Methicillin-resistant Staphylococcus Aureus Biofilm Formation Research Articles

Scutellaria baicalensis Polysaccharide-Mediated Green Synthesis of Smaller Silver Nanoparticles with Enhanced Antimicrobial and Antibiofilm Activity.

Silver nanoparticles (AgNPs) have attracted widespread attention in multidrug-resistant bacterial infections. However, the application of AgNPs synthesized by conventional methods is restricted by its high costs, toxicity, and poor stability. Herein, a water-soluble polysaccharide (Scutellaria baicalensis polysaccharide, SBP) rich in reducing sugars was used as both the reductant and stabilizer to greenly synthesize spherical AgNPs@SBP with smaller particle sizes (11.18 ± 2.50 nm) and higher negative zeta potential (-23.05 ± 2.76 mV), which was favorable to enhance its antimicrobial activity and improve pH and thermal stability. Besides, SBP facilitated the adhesion and penetration of AgNPs@SBP to methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Escherichia coli (CREC), thus significantly enhancing its antibacterial activity (increased by 32-fold and 64-fold, respectively). Likewise, AgNPs@SBP at a low concentration (7.8 μg/mL) could effectively penetrate and inhibit nearly 90% of MRSA and CREC biofilm formation. Antimicrobial mechanism studies showed that AgNPs@SBP could lead to more severe cell membrane damage and genetic material leakage by upregulating reactive oxygen species and depolarizing mitochondrial membrane potential, ultimately resulting in the apoptosis of bacteria. Overall, the wrapping of SBP significantly enhanced the antibacterial and antibiofilm activity of AgNPs, which possessed great potential in the prevention and treatment of multidrug-resistant bacterial infections.

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.

Sub-inhibitory concentrations of tetrabromobisphenol A induce the biofilm formation of methicillin-resistant Staphylococcus aureus.

Biofilm formation by methicillin-resistant Staphylococcus aureus (MRSA) on indwelling medical devices complicates the treatment of infection. Tetrabromobisphenol A (TBBPA), a synthetic, lipophilic, halogenated aromatic compound widely used as an additive in plastics and electronic products, has raised environmental concerns due to its potential for bioaccumulation. This study investigated the impact of sub-inhibitory concentrations of TBBPA on MRSA biofilm formation. Crystal violet staining and confocal laser scanning microscopy analysis demonstrated that 1/8 MIC (0.5µg/mL) of TBBPA significantly stimulated MRSA biofilm formation (P < 0.0001). MTT assays indicated that the metabolic activity within the biofilms increased by 15.60-40.85% compared to untreated controls. Dot blot immunoassay, autolysis assay, and extracellular DNA (eDNA) quantification further revealed TBBPA enhanced the production of polysaccharide intercellular adhesin (PIA) and eDNA, which are key biofilm components. Additionally, TBBPA was found to enhance the production of staphyloxanthin, facilitating MRSA survival under oxidative conditions and in human whole blood. RT-qPCR analysis showed that TBBPA significantly upregulated genes associated with biofilm formation (icaA, atlA, sarA), staphyloxanthin biosynthesis (crtM and sigB), and oxidative stress responses (sodA and katA). These findings suggest that TBBPA promotes MRSA biofilm development and enhances bacterial resistance to adverse conditions, thereby potentially exacerbating risks to human health.

Cinnamaldehyde targets SarA to enhance β-lactam antibiotic activity against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a current global public health problem due to its increasing resistance to the most recent antibiotic therapies. One critical approach is to develop ways to revitalize existing antibiotics. Here, we show that the phytogenic compound cinnamaldehyde (CIN) and β-lactam antibiotic combinations can functionally synergize and resensitize clinical MRSA isolates to β-lactam therapy and inhibit MRSA biofilm formation. Mechanistic studies indicated that the CIN potentiation effect on β-lactams was primarily the result of inhibition of the mecA expression by targeting the staphylococcal accessory regulator sarA. CIN alone or in combination with β-lactams decreased sarA gene expression and increased SarA protein phosphorylation that impaired SarA binding to the mecA promoter element and downregulated virulence genes such as those encoding biofilm, α-hemolysin, and adhesin. Perturbation of SarA-mecA binding thus interfered with PBP2a biosynthesis and this decreased MRSA resistance to β-lactams. Furthermore, CIN fully restored the anti-MRSA activities of β-lactam antibiotics in vivo in murine models of bacteremia and biofilm infections. Together, our results indicated that CIN acts as a β-lactam adjuvant and can be applied as an alternative therapy to combat multidrug-resistant MRSA infections.

