Structural analysis of avibactam-mediated activation of the bla and mec divergons in methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections cause significant mortality and morbidity globally. MRSA resistance to ß-lactam antibiotics is mediated by two divergons that control levels of a ß-lactamase, PC1, and a penicillin-binding protein poorly acylated by ß-lactam antibiotics, PBP2a. Expression of genes encoding these proteins is controlled by two integral membrane proteins, BlaR1 and MecR1, which both have an extracellular ß-lactam-binding sensor domain. Here, we solved the X-ray crystallographic structures of the BlaR1 and MecR1 sensor domains in complex with avibactam, a diazabicyclooctane ß-lactamase inhibitor at 1.6-2.0 Å resolution. Additionally, we show that S. aureus SF8300, a clinically relevant strain from the USA300 clone of MRSA, responds to avibactam by up-regulating the expression of the blaZ and pbp2a antibiotic-resistance genes, encoding PC1 and PBP2a, respectively. The BlaR1-avibactam structure of the carbamoyl-enzyme intermediate revealed that avibactam is bound to the active-site serine in two orientations ∼180° to each other. Although a physiological role of the observed alternative pose remains to be validated, our structural results hint at the presence of a secondary sulfate-binding pocket that could be exploited in the design of future inhibitors of BlaR1/MecR1 sensor domains or the structurally similar class D ß-lactamases. The MecR1-avibactam structure adopted a singular avibactam orientation similar to one of the two states observed in the BlaR1-avibactam structure. Given avibactam up-regulates expression of blaZ and pbp2a antibiotic resistance genes, we suggest further consideration and research is needed to explore what effects administering ß-lactam-avibactam combinations have on treating MRSA infections.

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.

Imidazolidine-4-one derivatives in the search for novel chemosensitizers of Staphylococcus aureus MRSA: synthesis, biological evaluation and molecular modeling studies.

A series of amine derivatives of 5-aromatic imidazolidine-4-ones (7-19), representing three subgroups: piperazine derivatives of 5-arylideneimidazolones (7-13), piperazine derivatives of 5-arylideneimidazolidine-2,4-dione (14-16) and primary amines of 5-naphthyl-5-methylimidazolidine-2,4-diones (17-19), was evaluated for their ability to improve antibiotics effectiveness in two strains of Gram-positive Staphylococcus aureus: ATCC 25923 (a reference strain) and MRSA (methicillin resistant S. aureus) HEMSA 5 (a resistant clinical isolate). The latter compounds (17-19) were obtained by 4-step synthesis using Bucherer-Bergs condensation, two-phase bromoalkylation and Gabriel reactions. The naphthalen derivative: (Z)-5-(naphthalen-2-ylmethylene)-2-(piperazin-1-yl)-3H-imidazol-4(5H)-one (10) was the most potent in combination with ß-lactam antibiotics and ciprofloxacin against the resistant strain. The high potency to increase efficacy of oxacillin was noted for (Z)-5-(anthracen-10-ylmethylene)-2-(piperazin-1-yl)-3H-imidazol-4(5H)one (12) too. In order to explain the mechanism of action of the compounds 10 and 12, docking studies with the use of crystal structures of a penicillin binding protein (PBP2a) and MecR1 were carried out. Their outcomes suggested that the most probable mechanism of action of the active compounds is the interaction with MecR1. Molecular dynamic experiments performed for the active compounds and compound 13 (structurally similar to 12) supported this hypothesis and provided possible explanation of activity dependencies of the tested compounds in terms of the restoration of antibiotic efficacy in S. aureus MRSA HEMSA 5.

Co-blockade of mecR1/blaR1 signal pathway to restore antibiotic susceptibility in clinical isolates of methicillin-resistant Staphylococcus aureus.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) is caused by the production of low-affinity penicillin-binding protein 2a and ß-lactamases, which are encoded by mecA and blaZ, respectively. Expressions of the two key genes are mutually regulated by MecI and BlaI. The aim of this study was to design specific anti-mecR1 and anti-blaR1 deoxyribozymes and identify the restoration of susceptibility in MRSA isolates with mecI or blaI or no deletions by interfering with the mutual regulation of mecA and blaZ. MATERIAL AND METHODS: Specific deoxyribozymes were designed by using the program RNA structure 4.6. RNA substrates were obtained by transcription in vitro and used to assess the target cleavage of DNAzymes. Transcription of mecR1-mecA and blaR1-blaZ was analysed by real time RT-PCR. The susceptibility of MRSA was tested. RESULTS: Specific deoxyribozymes showed efficient catalytic activity to each own substrate mecR1 or blaR1 in vitro and caused the reduction of mecR1 and blaR1 transcription in vivo. Furthermore, simultaneous administration of two DNAzymes to knockdown mecR1 and blaR1 resulted in increased susceptibility of all MRSA strains tested in this study. CONCLUSIONS: These results demonstrated that combined use of the two specific phosphorothioate deoxyribozymes could be a viable and promising strategy to restore the susceptibility of almost all MRSA clinical isolates.

