Presence of multiple van genes among glycopeptide non-susceptible Staphylococcus aureus exhibiting in vitro MIC creep phenomenon: A study from north-east India.

Background & objectives The global prevalence of vancomycin-resistant Staphylococcus aureus (VRSA) has increased two fold since 2010, accounting for 2.4 per cent of S. aureus infections. The emerging hVISA isolates and their increasing trends pose a serious therapeutic challenge. The present study investigated in vitro vancomycin and teicoplanin minimum inhibitory concentration (MIC) creep in S. aureus and assessed their revertants. Methods A total of 845 isolates were collected for this study, and 246 were confirmed as S. aureus. Molecular characterization of vancomycin resistance was carried out by PCR assay targeting genes types viz: vanA, vanB, vanC, vanC2/C3, vanD, vanE, and vanG. MIC was determined for vancomycin and teicoplanin by agar dilution method. MIC creep and revertant analysis were done by broth dilution method in the presence and absence of antibiotics. Results PCR assay confirmed 12 isolates were harboured vanA, followed by vanD (n=8) and vanB (n=7). The study showed 69 isolates were screened positive for glycopeptide non-susceptibility. While analyzing vancomycin MIC creep, four isolates showed a significant increase in MIC, whereas no creep phenomenon was observed for the rest. In the case of teicoplanin, seven isolates showed the MIC creep phenomenon. Revertant analysis of all the isolates that showed MIC creep phenomenon for vancomycin and teicoplanin reverted to their original MIC when the antibiotic pressure was withdrawn. Interpretation & conclusions In the present study setting, glycopeptide non-susceptibility was found in eight per cent of the isolates, and the present study found the occurrence of multiple van genes from isolates calculated from a single study center will impose a serious challenge in infection control and antibiotic policy. This study also underscores that heterogenic resistant isolates, upon exposure to vancomycin and teicoplanin at a minimum level, exhibited an increase in MIC, which will impact individuals receiving glycopeptide therapy.

Intracellular bactericidal activity and action mechanism of MDP1 antimicrobial peptide against VRSA and MRSA in human endothelial cells.

Introduction: Staphylococcus aureus is a prominent cause of postoperative infections, often persisting within host cells, leading to chronic infections. Conventional antibiotics struggle to eliminate intracellular S. aureus due to poor cell penetration. Antimicrobial peptides are a new hope for tackling intracellular bacteria. Accordingly, this study examines the antimicrobial peptide MDP1, derived from melittin, for its efficacy against intracellular S. aureus. Methods: In this study, the physiochemical properties (Prediction of three-dimensional structure, circular dichroism and helical wheel projection analysis) were investigated. Extracellular antibacterial activity and cytotoxicity of MDP1 were also assessed. The mechanism of interaction of MDP1 with S. aureus was evaluated by molecular dynamic simulation, atomic force and confocal microscopy. Bacterial internalization into an endothelial cell model was confirmed through culture and transmission electron microscopy. The effect of the peptide on intracellular bacteria was investigated by culture and epi-fluorescence microscopy. Results and discussion: 3D structural prediction proved the conformation of MDP1 as an α-helix peptide. Helical-wheel projection analysis indicated the proper orientation of hydrophobic amino acid residues for membrane interaction. CD spectroscopy of MDP1 showed that MDP1 in SDS 10 and 30 mM adopted 87 and 91% helical conformation. Atomic force and confocal microscopy assessments as well as molecular dynamics studies revealed the peptide-bacterial membrane interaction. MDP1, at the concentration of 0.32 µg mL-1, demonstrated a fold reduction of 21.7 ± 1.8, 1.7 ± 0.2, and 7.3 ± 0.8 in intracellular bacterial load for ATCC, VRSA, and MRSA, respectively. Molecular dynamics results demonstrate a preferential interaction of MDP1 with POPG/POPE membranes, primarily driven by electrostatic forces and hydrogen bonding. In POPC systems, two out of four MDP1 interacted effectively, while all four MDP1 engaged with POPG/POPE membranes. Gathering all data together, MDP1 is efficacious in the reduction of intracellular VRSA and MRSA proved by culture and epi-fluorescent microscopy although further studies should be performed to increase the intracellular activity of MDP1.

