Expression Of Penicillin-binding Protein 2a Research Articles

Glycyrrhizic Acid Nanoparticles Subside the Activity of Methicillin-Resistant Staphylococcus aureus by Suppressing PBP2a.

Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) are classified as high-risk infections that can lead to death, particularly among older individuals. Nowadays, plant nanoparticles such as glycyrrhizic acid are recognized as efficient bactericides against a wide range of bacterial strains. Recently, scientists have shown interest in plant extract nanoparticles, derived from natural sources, which can be synthesized into nanomaterials. Interestingly, glycyrrhizic acid is rich in antioxidants as well as antibacterial agents, and it exhibits no adverse effects on normal cells. In this study, glycyrrhizic acid nanoparticles (GA-NPs) were synthesized using the hydrothermal method and characterized through physicochemical techniques such as UV-visible spectrometry, DLS, zeta potential, and TEM. The antimicrobial activity of GA-NPs was investigated through various methods, including MIC assays, anti-biofilm activity assays, ATPase activity assays, and kill-time assays. The expression levels of mecA, mecR1, blaR1, and blaZ genes were measured by quantitative RT-qPCR. Additionally, the presence of the penicillin-binding protein 2a (PBP2a) protein of S. aureus and MRSA was evaluated by a Western blot assay. The results emphasized the fabrication of GA nanoparticles in spherical shapes with a diameter in the range of 40-50 nm. The data show that GA nanoparticles exhibit great bactericidal effectiveness against S. aureus and MRSA. The treatment with GA-NPs remarkably reduces the expression levels of the mecA, mecR1, blaR1, and blaZ genes. PBP2a expression in MRSA was significantly reduced after treatment with GA-NPs. Overall, this study demonstrates that glycyrrhizic acid nanoparticles have potent antibacterial activity, particularly against MRSA. This research elucidates the inhibition mechanism of glycyrrhizic acid, which involves the suppressing of PBP2a expression. This work emphasizes the importance of utilizing plant nanoparticles as effective antimicrobial agents against a broad spectrum of bacteria.

Methicillin Resistant Staphylococcus aureus: Molecular Mechanisms Underlying Drug Resistance Development and Novel Strategies to Combat.

Antimicrobial resistance (AMR) represents a major threat to global health. Infection caused by Methicillin-resistant Staphylococcus aureus (MRSA) is one of the well-recognized global public health problem globally. In some regions, as many as 90% of S. aureus infections are reported to be MRSA, which cannot be treated with standard antibiotics. WHO reports indicated that MRSA is circulating in every province worldwide, significantly increasing the risk of death by 64% compared to drug-sensitive forms of the infection which is attributed to its antibiotic resistance. The emergence and spread of antibiotic-resistant MRSA strains have contributed to its increased prevalence in both healthcare and community settings. The resistance of S. aureus to methicillin is due to expression of penicillin-binding protein 2a (PBP2a), which renders it impervious to the action of β-lactam antibiotics including methicillin. The other is through the production of beta-lactamases. Although the treatment options for MRSA are limited, there are promising alternatives to antibiotics to combat the infections. Innovative therapeutic strategies with wide range of activity and modes of action are yet to be explored. The review highlights the global challenges posed by MRSA, elucidates the mechanisms underlying its resistance development, and explores mitigation strategies. Furthermore, it focuses on alternative therapies such as bacteriophages, immunotherapy, nanobiotics, and antimicrobial peptides, emphasizing their synergistic effects and efficacy against MRSA. By examining these alternative approaches, this review provides insights into the potential strategies for tackling MRSA infections and combatting the escalating threat of AMR. Ultimately, a multifaceted approach encompassing both conventional and novel interventions is imperative to mitigate the impact of MRSA and ensure a sustainable future for global healthcare.

Catharanthus roseus (L.) G. Don counteracts the ampicillin resistance in multiple antibiotic-resistant Staphylococcus aureus by downregulation of PBP2a synthesis.

It is essential to revisit the global biodiversity, search for ethnopharmacologically relevant plants, and unveil their untapped potential to overcome the complications associated while treating infections triggered by multiple antibiotic-resistant Staphylococcus aureus. Catharanthus roseus (L.) G. Don of the Apocynaceae family is a medicinal plant used for remedial purposes against infectious diseases from ancient times. In this study, we intended to evaluate the mechanism by which the ethanolic extract of C. roseus root (EECRR) causes the reversal of ampicillin resistance in S. aureus. To achieve this goal, we have stained EECRR-treated S. aureus with acridine orange, analysed DNA damage by comet assay, and studied the alteration of plasmid band pattern and expression of penicillin-binding protein 2a (PBP2a) protein. Experiments revealed better S. aureus killing efficiency of EECRR at its minimum inhibitory concentration (MIC) doses due to DNA damage and reducing plasmid band intensities along with a decline in the expression of PBP2a in EECRR-treated cells at half-MIC dose. EECRR proved to be an efficient growth inhibitor of S. aureus that reduces the expression of PBP2a. Therefore, EECRR can also render ampicillin-resistant S. aureus susceptible to the antibiotic.