Relationship Between the Level of Methicillin Resistance and mecA, mecI, femA Genes Genes in Staphylococci / 감염

BACKGROUND: About 60~70% of hospital isolates of staphylococci are resistant to methicillin. The level of resistance varies from low to high depending upon the genetic background of the strains. The purpose of this study was twofold : (i) to understand the relationship between beta-lactamase and methicillin-resistance genes(mecA, mecI, mecR1, femA) and the level of resistance and (ii) to survey the distribution of mec regulator genes(mec I, mecR1) among methicillin-resistant staphylococci. METHODS: Eighty-three isolates of Staphylococus aureus and 29 of coagulase-negative staphylococci(CNS) at Catholic University Hospital were examined. The level of methicillin resistance was studied using disk diffusion test and agar dilution test. MecA, mecI, mecR1, and femA genes detected by polymerase chain reaction. RESULTS: beta-lactamase production was significantly high in S. aureus and CNS isolates with low-level resistance. MecA and mecR1 genes amplification correlated with the level of resistance in S. aureus and CNS isolates. There was no correlation between the level of resistance and mecI and fem A genes amplification in S. aureus and CNS isolates. Methicillin- resistant S. aureus isolates showed more variety in mec regulator region than methicillin-resistant CNS isolates. CONCLUSION: From this study, we conclude that mecR1 gene could be considered as one of the important factors influencing the level of methicillin resistance in staphylococcal strains.

Acquisition of methicillin resistance and progression of multiantibiotic resistance in methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) produces specific penicillin-binding protein, PBP2', which shows remarkably low affinities to most beta-lactam antibiotics except those such as penicillin G and ampicillin. The region surrounding mecA has been called additional DNA or mec and is thought to be of extraspecies origin. From the study of mec, we found that mec is a novel mobile genetic element and designated as staphylococcal cassette chromosome mec (SCCmec). There are three types of SCCmec. In the past decades, MRSA has become resistant to many antibiotics, such as carbapenems, new quinolones, and minocycline etc. It seems to be a characteristic of MRSA to acquire multi-resistance by accumulating multiple resistance genes around the mecA gene inside SCCmec.

Interaction of the mec Regulator Genes in Methicillin Resistant Staphylococcus

PURPOSE: Low-affinity penicillin-binding protein PBP 2a encoded by mecA is closely related to methicillin resistance in staphylococci, and the expression of PBP 2a is controlled by regulator elements encoded by mecR1 and mecI. Deletion or mutation which occurred in mecI is considered to be associated with constitutive production of PBP 2a. We investigated the distribution of mec regulator genes and the presence of the mutations in mecI among mecA gene-positive methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase negative Staphylococcus (MRCNS) strains. METHODS: A total of 28 MRSA and 26 MRCNS clinical strains were isolated at Chung-Ang University Hospital. The distribution of mec regulator genes and the presence of mutations in mecI were analyzed using polymerase chain reaction and direct sequencing. RESULTS: In 28 MRSA and 26 MRCNS, only mecR1A-positive pattern (type lll) was detected in 53.6% of MRSA and 46.4% of MRCNS. The mecR1 (mecR1A and mecR1B) and mecI-positive pattern (type l) were detected in 42.3% of MRSA and 38.5% of MRCNS. In 19.2% of MRCNS was type lV in which no mec regulator genes were detected. Our results showed that a greater genomic variation existed in MRCNS than a MRSA. Results in direct sequencing of mecI revealed that mecI gene tested in our study did not harbour mutations and deletions. There was no correlation between the level of resistance and the presence or absence of mec regulator genes. CONCLUSION: The induction of methicillin resistance and the variability of phenotypic expression of methicillin resistance suggested that additional factors on the chromosome are involved.