 16 phenolic compounds against MRSA, four of which exhibited antibacterial activity. Their antibacterial activities increased in a dose-dependent manner but showed different responses with the extension of treatment time. Trialdehyde phloroglucinol (TPG) and 2-nitrophloroglucinol (NPG) maintained stable antibacterial activity; however, that of dichlorophenol and myricetin decreased rapidly over 24 hr of treatment. Checkerboard and time-kill assays indicated that TPG and NPG exhibited strong synergistic antibacterial activities with penicillin or bacitracin. Microscopic observation and membrane integrity analysis showed that the combination of TPG and penicillin destroyed the MRSA cell membrane, resulting in the leakage of intracellular biomacromolecules, marked changes in surface zeta potential, and the collapse of membrane potential. Moreover, the combination significantly decreased penicillinase activity and penicillin-binding protein 2a mRNA expression, inhibiting MRSA growth. Taken together, these results demonstrated that the combination of the phloroglucinol derivative TPG and penicillin has significant synergistic anti-MRSA activity and can serve as a potential therapeutic strategy to treat MRSA infections.

Proton Motive Force Inhibitors Are Detrimental to Methicillin-Resistant Staphylococcus aureus Strains.

ABSTRACTMethicillin-resistant Staphylococcus aureus (MRSA) strains are tolerant of conventional antibiotics, making them extremely dangerous. Previous studies have shown the effectiveness of proton motive force (PMF) inhibitors at killing bacterial cells; however, whether these agents can launch a new treatment strategy to eliminate antibiotic-tolerant cells mandates further investigation. Here, using known PMF inhibitors and two different MRSA isolates, we showed that the bactericidal potency of PMF inhibitors seemed to correlate with their ability to disrupt PMF and permeabilize cell membranes. By screening a small chemical library to verify this correlation, we identified a subset of chemicals (including nordihydroguaiaretic acid, gossypol, trifluoperazine, and amitriptyline) that strongly disrupted PMF in MRSA cells by dissipating either the transmembrane electric potential (ΔΨ) or the proton gradient (ΔpH). These drugs robustly permeabilized cell membranes and reduced MRSA cell levels below the limit of detection. Overall, our study further highlights the importance of cellular PMF as a target for designing new bactericidal therapeutics for pathogens.IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) emerged as a major hypervirulent pathogen that causes severe health care-acquired infections. These pathogens can be multidrug-tolerant cells, which can facilitate the recurrence of chronic infections and the emergence of diverse antibiotic-resistant mutants. In this study, we aimed to investigate whether proton motive force (PMF) inhibitors can launch a new treatment strategy to eliminate MRSA cells. Our in-depth analysis showed that PMF inhibitors that strongly dissipate either the transmembrane electric potential or the proton gradient can robustly permeabilize cell membranes and reduce MRSA cell levels below the limit of detection.

18β-Glycyrrhetinic Acid Induces Metabolic Changes and Reduces Staphylococcus aureus Bacterial Cell-to-Cell Interactions.

The rise in bacterial resistance to common antibiotics has raised an increased need for alternative treatment strategies. The natural antibacterial product, 18β-glycyrrhetinic acid (GRA) has shown efficacy against community-associated methicillin-resistant Staphylococcus aureus (MRSA), although its interactions against planktonic and biofilm modes of growth remain poorly understood. This investigation utilized biochemical and metabolic approaches to further elucidate the effects of GRA on MRSA. Prolonged exposure of planktonic MRSA cell cultures to GRA resulted in increased production of staphyloxanthin, a pigment known to exhibit antioxidant and membrane-stabilizing functions. Then, 1D 1H NMR analyses of intracellular metabolite extracts from MRSA treated with GRA revealed significant changes in intracellular polar metabolite profiles, including increased levels of succinate and citrate, and significant reductions in several amino acids, including branch chain amino acids. These changes reflect the MRSA response to GRA exposure, including potentially altering its membrane composition, which consumes branched chain amino acids and leads to significant energy expenditure. Although GRA itself had no significant effect of biofilm viability, it seems to be an effective biofilm disruptor. This may be related to interference with cell–cell aggregation, as treatment of planktonic MRSA cultures with GRA leads to a significant reduction in micro-aggregation. The dispersive nature of GRA on MRSA biofilms may prove valuable for treatment of such infections and could be used to increase susceptibility to complementary antibiotic therapeutics.

Leucyl-tRNA Synthetase Inhibitor, D-Norvaline, in Combination with Oxacillin, Is Effective against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium that causes severe diseases in humans. For decades, MRSA has acquired substantial resistance against conventional antibiotics through regulatory adaptation, thereby posing a challenge for treating MRSA infection. One of the emerging strategies to combat MRSA is the combinatory use of antibacterial agents. Based on the dramatic change in phospholipid fatty acid (PLFA) composition of MRSA in previous results, this study investigated branched-chain amino acid derivatives (precursors of fatty acid synthesis of cell membrane) and discovered the antimicrobial potency of D-norvaline. The compound, which can act synergistically with oxacillin, is among the three leucine-tRNA synthetase inhibitors with high potency to inhibit MRSA cell growth and biofilm formation. PLFA analysis and membrane properties revealed that D-norvaline decreased the overall amount of PLFA, increasing the fluidity and decreasing the hydrophobicity of the bacterial cell membrane. Additionally, we observed genetic differences to explore the response to D-norvaline. Furthermore, deletion mutants and clinically isolated MRSA strains were treated with D-norvaline. The study revealed that D-norvaline, with low concentrations of oxacillin, was effective in killing several MRSA strains. In summary, our findings provide a new combination of aminoacyl-tRNA synthetase inhibitor D-norvaline and oxacillin, which is effective against MRSA.