Methicillin-Resistant Staphylococcus aureus Infection and its Health Perspective: A Review.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is a potential threat globally since it is associated with high morbidity and mortality. In addition, the ability of MRSA to develop resistance and adapt to various environments makes it exceptional from other bacterial strains. Effective management is best determined by the site of infection. OBJECTIVES: This study aims to summarize and assess the epidemiology of MRSA, resistance, detection of MRSA in humans, animals, and food products, treatment employed, and combination therapy. METHODS: For the present review, we collected data from PubMed, Embase, Web of Science, BioMed Central, Medline, Encyclopedia of Life Sciences, Scopus, Cochrane Library, and ScienceDirect that report the epidemiology of MRSA, drug resistance in MRSA, spread of MRSA infection, diagnosis of infection, existing and emerging remedies of MRSA infections. Collected data were analyzed and represented in this article with the help of Figures and Tables. RESULTS: S. aureus resistance to vancomycin is because of genetic adaptation and also due to the widespread and indiscriminate use of antibiotics in the treatment of MRSA infection. Specifically, infections related to vancomycin-resistant S. aureus are life-threatening and difficult to treat. MRSA epidemiology with the recognition of community-acquired-MRSA transmission between livestock and humans is also reported and is alarming. Multiple studies suggested that early detection of MRSA colonization and elimination of carriage can help reduce the risk of subsequent infection. Specifically, PCR-based screening from different body sites offers the highest overall sensitivity for the detection of MRSA carriage. CONCLUSION: Screening novel mutants and methods of transmission in each environment will assist in managing MRSA. Further, effective MRSA control in all clinical setups is required with the avoidance of uncontrolled antibiotic usage.

Insights into the molecular basis of reduced vancomycin susceptibility among three prominent Staphylococcus aureus clonal complexes.

Staphylococcus aureus is a leading cause of healthcare-associated infections globally. Vancomycin-resistant S. aureus (VRSA), those with high-level resistance [minimum inhibitory concentration (MIC) of 16-32 µg/mL vancomycin], are uncommon, whereas vancomycin-intermediate S. aureus (VISA; MIC of 4-8 µg/mL), are isolated more frequently and develop during long-term and/or repeated use of the antibiotic. VISA can be difficult to eradicate and infections may persist. Our knowledge of mechanisms that underlie the development of VISA is incomplete. We used a genomics approach to investigate the VISA phenotype in three prominent S. aureus lineages. All VISA clinical isolates tested had increased cell wall thickness compared with vancomycin-susceptible S. aureus strains. Growth rates of clonal complex (CC) 5, CC8, and CC45 clinical isolates were reduced in 2 µg/mL vancomycin compared to media alone. Culture in 2 and 4 µg/mL vancomycin sequentially for two weeks reduced susceptibility to daptomycin, televancin, tigecycline, and vancomycin in a majority of CC5, CC8, and CC45 isolates tested. We identified alleles reported previously to contribute to the VISA phenotype, but unexpectedly, these alleles were unique to each CC. A subtherapeutic concentration of vancomycin elicited changes in the VISA transcriptome-common and unique-among the three CCs tested. Multiple genes, including those encoding a glycerate kinase, an M50 family metallopeptidase, and an uncharacterized membrane protein, were upregulated among all three lineages and not reported previously as associated with VISA. Although there are lineage-specific changes in DNA sequence, our findings suggest changes in the VISA transcriptome constitute a general response to stress that confers reduced susceptibility to multiple antibiotics. IMPORTANCE: Our understanding of the mechanisms that underlie the development of vancomycin-intermediate Staphylococcus aureus (VISA) is incomplete. To provide a more comprehensive view of this process, we compared genome sequences of clonal complex (CC) 5, CC8, and CC45 VISA clinical isolates and measured changes in the transcriptomes of these isolates during culture with a subtherapeutic concentration of vancomycin. Notably, we identified differentially expressed genes that were lineage-specific or common to the lineages tested, including genes that have not been previously reported to contribute to a VISA phenotype. Changes in gene expression were accompanied by reduced growth rate, increased cell wall thickness, and reduced susceptibility to daptomycin, televancin, tigecycline, and vancomycin. Our results provide support to the idea that changes in gene expression contribute to the development of VISA among three CCs that are a prominent cause of human infections.

Repeated Exposure of Vancomycin to Vancomycin-Susceptible Staphylococcus aureus (VSSA) Parent Emerged VISA and VRSA Strains with Enhanced Virulence Potentials.

The emergence of resistance against the last-resort antibiotic vancomycin in staphylococcal infections is a serious concern for human health. Although various drug-resistant pathogens of diverse genetic backgrounds show higher virulence potential, the underlying mechanism behind this is not yet clear due to variability in their genetic dispositions. In this study, we investigated the correlation between resistance and virulence in adaptively evolved isogenic strains. The vancomycin-susceptible Staphylococcus aureus USA300 was exposed to various concentrations of vancomycin repeatedly as a mimic of the clinical regimen to obtain mutation(s)-accrued-clonally-selected (MACS) strains. The phenotypic analyses followed by expression of the representative genes responsible for virulence and resistance of MACS strains were investigated. MACS strains obtained under 2 and 8 µg/ml vancomycin, named Van2 and Van8, respectively; showed enhanced vancomycin minimal inhibitory concentrations (MIC) to 4 and 16 µg/ml, respectively. The cell adhesion and invasion of MACS strains increased in proportion to their MICs. The correlation between resistance and virulence potential was partially explained by the differential expression of genes known to be involved in both virulence and resistance in MACS strains compared to parent S. aureus USA300. Repeated treatment of vancomycin against vancomycin-susceptible S. aureus (VSSA) leads to the emergence of vancomycin-resistant strains with variable levels of enhanced virulence potentials.