Micrococcal nuclease regulates biofilm formation and dispersal in methicillin-resistant Staphylococcus aureus USA300.

Biofilm formation is an important virulence factor for methicillin-resistant Staphylococcus aureus (MRSA). The extracellular matrix of MRSA biofilms contains significant amounts of double-stranded DNA that hold the biofilm together. MRSA cells secrete micrococcal nuclease (Nuc1), which degrades double-stranded DNA. In this study, we used standard methodologies to investigate the role of Nuc1 in MRSA biofilm formation and dispersal. We quantified biofilm formation and extracellular DNA (eDNA) levels in broth and agar cultures. In some experiments, cultures were supplemented with sub-MIC amoxicillin to induce biofilm formation. Biofilm erosion was quantitated by culturing biofilms on rods and enumerating detached colony-forming units (CFUs), and biofilm sloughing was investigated by perfusing biofilms cultured in glass tubes with fresh broth and measuring the sizes of the detached cell aggregates. We found that an MRSA nuc1- mutant strain produced significantly more biofilm and more eDNA than a wild-type strain, both in the absence and presence of sub-MIC amoxicillin. nuc1- mutant biofilms grown on rods detached significantly less than wild-type biofilms. Detachment was restored by exogenous DNase or complementing the nuc1- mutant. In the sloughing assay, nuc1- mutant biofilms released cell aggregates that were significantly larger than those released by wild-type biofilms. Our results suggest that Nuc1 modulates biofilm formation, biofilm detachment, and the sizes of detached cell aggregates. These processes may play a role in the spread and subsequent survival of MRSA biofilms during biofilm-related infections.IMPORTANCEInfections caused by antibiotic-resistant bacteria known as methicillin-resistant Staphylococcus aureus (MRSA) are a significant problem in hospitals. MRSA forms adherent biofilms on implanted medical devices such as catheters and breathing tubes. Bacteria can detach from biofilms on these devices and spread to other parts of the body such as the blood or lungs, where they can cause life-threatening infections. In this article, researchers show that MRSA secretes an enzyme known as thermonuclease that causes bacteria to detach from the biofilm. This is important because understanding the mechanism by which MRSA detaches from biofilms could lead to the development of procedures to mitigate the problem.

Multifunctional application of seagrass-derived rosmarinic acid in mitigating biofilm and quorum-sensing virulence transcripts of methicillin-resistant Staphylococcus aureus

Anti-microbial drug resistance is a primary public health concern, and the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) infections is gradually increasing. Considering the folk medicinal value of the seagrass Cymodocea rotundata and its antioxidant potential, the aim of this study was to assess C. rotundata for bioactive constituents that mitigate biofilm formation and quorum-sensing (QS) mechanism of MRSA. Different solvent extracts (chloroform, ethyl acetate, methanol, and distilled water) of C. rotundata were used, of which the methanolic extract exhibited maximum biofilm inhibition with complete cell lysis in 2 h. Column fractionation of the methanolic extract was performed, and 22 fractions were obtained. Fraction 7 exhibited maximum anti-biofilm potential, and the lead molecule was separated using thin-layer chromatography and characterized with high-performance liquid chromatography and nuclear magnetic resonance spectroscopy. The active constituent was identified as rosmarinic acid (RA), and the minimum inhibitory concentration was found to be 61.5 µg/mL; RA inhibited MRSA virulence factors such as protease by 89.37 %, lipase by 87.69 %, α-hemolysin by 91.52 %, and exopolysaccharides by 81.79 %, with an effective anti-adherence potential. Moreover, the lead molecule RA exhibited significant downregulation of QS-mediated virulence genes such as agrA, sarA, RNAIII, fnbA, fnbB, icaA, and icaD. Thus, seagrass C. rotundata has bioactive compounds with anti-microbial potential, and RA was found to control MRSA infection.

PH-Responsive Co-Assembled Peptide Hydrogel to Inhibit Drug-Resistant Bacterial Infection and Promote Wound Healing.