Urea-Based Ligand as an Efflux Pump Inhibitor: Warhead to Counter Ciprofloxacin Resistance and Inhibit Collagen Adhesion by MRSA

Methicillin-resistant Staphylococcus aureus (MRSA) is a frontline human pathogen in which efflux pump activity confers high levels of antibiotic-resistance and poses a therapeutic challenge in the clinics. The present study illustrates the potential of urea-based ligand as an efflux pump inhibitor (EPI) in order to restore the efficacy of ciprofloxacin (CPX) against MRSA. Among eight structurally varying urea-based ligands, the ligand C8 could significantly inhibit efflux pump activity in the clinical MRSA strain S. aureus 4s and was superior to the known EPI reserpine. In combinatorial treatment, C8 enhanced cellular accumulation of CPX, rendered a 16× decrease in the MIC of CPX, and restored the susceptibility of S. aureus 4s to CPX. Notably, C8 downregulated the expression of norA gene coding for the efflux pump in MRSA and treatment with 10 μM C8 and 2.0 μM CPX prevented emergence of the CPX resistance trait and suppressed MRSA cell growth till 120 generations. For potential anti-MRSA therapy, C8-loaded poly(d,l-lactide-co-glycolide) nanocarrier (C8-PNC) was generated, which facilitated facile release of C8 in physiologically relevant fluid. C8-PNC (loaded with 50 μM C8) rendered efflux pump inhibition and eliminated MRSA in combination with only 2.0 μM CPX. Treatment with the non-toxic C8-PNC (loaded with 50 μM C8) and CPX (2.0 μM) also hindered MRSA adhesion on collagen manifold higher as compared to cells treated with 32 μM CPX and significantly downregulated norA gene expression in non-adhered MRSA cells. The urea-based ligand presented herein is a promising biocompatible therapeutic material for effective mitigation of MRSA infections.

Penicillin-binding proteins (PBPs) determine antibiotic action in Langmuir monolayers as nanoarchitectonics mimetic membranes of methicillin-resistant Staphylococcus aureus

The membrane of methicillin-resistant Staphylococcus aureus (MRSA) contains penicillin-binding proteins (PBPs) in the phospholipidic bilayer, with the protein PBP2a being linked with the resistance mechanism. In this work we confirm the role of PBP2a with molecular-level information obtained with Langmuir monolayers as cell membrane models. The MRSA cell membrane was mimicked with a mixed monolayer of dipalmitoyl phosphatidyl glycerol (DPPG) and cardiolipin (CL), also incorporating PBP2a. The surface pressure-area isotherms and the Brewster angle microscopy (BAM) images for these mixed monolayers were significantly affected by the antibiotic meropenem, which is PBP2a inhibitor. The meropenem effects were associated with the presence of PBP2a, as they were absent in the Langmuir monolayers without PBP2a. The relevance of PBP2a was confirmed with results where the antibiotic methicillin, known to be unsuitable to kill MRSA, had the same effects on mixed DPPG/CL and DPPG/CL-PBP2a monolayers since it prevented PBP2a from incorporating in the monolayer. The biological implication of the findings presented here is that a successful antibiotic against MRSA should be able to interact with PBP2a, but in the membrane.

In Vitro Antibacterial Experiments of Qixingjian Decoction and Its Synergistic Interaction with Oxacillin against Clinical Isolates of Methicillin-Resistant Staphylococcus aureus.

Background With the widespread use and abuse of antimicrobial drugs, the problem of bacterial resistance is becoming increasingly prominent. The clinical detection rate of drug-resistant bacteria is increasing year by year, so there is an urgent need to develop new antimicrobial drugs. Qixingjian Decoction (QXJT) is a formula commonly used in Chinese medicine for the treatment of sepsis caused by acute purulent infections of the face, hands, and feet. There are many compounds with antimicrobial effects that are available, but little is known about their mode of action. In this study, we mainly evaluated the antimicrobial activity of QXJT and explored its synergistic interaction with oxacillin (OX) and the mechanism of its antimicrobial activity. Methods The antimicrobial activity of QXJT against methicillin-resistant Staphylococcus aureus (MRSA) was determined by the microdilution method, the broth macrodilution method, and the time-kill curve method. The main compounds in QXJT were analyzed by ultra-performance liquid chromatography. The synergistic interaction of QXJT and oxacillin (OX) was determined by checkerboard assay, and the antimicrobial mechanism of QXJT, OX, and QXJT + OX was evaluated by transmission electron microscopy (TEM) technique. The expression of MRSA superantigen virulence factors (sea, seb, and tst), and drug resistance gene (mecA) was detected to provide a new strategy for new antibiotic drugs. Results QXJT exhibited antimicrobial activity against both clinical isolates of MRSA, MICs ranging from 18.75 to 37.5 mg/mL. Active substances such as Scutellarein, Scutellarin, Apigenin, and Wogonin 7-O-glucuronide were detected in the phytochemical analysis that may be associated with the antimicrobial activity of QXJT. The synergistic effect of QXJT and OX was determined by checkerboard assay (FICI = 0.5), and TEM images showed that QXJT could cause the disruption of MRSA cell wall, and QXJT + OX could produce greater disruption of MRSA cell wall, elucidating the synergistic effect of the two together on cell wall disruption by microscopic mechanisms. Our study shows that the combination of QXJT and OX can inhibit the expression of MRSA virulence factor, reduce the virulence of MRSA, and have no significant effect on the expression of MRSA resistance gene mecA. Conclusion The results of this study provide scientific experimental data for the traditional application of QXJT and initially explore the mechanism of action of QXJT combined with OX.