Pyridine-2,6-Dicarboxamide Proligands and their Cu(II)/Zn(II) Complexes Targeting Staphylococcus Aureus for the Attenuation of In Vivo Dental Biofilm.

In the pursuit to combat stubborn bacterial infections, particularly those stemming from gram-positive bacteria, this study is an attempt to craft a precision-driven platform characterized by unparalleled selectivity, specificity, and synergistic antimicrobial mechanisms. Leveraging remarkable potential of metalloantibiotics in antimicrobial applications, herein, this work rationally designs, synthesizes, and characterizes a new library of Pyridine-2,6-dicarboxamide ligands and their corresponding transition metal Cu(II)/Zn(II) complexes. The lead compound L11 demonstrates robust antibacterial properties against Staphylococcus aureus (Minimum Inhibitory Concentration (MIC) = 2-16 µg mL-1), methicillin and vancomycin-resistant S. aureus (MIC = 2-4 µg mL-1) and exhibit superior antibacterial activity when compared to FDA-approved vancomycin, the drug of last resort. Additionally, the compound exhibits notable antimicrobial efficacy against resistant enterococcus strains (MIC = 2-8 µg mL-1). To unravel mechanistic profile, advanced imaging techniques including SEM and AFM are harnessed, collectively suggesting a mechanistic pathway involving cell wall disruption. Live/dead fluorescence studies further confirm efficacy of L11 and its complexes against S. aureus membranes. This translational exploration extends to a rat model, indicating promising in vivo therapeutic potential. Thus, this comprehensive research initiative has capabilities to transcends the confines of this laboratory, heralding a pivotal step toward combatting antibiotic-resistant pathogens and advancing the frontiers of metalloantibiotics-based therapy with a profound clinical implication.

Rapid eradication of vancomycin and methicillin-resistant Staphylococcus aureus by MDP1 antimicrobial peptide coated on photocrosslinkable chitosan hydrogel: in vitro antibacterial and in silico molecular docking studies.

Introduction: Antibiotic resistance and weak bioavailability of antibiotics in the skin due to systemic administration leads to failure in eradication of vancomycin- and methicillin-resistant Staphylococcus aureus (VRSA and MRSA)-associated wound infections and subsequent septicemia and even death. Accordingly, this study aimed at designing a photocrosslinkable methacrylated chitosan (MECs) hydrogel coated by melittin-derived peptide 1 (MDP1) that integrated the antibacterial activity with the promising skin regenerative capacity of the hydrogel to eradicate bacteria by burst release strategy. Methods: The MECs was coated with MDP1 (MECs-MDP1), characterized, and the hydrogel-peptide interaction was evaluated by molecular docking. Antibacterial activities of MECs-MDP1 were evaluated against VRSA and MRSA bacteria and compared to MECs-vancomycin (MECs-vanco). Antibiofilm activity of MECs-MDP1 was studied by our novel 'in situ biofilm inhibition zone (IBIZ)' assay, and SEM. Biocompatibility with human dermal fibroblast cells (HDFs) was also evaluated. Results and Discussion: Molecular docking showed hydrogen bonds as the most interactions between MDP1 and MECs at a reasonable affinity. MECs-MDP1 eradicated the bacteria rapidly by burst release strategy whereas MECs-vanco failed to eradicate them at the same time intervals. Antibiofilm activity of MECs-MDP1 were also proved successfully. As a novel report, molecular docking analysis has demonstrated that MDP1 covers the structure of MECs and also binds to lysozyme with a reasonable affinity, which may explain the inhibition of lysozyme. MECs-MDP1 was also biocompatible with human dermal fibroblast skin cells, which indicates its safe future application. The antibacterial properties of a photocrosslinkable methacrylated chitosan-based hydrogel coated with MDP1 antimicrobial peptide were successfully proved against the most challenging antibiotic-resistant bacteria causing nosocomial wound infections; VRSA and MRSA. Molecular docking analysis revealed that MDP1 interacts with MECs mainly through hydrogen bonds with reasonable binding affinity. MECs-MDP1 hydrogels eradicated the planktonic state of bacteria by burst release of MDP1 in just a few hours whereas MECs-vanco failed to eradicate them. inhibition zone assay showed the anti-biofilm activity of the MECs-MDP1 hydrogel too. These findings emphasize that MECs-MDP1 hydrogel would be suggested as a biocompatible wound-dressing candidate with considerable and rapid antibacterial activities to prevent/eradicate VRSA/MRSA bacterial wound infections.

Chitosan-Loaded Lagenaria siceraria and Thymus vulgaris Potentiate Antibacterial, Antioxidant, and Immunomodulatory Activities against Extensive Drug-Resistant Pseudomonas aeruginosa and Vancomycin-Resistant Staphylococcus aureus: In Vitro and In Vivo Approaches.