Drug-resistant bacterial infection and biofilm formation are the key inhibitors of wound healing, and new strategies are urgently needed to address these issues. In this study, we designed a pH-responsive co-assembled peptide hydrogel to inhibit Methicillin-resistant Staphylococcus aureus (MRSA) infection and promote wound healing. We synthesized a cationic short peptide (Nap-FFKKK) and a co-assembled hydrogel with curcumin at pH ∼ 7.8. The loaded curcumin was continuously released in a weak acid environment (pH ∼ 5.5). The lysine-rich cationic peptide inhibited biofilm formation in MRSA via electrostatic interaction with the negatively charged bacterial cell surface and, thus, provided a reinforcing antibacterial effect with curcumin. In vitro antibacterial experiments showed that the co-assembled system considerably reduced the minimum inhibitory concentration of curcumin against MRSA by 10-fold and promoted wound healing in a mouse model of MRSA-infected wounds. This study provides a simple and promising strategy to treat drug-resistant bacterial infections in wounds.

Ginkgo biloba L. exocarp petroleum ether extract inhibits methicillin-resistant Staphylococcus aureus by modulating ion transport, virulence, and biofilm formation in vitro and in vivo

Ethnopharmacological relevanceAs reported in the Ancient Chinese Medicinal Books, Ginkgo biloba L. fruit has been used as a traditional Chinese medicine for the treatment asthma and cough or as a disinfectant. Our previous study demonstrated that G. biloba exocarp extract (GBEE), an extract of a traditional Chinese herb, inhibits the formation of methicillin-resistant Staphylococcus aureus (MRSA) biofilms. However, GBEE is a crude extract that contains many components, and the underlying mechanisms of purified GBEE fractions extracted with solvents of different polarities are unknown. Aim of the studyThis study aimed to investigate the different components in GBEE fractions extracted with solvents of different polarities and their antibacterial effects and mechanisms against MRSA and Staphylococcus haemolyticus biofilms both in vitro and in vivo. MethodsThe components in different fractions were detected by high-performance liquid chromatography–high resolution mass spectrometry (HPLC–HRMS). Microbroth dilution assays and time growth curves were used to determine the antibacterial effects of the fractions on 15 clinical bacterial isolates. Crystal violet staining, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were utilized to identify the fractions that affected bacterial biofilm formation. The potential MRSA targets of the GBEE fraction obtained with petroleum ether (PE), denoted GBEE-PE, were screened by transcriptome sequencing, and the gene expression profile was verified by quantitative polymerase chain reaction (qPCR). ResultsHPLC–HRMS analysis revealed that the four GBEE fractions (extracted with petroleum ether, ethyl acetate, n-butanol, and water) contained different ginkgo components, and the antibacterial effects decreased as the polarity of the extraction solvent increased. The antibacterial activity of GBEE-PE was greater than that of the GBEE fraction extracted with ethyl acetate (EA). GBEE-PE improved H. illucens survival and reduced MRSA colonization in model mouse organs. Crystal violet staining and SEM and TEM analyses revealed that GBEE-PE inhibited MRSA and S. haemolyticus biofilm formation. Transcriptional analysis revealed that GBEE-PE inhibits MRSA biofilms by altering ion transport, cell wall metabolism and virulence-related gene expression. In addition, the LO2 cell viability and H. illucens toxicity assay data showed that GBEE-PE at 20 mg/kg was nontoxic. ConclusionThe GBEE fractions contained different components, and their antibacterial effects decreased with increases in the polarity of the extraction solvent. GBEE-PE limited MRSA growth and biofilm formation by affecting ion transport, cell wall synthesis, and virulence-related pathways. This research provides a more detailed overview of the mechanism by which GBEE-PE inhibits MRSA both in vitro and in vivo and suggests that GBEE-PE is a new prospective antimicrobial with the potential to be used in MRSA therapeutics in the future.

Peptide recovery from chicken feather keratin and their anti-biofilm properties against methicillin-resistant Staphylococcus aureus (MRSA).

Bacteria have the potential to adhere to abiotic surfaces, which has an undesirable effect in the food industry because they can survive for sustained periods through biofilm formation. In this study, an antibacterial peptide (ABP), with a molecular mass of 3861 Da, was purified from hydrolyzed chicken feathers using a locally isolated keratinolytic bacterium, namely Rhodococcus erythropolis, and its antibacterial and antibiofilm potential were investigated against planktonic and biofilm cells of Methicillin-Resistant Staphylococcus Aureus (MRSA). The results demonstrated that purified ABP showed the growth inhibition of MRSA cells with the minimum inhibitory concentration (MIC) of 45µg/ml and disrupted MRSA biofilm formation at a concentration of 200 ug/ml, which results were confirmed by scanning electron micrograph (SEM). Moreover, the secondary structures of the peptide were assessed as part of the FTIR analysis to evaluate its mode of action. ExPASy tools were used to predict the ABP sequence, EPCVQUQDSRVVIQPSPVVVVTLPGPILSSFPQNTA, from a chicken feather keratin sequence database following in silico digestion by trypsin. Also, ABP had 54.29% hydrophobic amino acids, potentially contributing to its antimicrobial activity. The findings of toxicity prediction of the peptide by the ToxinPred tool revealed that ABP had non-toxic effects. Thus, these results support the potential of this peptide to be used as an antimicrobial agent for the treatment or prevention of MRSA biofilm formation in feed, food, or pharmaceutical applications.