Molecular Determinants of β-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 β-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 β-lactam resistance in MRSA. Although several genetic factors that modulate β-lactam resistance have been identified, it remains unclear how they alter PBP2a expression and affect antibiotic resistance. This review describes the molecular determinants of β-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 β-lactam resistance. Understanding the molecular determinants of β-lactam resistance can aid in developing novel strategies to combat MRSA.

Rapid and duplex detection of MRSA using SERS-based molecular beacons

The presence of the mecA gene and penicillin-binding protein 2a (PBP2a) plays an important role in the antibiotic resistance of methicillin-resistant S. aureus (MRSA). We have developed a method for duplex detection of transmembrane PBP2a expression and the mecA gene using the plasmonic decay properties of Surface-enhanced Raman scattering (SERS) and the unique properties of beacon molecules. The duplex SERS-based test was more specific, sensitive, and rapid than polymerase chain reaction for genetic materials or ELISA for PBP2a expression, and it has a limit of detection that can detect as little as 27 PBP2a-expressed S. aureus and 8.5 pM of mecA. The efficacy of the duplexing test was demonstrated by the observed receiver operating characteristics (ROC) with its area under the curve (AUC) of 0.92 for MRSA-spiked samples. Additionally, the sensor can be developed and integrated with a portable Raman system for on-site detections.

Molecular Basis of Non-β-Lactam Antibiotics Resistance in Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most successful human pathogens with the potential to cause significant morbidity and mortality. MRSA has acquired resistance to almost all β-lactam antibiotics, including the new-generation cephalosporins, and is often also resistant to multiple other antibiotic classes. The expression of penicillin-binding protein 2a (PBP2a) is the primary basis for β-lactams resistance by MRSA, but it is coupled with other resistance mechanisms, conferring resistance to non-β-lactam antibiotics. The multiplicity of resistance mechanisms includes target modification, enzymatic drug inactivation, and decreased antibiotic uptake or efflux. This review highlights the molecular basis of resistance to non-β-lactam antibiotics recommended to treat MRSA infections such as macrolides, lincosamides, aminoglycosides, glycopeptides, oxazolidinones, lipopeptides, and others. A thorough understanding of the molecular and biochemical basis of antibiotic resistance in clinical isolates could help in developing promising therapies and molecular detection methods of antibiotic resistance.

Eleutheroside K isolated from Acanthopanax henryi (Oliv.) Harms suppresses methicillin resistance of Staphylococcus aureus.

Acanthopanax (A.) henryi (Oliv.) Harms contain many bioactive compounds commonly used in traditional Chinese medicine. The objective of the present study was to investigate the antibacterial activity of the single constituent, Eleutheroside K (ETSK) isolated from the leaves of A. henryi (Oliv.) Harms, against methicillin-resistant Staphylococcus (S.) aureus (MRSA). Broth microdilution assay was used to measure the minimal inhibitory concentration (MIC) and the MIC values of ETSK against eight clinical S. aureus strains were all 50µgml-1 . At sub-inhibitory concentrations, a synergistic effect between oxacillin (OXA) and ETSK was confirmed using checkerboard dilution assay and time-kill curve analysis. The bacteriostatic effect became more pronounced when ETSK was used in combination with detergent (Triton X-100) or ATPase inhibitor (N, N'-dicyclohexylcarbodiimide). According to western blot analysis, the down-regulated expression of Penicillin-binding protein 2a (PBP2a) further validated that the bacterial activity was inhibited when treated with ETSK in a dose-dependent manner. Results based on our study verified that ETSK significantly suppressed MRSA infections and emphasized the potential application of ETSK as a novel anti-MRSA natural drug.

Cationic Branched Polyethylenimine (BPEI) Disables Antibiotic Resistance in Methicillin-Resistant Staphylococcus epidermidis (MRSE).

Staphylococcus epidermidis is one of the most prevalent prokaryotic species on human skin and mucosal membranes that constitute the commensal flora. S. epidermidis has become one of the most common causes of primary bacteremia. Infections are difficult to diagnose because the pathogen has natural niches on human skin and the ability to adhere to inanimate surfaces via biofilms. Alarmingly, S. epidermidis has acquired resistance to many antibiotics, which presents a danger to human health. Known as methicillin-resistant S. epidermidis (MRSE), most clinical isolates of MRSE in North America exhibit β-lactam resistance primarily due to the presence of mecA, a gene that bestows β-lactam antibiotic resistance in a manner similar to methicillin-resistant Staphylococcus aureus (MRSA). MecA encodes for expression of penicillin-binding protein 2a (PBP2a), which is absent in β-lactam susceptible strains of S. epidermidis. We can disable this resistance factor in MRSE with 600-Da branched polyethylenimine (BPEI). Cationic BPEI targets anionic wall teichoic acid (WTA), an essential cofactor for proper functioning of PBP2a. We found that BPEI synergizes the activity of β-lactam antibiotics against MRSE. Growth curves suggest that the combination of BPEI and oxacillin is bactericidal. Electron micrographs indicate abnormalities in the cellular septa and cell walls of treated samples. Therefore, first-line clinical treatments can be effective against MRSE when used in combination with BPEI.