Antimicrobial resistance poses considerable issues for current clinical care, so the modified use of antimicrobial agents and public health initiatives, coupled with new antimicrobial approaches, may help to minimize the impact of multidrug-resistant (MDR) bacteria in the future. This study aimed to evaluate the antimicrobial, antioxidant, and immunomodulatory activities of Lagenaria siceraria, Thymus vulgaris, and their chitosan nanocomposites against extensive drug-resistant (XDR) Pseudomonas aeruginosa and vancomycin-resistant Staphylococcus aureus (VRSA) using both in vitro and in vivo assays. The in vitro antimicrobial susceptibilities of P. aeruginosa and VRSA strains revealed 100% sensitivity to imipenem (100%). All P. aeruginosa strains were resistant to cefoxitin, cefepime, trimethoprim + sulfamethoxazole, and fosfomycin. However, S. aureus strains showed a full resistance to cefoxitin, amoxicillin, ampicillin, erythromycin, chloramphenicol, and fosfomycin (100% each). Interestingly, all S. aureus strains were vancomycin-resistant (MIC = 32-512 µg/mL), and 90% of P. aeruginosa and S. aureus strains were XDR. The antimicrobial potential of Lagenaria siceraria and Thymus vulgaris nanocomposites with chitosan nanoparticles demonstrated marked inhibitory activities against XDR P. aeruginosa and VRSA strains with inhibition zones' diameters up to 50 mm and MIC values ranging from 0.125 to 1 µg/mL and 1 to 8 µg/mL, respectively. The results of the in vivo approach in male Sprague Dawley rats revealed that infection with P. aeruginosa and S. aureus displayed significant changes in biochemical, hematological, and histopathological findings compared to the negative control group. These values returned to the normal range after treatment by chitosan nanoparticles, either loaded with Lagenaria siceraria or Thymus vulgaris. Real-time quantitative polymerase chain reaction (RT-qPCR) findings presented significant upregulation of the relative expression of the IL10 gene and downregulation of the IFNG gene throughout the experimental period, especially after treatment with chitosan nanoparticles loaded either with Lagenaria siceraria or Thymus vulgaris in comparison to the positive control groups. In conclusion, this is the first report suggesting the use of Lagenaria siceraria and Thymus vulgaris nanocomposites with chitosan nanoparticles as a promising contender for combating XDR P. aeruginosa and VRSA infections as well as a manager for inflammatory situations and oxidative stress-related disorders.

Emerging novel sequence types of Staphylococcus aureus in Pakistan.

BACKGROUND: Despite an increasing incidence of Staphylococcus aureus infection and dissemination in Pakistan, the epidemiology of different Staphylococcus aureus research clones has been the subject of only a small number of investigations. By analyzing the collected data sequence, this study was designed to study the epidemiology of Staphylococcus aureus in the area using multilocus sequence typing (MLST). METHODS: A total of 1015 staphylococcus strains collected from the city's tertiary care facilities were biochemically screened, followed by antimicrobial susceptibility testing against a panel of 13 antibiotics. Analyzed methicillin-resistant Staphylococcus aureus (MRSA) was subjected to molecular characterization using multilocus sequence typing (MLST), clonal complex analysis, recombination testing, and phylogenetic analysis. RESULTS: Approximately 421 bacteria were verified as Staphylococcus aureus by biochemical analysis. 57% of the isolates exhibited multidrug resistance, of which 89% were found to be methicillin-resistant Staphylococcus aureus (MRSA). MLST results in a total of 39 sequence types (ST) and 5 clonal complexes (CC), out of which twenty-two STs were newly documented worldwide. The most common CC identified was CC8. The direct sequencing data also revealed significant shifts at MLST loci, with point mutations resulting in the aroE-343 and tpi-278 alleles. CONCLUSIONS: This study concludes that there is high diversity in the locally circulating clones of Staphylococcus aureus present in nature and that they are defined by their geographic epidemiology. These findings have practical implications for public health, including the need for tailored infection control strategies, antibiotic stewardship, global surveillance, and a deeper understanding of bacterial evolution.

Molecular distribution of biocide resistance genes and susceptibility to biocides among vancomycin resistant Staphylococcus aureus (VRSA) isolates from intensive care unit (ICU) of cardiac hospital- A first report from Pakistan.

Background: The study was conducted with the aim to investigate the VRSA isolates in terms of their susceptibility to routinely used biocides influenced by the co-occurrence of biocide resistant gene (BRGs) and efflux pumps genes. Methodology: Frequently touched surfaces within intensive care unit (ICU) of cardiac hospital were classified into three primary sites i.e., structure, machines and miscellaneous. Over a period of six months (January 2021 to July 2021) twenty three swabs samples were collected from these sites. Subsequently, these samples underwent both phenotypic and molecular methods for VRSA isolation and identification. Susceptibility and efficacy testing of biocides (benzalkonium chloride (BAC), cetrimide (CET) and chlorhexidine gluconate (CHG)) were evaluated using microdilution broth and suspension method. Furthermore, specific primers were used for singleplex PCR targeting BRGs (cepA, qacA, and qacE) and efflux pump (norA, norB, norC, sepA, mepA and mdeA) associated genes. Results: We found that 72.2 % S. aureus demonstrate the presence of vanA or vanB genes with no significant difference among three sites (p > 0.05). cepA is the most dominant BRGs followed by qacA and qacE from structure site as compared to other sites (p < 0.05). BAC showed reduced biocide susceptibility and MIC50. There was no significant difference between presence or absence of BRGs and high MIC values of VRSA isolates from all three sites. However, efflux pump genes (EFPGs) particularly norA and norA + sepA had a significant association with BRGs and reduced biocide. Conclusion: BAC is the most effective disinfectant against VRSA. Proper and controlled use of BAC is required to overcome the VRSA contamination. We recommend continuous monitoring of the BRGs prevalence for better prevention of microorganism dissemination and infection control in hospitals.