Inhibition of penicillin-binding protein 2a (PBP2a) in methicillin resistant Staphylococcus aureus (MRSA) by combination of ampicillin and a bioactive fraction from Duabanga grandiflora.

BackgroundThe inhibition of penicillin-binding protein 2a (PBP2a) is a promising solution in overcoming resistance of methicillin resistance Staphylococcus aureus (MRSA). A potential approach in achieving this is by combining natural product with currently available antibiotics to restore the activity as well as to amplify the therapeutic ability of the drugs. We studied inhibition effects of a bioactive fraction, F-10 (isolated from the leaves of Duabanga grandiflora) alone and in combination with a beta-lactam drug, ampicillin on MRSA growth and expression of PBP2a. Additionally, phytochemical analysis was conducted on F-10 to identify the classes of phytochemicals present.MethodsFractionation of the ethyl acetate leaf extract was achieved by successive column chromatography which eventually led to isolation of an active fraction, F-10. Both extract and F-10 were analyzed for the presence of major classes of phytochemicals in addition to obtaining a high performance liquid chromatography (HPLC) profile to reveal the complexity of the fraction F-10. Broth microdilution method was employed to determine minimum inhibitory concentration (MIC) of the extract and fractions against MRSA. Evaluation of synergistic activity of the active fraction with ampicillin was determined using checkerboard methodand kinetic growth experiments. Effect of combination treatments on expression of PBP2a, a protein that confers resistance to beta-lactam antibiotics, was elucidated with the Western blot assay.ResultsMIC of F-10 against MRSA was 750 mg/L which showed an improved activity by 4-fold compared to its crude extract (MIC = 3000 mg/L). Phytochemical analysis revealed occurrence of tannins, saponin, flavonoids, sterols, and glycosides in F10 fraction. In FIC index interpretation, the most synergistic activity was achieved for combinations of 1/64 × MIC ampicillin + 1/4 × MIC F-10. The combination also evidently inhibited MRSA growth in kinetic growth curve assay. As a result of this synergistic interaction, MIC of ampicillin against MRSA was reduced to 0.78 mg/L (64-fold) from initial value of 50 mg/L. Western blot analysis suggested inhibition of PBP2a in MRSA cultures grown in synergistic combination treatment in which no PBP2a band was expressed.ConclusionsThe results demonstrated synergism between fraction F-10 of D. grandiflora with ampicillin in suppressing MRSA growth via PBP2a inhibition.

The mechanism of antibacterial activity of phlorofucofuroeckol-A against methicillin-resistant Staphylococcus aureus.

To find more effective ways of overcoming methicillin-resistant Staphylococcus aureus (MRSA), there has been considerable interest in the use of marine-derived constituents as alternatives to control pathogenic microorganisms. In this study, we investigated whether phlorofucofuroeckol-A (PFF) isolated from the edible brown alga Eisenia bicyclis suppressed production or function of penicillin-binding protein 2a (PBP2a). The antimicrobial mode of action of PFF in MRSA was identified by measuring cell membrane integrity and using the time-kill curve method. We attempted to determine the antimicrobial effects of PFF on the expression level of the resistance determinants mecA and its regulatory genes mecI and mecR1 in MRSA by reverse transcriptase polymerase chain reaction. PFF suppressed mecI, mecR1, and mecA gene expression in a dose-dependent manner. In addition, we revealed PFF mediates the suppressive effect of PBP2a expression in MRSA by Western blot analysis. PFF suppressed production of the PBP2a protein, suggesting that PFF probably acts by controlling the methicillin resistance-associated genes involved in the cell wall and production of PBP2a. These results demonstrate that PFF isolated from E. bicyclis significantly suppressed the expression of the methicillin resistance-associated genes and production of PBP2a, which is considered the primary cause of methicillin resistance.

Penicillin‐binding protein 2a of methicillin‐resistant Staphylococcus aureus

High-level resistance to β-lactam antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) is due to expression of penicillin-binding protein 2a (PBP2a), a transpeptidase that catalyzes cell-wall crosslinking in the face of the challenge by β-lactam antibiotics. The activity of this protein is regulated by allostery at a site 60 Å distant from the active site, where crosslinking of cell wall takes place. This review discusses the state of knowledge on this important enzyme of cell-wall biosynthesis in MRSA.

Beta-lactam resistance in Staphylococcus aureus: the adaptive resistance of a plastic genome.

Staphylococci have two mechanisms for resistance to beta-lactam antibiotics. One is the production of beta-lactamases, enzymes that hydrolytically destroy beta-lactams. The other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by beta-lactam antibiotics. Strains of S. aureus exhibiting either beta-lactamase or PBP 2a-directed resistance (or both) have established a considerable ecological niche among human pathogens. The emergence and subsequent spread of bacterial strains designated as methicillin-resistant S. aureus (MRSA), from the 1960s to the present, has created clinical difficulties for nosocomial treatment on a global scale. The recent variants of MRSA that are resistant to glycopeptide antibiotics (such as vancomycin) have ushered in a new and disconcerting chapter in the evolution of this organism.

Is the Cefoxitin Disk Test Reliable Enough To Detect Oxacillin Resistance in Coagulase-Negative Staphylococci?

Is the Cefoxitin Disk Test Reliable Enough To Detect Oxacillin Resistance in Coagulase-Negative Staphylococci?

Resistance to β-Lactam Antibiotics and Its Mediation by the Sensor Domain of the Transmembrane BlaR Signaling Pathway in Staphylococcus aureus

Staphylococci, a leading cause of infections worldwide, have devised two mechanisms for resistance to beta-lactam antibiotics. One is production of beta-lactamases, hydrolytic resistance enzymes, and the other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by beta-lactam antibiotics. The beta-lactam sensor-transducer (BlaR), an integral membrane protein, binds beta-lactam antibiotics on the cell surface and transduces the information to the cytoplasm, where gene expression is derepressed for both beta-lactamase and penicillin-binding protein 2a. The gene for the sensor domain of the sensor-transducer protein (BlaR(S)) of Staphylococcus aureus was cloned, and the protein was purified to homogeneity. It is shown that beta-lactam antibiotics covalently modify the BlaR(S) protein. The protein was shown to contain the unusual carboxylated lysine that activates the active site serine residue for acylation by the beta-lactam antibiotics. The details of the kinetics of interactions of the BlaR(S) protein with a series of beta-lactam antibiotics were investigated. The protein undergoes acylation by beta-lactam antibiotics with microscopic rate constants (k(2)) of 1-26 s(-1), yet the deacylation process was essentially irreversible within one cell cycle. The protein undergoes a significant conformational change on binding with beta-lactam antibiotics, a process that commences at the preacylation complex and reaches its full effect after protein acylation has been accomplished. These conformational changes are likely to be central to the signal transduction events when the organism is exposed to the beta-lactam antibiotic.

Mechanisms of methicillin resistance in staphylococci.

The continuously high prevalence of methicillin-resistant staphylococci (MRS) throughout the world is a constant threat to public health, owing to the multiresistant characteristics of these bacteria. Methicillin resistance is phenotypically associated with the presence of the penicillin-binding protein 2a (PBP2a) not present in susceptible staphylococci. This protein has a low binding affinity for beta-lactam antibiotics. It is a transpeptidase which may take over cell wall synthesis during antibiotic treatment when normally occurring PBPs are inactivated by ligating beta-lactams. PBP2a is encoded by the mecA gene, which is located in mec, a foreign DNA region. Expression of PBP2a is regulated by proteins encoded by the plasmid-borne blaR1-bla1 inducer-repressor system and the corresponding genomic mecRl-mecl system. The blaRl-blal products are important both for the regulation of beta-lactamase and for mecA expression. Methicillin resistance is influenced by a number of additional factors, e.g. the products of the chromosomal fem genes which are important in the synthesis of normal peptidoglycan precursor molecules. Inactivation of fem-genes results in structurally deficient precursors which are not accepted as cell wall building blocks by the ligating PBP2a transpeptidase during antibiotic treatment. This may result in reduced resistance to beta-lactam antibiotics. Inactivation of genes affecting autolysis has shown that autolytic enzymes are also of importance in the expression of methicillin resistance. Methicillin resistance has evolved among earth microorganisms for protection against exogenous or endogenous antibiotics. Presumably the mec region was originally transferred from coagulase negative staphylococci (CNS) to Staphylococcus aureus (SA). A single or a few events of this kind with little subsequent interspecies transfer had been anticipated. However, recent data suggest a continuous horizontal acquisition by S. aureus of mec, being unidirectional from CNS to SA. Methicillin resistance may also be associated with mechanisms independent of mecA, resulting in borderline methicillin resistance. These mechanisms include beta-lactamase hyperproduction, production of methicillinases, acquisition of structurally modified normal PBPs, or the appearance of small colony variants of SA. Most MRS are multiresistant, and the mec region may harbour several resistance determinants, resulting in a clustering of resistance genes within this region.