Structural and Functional Analysis of Urease Accessory Protein E from Vancomycin-Resistance Staphylococcus aureus MU50 Strain.

BACKGROUND: An increasing prevalence of biofilm forming strains by vancomycinresistance Staphylococcus aureus (VRSA) is one of the most important causes of antimicrobial resistance. VRSA possesses various regulatory factors to form and sustain biofilm in biotic or abiotic conditions. Among them, ureolytic activity is an important factor in the stabilization of biofilms by neutralizing the acidic environment. Various urease accessory proteins are required to activate the urease enzyme inside the biofilm. OBJECTIVE: To optimize the cloning, expression and purification of urease accessory protein E from VRSA for determination of the secondary structure, and functional characterization by using Berthelot's method. METHODS: BAB58453.1 gene (which encodes possible urease accessory protein E), having 38% similarity to Bacillus pasteurii UreE protein, was cloned, expressed, and purified by single-step affinity chromatography for performing secondary structural studies using circular dichroism spectroscopy, and functional analysis using Berthelot's and crystal violet assay. RESULTS: Structure elucidation using NMR and circular dichroism spectroscopy techniques revealed that UreE protein has a partially foldedα-helical structure. Using Berthelot's method, it was identified that the purified UreE protein has enhanced urease enzyme activity, in comparison to the control. From the results of Berthelot's and crystal violet assays, it was deduced that the selected gene (UreE protein) plays a key role in enhancing urease enzyme activity and contributes to biofilm stability. CONCLUSION: Structural studies on VRSA urease accessory proteins could aid in the identification of new drug targets or the development of effective antibiofilm strategies (in combination with other drug targets) against infections caused by biofilm-producing strains.

Simulation of optimal dose regimens of photoactivated curcumin for antimicrobial resistance pneumonia in COVID-19 patients: A modeling approach.

Background: Secondary antimicrobial resistance bacterial (AMR) pneumonia could lead to an increase in mortality in COVID-19 patients, particularly of geriatric patients with underlying diseases. The comedication of current medicines for AMR pneumonia with corticosteroids may lead to suboptimal treatment or toxicities due to drug-drug interactions (DDIs). Objective: This study aimed to propose new promising dosage regimens of photoactivated curcumin when co-administered with corticosteroids for the treatment of antimicrobial resistance (AMR) pneumonia in COVID-19 patients. Methods: A whole-body physiologically-based pharmacokinetic (PBPK) with the simplified lung compartments model was built and verified following standard model verification (absolute average-folding error or AAFEs). The pharmacokinetic properties of photoactivated were assumed to be similar to curcumin due to minor changes in physiochemical properties of compound by photoactivation. The acceptable AAFEs values were within 2-fold. The verified model was used to simulate new regimens for different formulations of photoactivated curcumin. Results: The AAFEs was 1.12-fold. Original formulation (120 mg once-daily dose) or new intramuscular nano-formulation (100 mg with a release rate of 10/h given every 7 days) is suitable for outpatients with MRSA pneumonia to improve patient adherence. New intravenous formulation (2000 mg twice-daily doses) is for hospitalized patients with both MRSA and VRSA pneumonia. Conclusion: The PBPK models, in conjunction with MIC and applied physiological changes in COVID-19 patients, is a potential tool to predict optimal dosage regimens of photoactivated curcumin for the treatment of co-infected AMR pneumonia in COVID-19 patients. Each formulation is appropriate for different patient conditions and pathogens.

Introduction of Novel Drug Targets against Staphylococcus aureus and Proposing Putative Inhibitors against Adenine N1 (m1A22)-tRNA Methyltransferase (TrmK) using Computer-aided Drug Discovery.

BACKGROUND: Nowadays, the emergence of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) strains has dramatically restricted the treatment options against this microorganism. AIM: In this study, we aimed to discover new drug targets and inhibitors against S. aureus. METHODS: This study consists of two major sections. In the upstream evaluation, after a comprehensive coreproteome analysis, essential cytoplasmic proteins with no similarity to the human proteome were selected. Then the S. aureus metabolome-specific proteins were selected, and novel drug targets were identified using the DrugBank database. In the downstream analysis, a structure-based virtual screening approach was performed to reveal potential hit compounds against adenine N1 (m(m1A22)-tRNA methyltransferase (TrmK) using the StreptomeDB library and AutoDock Vina software. The compounds with a binding affinity > -9 kcal/mol were analyzed based on ADMET properties. Finally, the hit compounds were selected based on Lipinski's rule of five (RO5). RESULTS: Three proteins, including glycine glycosyltransferase (FemA), TrmK, and heptaprenyl pyrophosphate synthase subunit A (HepS1), were selected as feasible and promising drug targets based on PDB file availability and their essential role in the survival of the S. aureus. Finally, seven hit compounds, including Nocardioazine_ A, Geninthiocin_D, Citreamicin_delta, Quinaldopeptin, Rachelmycin, Di-AFN_A1 and Naphthomycin_ K were introduced against the binding cavity of TrmK, as a feasible drug target. CONCLUSION: The results of this study provided three feasible drug targets against S. aureus. In the following, seven hit compounds were introduced as potential inhibitors of TrmK, and Geninthiocin_D was identified as the most desirable agent. However, in vivo and in vitro investigations are needed to confirm the inhibitory effect of these agents on S. aureus.

Evidence and Molecular Characterization of Multidrug Resistant Staphylococcus aureus Isolated From Equines in Pakistan.

The purpose of the current study was to investigate the nasal colonization and drug resistance profile of Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and vancomycin-resistant S. aureus (VRSA) in donkeys (n =63), mules (n = 42), and horses (n = 98). MRSA and VRSA were confirmed based on phenotypic and molecular methods, followed by phylogenetic analysis. Furthermore, the association of various animal and management-based risk factors with S. aureus colonization was also evaluated. The presence of nuc gene on polymerase chain reaction showed an overall prevalence of 42.86% of S. aureus in equines. Based on Kirby-Bauer disc diffusion test, 26.44% of isolates showed resistance to vancomycin. Further, mecA and vanB genes were targeted which showed an overall 14.94% and 13.79% prevalence of methicillin and vancomycin-resistant isolates, respectively. The phylogenetic analysis revealed a significant variation of the study isolates with each other and with already reported sequences of mecA and vanB genes. Risk factor analysis revealed that raising purpose (P = .011), work intensity (P < .001), stocking density (P = .006), presence of other livestock animals in surroundings (P = .043), and common drinking water source (P = .023) as significant. Antimicrobial susceptibility testing of MRSA and VRSA isolates showed high resistance to various commonly used antibiotics. Furthermore, all the tested isolates showing resistance to three or more than three antibiotics were considered multiple drug-resistant. The current study manifests the molecular evidence of MRSA and VRSA isolated from equines in Pakistan which will help to address the emerging issue of multidrug resistance in S. aureus in equines and emphasizes the need for possible measures to tackle this issue.

A Convenient Diels-Alder Approach toward Potential Polyketide-like Antibiotics Using α-Activated α,ß-Unsaturated 4,4-Dimethyl-1-tetralones as Dienophiles.

Making use of a Diels-Alder approach based on various α,ß-unsaturated 2-carbomethoxy-4,4-dimethyl-1-tetralones as novel dienophiles, the corresponding polycyclic adducts could be efficiently synthesized in good to high yields (74~99%) in the presence of Lewis acid (e.g., SnCl4). Accordingly, a synthetically useful platform is established to provide a focused aromatic polyketide-like library for screening of potential natural and non-natural antimicrobial agents.

Genomic Analysis of Vancomycin-Resistant Staphylococcus aureus Isolates from the 3rd Case Identified in the United States Reveals Chromosomal Integration of the vanA Locus.

Vancomycin-resistant Staphylococcus aureus (VRSA) is a human pathogen of significant public health concern. Although the genome sequences of individual VRSA isolates have been published over the years, very little is known about the genetic changes of VRSA within a patient over time. A total of 11 VRSA, 3 vancomycin-resistant enterococci (VRE), and 4 methicillin-resistant S. aureus (MRSA) isolates, collected over a period of 4.5 months in 2004 from a patient in a long-term-care facility in New York State, were sequenced. A combination of long- and short-read sequencing technologies was used to obtain closed assemblies for chromosomes and plasmids. Our results indicate that a VRSA isolate emerged as the result of the transfer of a multidrug resistance plasmid from a coinfecting VRE to an MRSA isolate. The plasmid then integrated into the chromosome via homologous recombination mediated between two regions derived from remnants of transposon Tn5405. Once integrated, the plasmid underwent further reorganization in one isolate, while two others lost the staphylococcal cassette chromosome mec element (SCCmec) determinant that confers methicillin-resistance. The results presented here explain how a few recombination events can lead to multiple pulsed-field gel electrophoresis (PFGE) patterns that could be mistaken for vastly different strains. A vanA gene cluster that is located on a multidrug resistance plasmid that is integrated into the chromosome could result in the continuous propagation of resistance, even in the absence of selective pressure from antibiotics. The genome comparison presented here sheds light on the emergence and evolution of VRSA within a single patient that will enhance our understanding VRSA genetics. IMPORTANCE High-level vancomycin-resistant Staphylococcus aureus (VRSA) began to emerge in the United States in 2002 and has since then been reported worldwide. Our study reports the closed genome sequences of multiple VRSA isolates obtained in 2004 from a single patient in New York State. Our results show that the vanA resistance locus is located on a mosaic plasmid that confers resistance to multiple antibiotics. In some isolates, this plasmid integrated into the chromosome via homologous recombination between two ant(6)-sat4-aph(3') antibiotic resistance loci. This is, to our knowledge, the first report of a chromosomal vanA locus in VRSA; the effect of this integration event on MIC values and plasmid stability in the absence of antibiotic selection remains poorly understood. These findings highlight the need for a better understanding of the genetics of the vanA locus and plasmid maintenance in S. aureus to address the increase of vancomycin resistance in the health care setting.

Molecular Basis of Methicillin and Vancomycin Resistance in Staphylococcus aureus from Cattle, Sheep Carcasses and Slaughterhouse Workers.

Staphylococcus aureus (S. aureus) is a serious infection-causing pathogen in humans and animal. In particular, methicillin-resistant S. aureus (MRSA) is considered one of the major life-threatening pathogens due to its rapid resistance to several antibiotics in clinical practice. MRSA strains have recently been isolated in a number of animals utilized in food production processes, and these species are thought to be the important sources of the spread of infection and disease in both humans and animals. The main objective of the current study was to assess the prevalence of drug-resistant S. aureus, particularly vancomycin-resistant S. aureus (VRSA) and MRSA, by molecular methods. To address this issue, a total of three hundred samples (200 meat samples from cattle and sheep carcasses (100 of each), 50 hand swabs, and 50 stool samples from abattoir workers) were obtained from slaughterhouses in Egypt provinces. In total, 19% S. aureus was isolated by standard culture techniques, and the antibiotic resistance was confirmed genotypically by amplification nucA gen. Characteristic resistance genes were identified by PCR with incidence of 31.5%, 19.3%, 8.7%, and 7% for the mecA, VanA, ermA, and tet L genes, respectively, while the aac6-aph gene was not found in any of the isolates. In this study, the virulence genes responsible for S. aureus' resistance to antibiotics had the highest potential for infection or disease transmission to animal carcasses, slaughterhouse workers, and meat products.

Tackling strong biofilm and multi-virulent vancomycin-resistant Staphylococcus aureus via natural alkaloid-based porous nanoparticles: perspective towards near future eradication.

Introduction: As a growing direction, nano-based therapy has become a successful paradigm used to address the phytogenic delivery-related problems in overcoming multivirulent vancomycin-resistant Staphylococcus aureus (VRSA) infection. Methods: Hence, our aim was to develop and assess a novel nanocarrier system (mesoporous silica nanoparticles, MPS-NPs) for free berberine (Free-BR) as an antimicrobial alkaloid against strong biofilm-producing and multi-virulent VRSA strains using in vitro and in vivo mouse model. Results and discussion: Our outcomes demonstrated vancomycin resistance in 13.7% of Staphylococcus aureus (S. aureus) strains categorized as VRSA. Notably, strong biofilm formation was observed in 69.2% of VRSA strains that were all positive for icaA gene. All strong biofilm-producing VRSA strains harbored a minimum of two virulence genes comprising clfA and icaA with 44.4% of them possessing all five virulence genes (icaA, tst, clfA, hla, and pvl), and 88.9% being multi-virulent. The study findings affirmed excellent in vitro antimicrobial and antibiofilm properties of BR-loaded MPS-NPs. Real-time quantitative reverse transcription PCR (qRT-PCR) assay displayed the downregulating role of BR-loaded MPS-NPs on strong biofilm-producing and multi-virulent VRSA strains virulence and agr genes in both in vitro and in vivo mice models. Additionally, BR-loaded MPS-NPs supplementation has a promising role in attenuating the upregulated expression of pro-inflammatory cytokines' genes in VRSA-infected mice with attenuation in pro-apoptotic genes expression resulting in reduced VRSA-induced apoptosis. In essence, the current study recommends the future scope of using BR-loaded MPS-NPs as auspicious alternatives for antimicrobials with tremendous antimicrobial, antibiofilm, anti-quorum sensing (QS), and anti-virulence effectiveness against problematic strong biofilm-producing and multi-virulent VRSA-associated infections.

A hope for ineffective antibiotics to return to treatment: investigating the anti-biofilm potential of melittin alone and in combination with penicillin and oxacillin against multidrug resistant-MRSA and -VRSA.

Background: The emergence and rapid spread of multi-drug resistant (MDR) bacterial strains, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA), have posed a significant challenge to the medical community due to their ability to form biofilm and develop resistance to common antibiotics. Traditional antibiotics that were once effective in treating bacterial infections are now becoming increasingly ineffective, leading to severe consequences for patient outcomes. This concerning situation has called for urgent research to explore alternative treatment strategies. Recent studies have shown that antimicrobial peptides (AMPs) hold promise as effective agents against biofilm-associated drug-resistant infections as well as to enhance the efficacy of conventional antibiotics. Accordingly, we aimed to investigate the antimicrobial and antibiofilm effects of melittin AMP, both alone and in combination with penicillin and oxacillin, against biofilm-forming MDR-MRSA and -VRSA. Methods: In this study, we investigated the kinetics of biofilm formation and assessed various parameters related to the antimicrobial and antibiofilm efficacy of melittin and antibiotics, both alone and in combination, against MDR-MRSA and -VRSA. The antimicrobial parameters included the Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC), Fractional Inhibitory Concentration Index (FICi), Fractional Bactericidal Concentration Index (FBCi), and the antibiofilm activity of melittin and antibiotics indicated by the Minimum Biofilm Inhibitory Concentration (MBIC), Minimal Biofilm Eradication Concentration (MBEC), Fractional Biofilm Inhibitory Concentration Index (FBICi), and Fractional Biofilm Eradication Concentration Index (FBECi). Results: The MIC results showed that all S. aureus isolates were resistant to penicillin (≥0.25 µg/mL), and 66% of isolates were resistant to oxacillin. The geometric means of the MIC values for penicillin, oxacillin, and melittin were 19.02, 16, and 1.62 µg/ml, respectively, and the geometric means of the MBC values for penicillin, oxacillin, and melittin were 107.63, 49.35, and 5.45 µg/ml, respectively. The study revealed that the combination indexes of melittin-penicillin and melittin-oxacillin, as determined by FIC values against all isolates, were 0.37 and 0.03, respectively. Additionally, melittin-penicillin and melittin-oxacillin exhibited combination indexes based on FBC values against all isolates at 1.145 and 0.711, respectively. Besides, melittin inhibited the biofilm formation of all S. aureus isolates, with MBIC values ranging from 10 to 1.25 µg/mL, and MBEC values ranging from 40 to 10 µg/mL. Generally, the combination indexes of melittin-penicillin and melittin-oxacillin, determined using FBIC values against all isolates, were 0.23 and 0.177, respectively. Moreover, melittin-penicillin and melittin-oxacillin typically had combination indexes based on FBEC values against all isolates at 5 and 2.97, respectively. Conclusion: In conclusion, our study provides evidence that melittin is effective against both planktonik and biofilm forms of MRSA and VRSA and exhibits significant synergistic effects when combined with antibiotics. These results suggest that melittin and antibiotics could be a potential candidate for further investigation for in vivo infections caused by MDR S. aureus. Furthermore, melittin has the potential to restore the efficacy of penicillin and oxacillin antibiotics in the treatment of MDR infections. Applying AMPs, like melittin, to revive beta-lactam antibiotics against MRSA and VRSA is an innovative approach against antibiotic-resistant bacteria. Further research is needed to optimize dosage and understand melittin mechanism and interactions with beta-lactam antibiotics for successful clinical applications.

Clinical and economic burden of bacteremia due to multidrug-resistant organisms in Korea: a prospective case control study.

OBJECTIVES: The socioeconomic and clinical burden of multidrug-resistant organisms (MDRO), including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), multidrug-resistant Acinetobacter baumannii (MRAB), multidrug-resistant Pseudomonas aeruginosa (MRPA), and carbapenem-resistant Enterobacteriaceae (CRE) have not yet been adequately addressed. METHODS: We prospectively searched for MDRO bacteremia cases with matched controls from 10 hospitals across Korea during a 6-month period in 2017. Patients were classified into the MDRO, susceptible organism, and no-infection groups. The corresponding susceptible or no-infection controls had been selected according to predefined criteria. We collected clinical information and estimated the total additional medical cost due to MDRO infections using the multistate model. RESULTS: During the 6-month period, a total of 486 MDRO bacteremia cases (260, 87, 18, 20, and 101 cases of MRSA, MRAB, MRPA, CRE, and VRE, respectively) were identified. The 90-d mortality rates were 30.4%, 63.2%, 16.7%, 55.0%, and 47.5%, respectively. The additional costs caused by bacteremia were $15 768, $35 682, $39 908, $72 051, and $33 662 per MDRO type, respectively. Based on these 6-month data, the estimated annual number of bacteremia cases due to these five MDRO in Korea were 7979 (4070, 1396, 218, 461, and 1834 cases, respectively). Overall, this caused an estimated 3280 (1237, 882, 36, 254, and 871, respectively) deaths and cost $294 505 002 ($84 707 359, $74 387 364, $10 344 370, $45 850 215, and $79 215 694, respectively) (range $170,627,020-$416,094,679) in socioeconomic loss. CONCLUSIONS: A tremendous clinical and economic burden is caused by MDRO bacteremia compared with antibiotic-susceptible and no-infection groups. Substantial investment and efforts by related government agencies and medical staffs are needed.