Piperine, a phytochemical prevents the biofilm city of methicillin-resistant Staphylococcus aureus: A biochemical approach to understand the underlying mechanism

Methicillin-resistant Staphylococcus aureus (MRSA), a drug-resistant human pathogen causes several nosocomial as well as community-acquired infections involving biofilm machinery. Hence, it has gained a wide interest within the scientific community to impede biofilm-induced MRSA-associated health complications. The current study focuses on the utilization of a natural bioactive compound called piperine to control the biofilm development of MRSA. Quantitative assessments like crystal violet, total protein recovery, and fluorescein-di-acetate (FDA) hydrolysis assays, demonstrated that piperine (8 and 16 μg/mL) could effectively compromise the biofilm formation of MRSA. Light and scanning electron microscopic image analysis confirmed the same. Further investigation revealed that piperine could reduce extracellular polysaccharide production by down-regulating the expression of icaA gene. Besides, piperine could reduce the cell-surface hydrophobicity of MRSA, a crucial factor of biofilm formation. Moreover, the introduction of piperine could interfere with microbial motility indicating the interaction of piperine with the quorum-sensing components. A molecular dynamics study showed a stable binding between piperine and AgrA protein (regulator of quorum sensing) suggesting the possible meddling of piperine in quorum-sensing of MRSA. Additionally, the exposure to piperine led to the accumulation of intracellular reactive oxygen species (ROS) and potentially heightened cell membrane permeability in inhibiting microbial biofilm formation. Besides, piperine could reduce the secretion of diverse virulence factors from MRSA. Further exploration revealed that piperine interacted with extracellular DNA (e-DNA), causing disintegration by weakening the biofilm architecture. Conclusively, this study suggests that piperine could be a potential antibiofilm molecule against MRSA-associated biofilm infections.

Biofilm Producing Methicillin-Resistant Staphylococcus aureus (MRSA) Infections in Humans: Clinical Implications and Management.

Since its initial description in the 1960s, methicillin-resistant Staphylococcus aureus (MRSA) has developed multiple mechanisms for antimicrobial resistance and evading the immune system, including biofilm production. MRSA is now a widespread pathogen, causing a spectrum of infections ranging from superficial skin issues to severe conditions like osteoarticular infections and endocarditis, leading to high morbidity and mortality. Biofilm production is a key aspect of MRSA's ability to invade, spread, and resist antimicrobial treatments. Environmental factors, such as suboptimal antibiotics, pH, temperature, and tissue oxygen levels, enhance biofilm formation. Biofilms are intricate bacterial structures with dense organisms embedded in polysaccharides, promoting their resilience. The process involves stages of attachment, expansion, maturation, and eventually disassembly or dispersion. MRSA's biofilm formation has a complex molecular foundation, involving genes like icaADBC, fnbA, fnbB, clfA, clfB, atl, agr, sarA, sarZ, sigB, sarX, psm, icaR, and srtA. Recognizing pivotal genes for biofilm formation has led to potential therapeutic strategies targeting elemental and enzymatic properties to combat MRSA biofilms. This review provides a practical approach for healthcare practitioners, addressing biofilm pathogenesis, disease spectrum, and management guidelines, including advances in treatment. Effective management involves appropriate antimicrobial therapy, surgical interventions, foreign body removal, and robust infection control practices to curtail spread within healthcare environments.

A Preliminary Study on the Effect of Deferoxamine on the Disruption of Bacterial Biofilms and Antimicrobial Resistance.

Antiviral therapy approaches have become significant strategies to combat antibiotic resistance. Metal ions, particularly iron, play crucial roles in metabolic activities and virulence of bacteria. Loading iron into siderophore molecules could potentially circumvent antimicrobial resistance. This study aimed to evaluate the antibiofilm and antimicrobial effects of deferoxamine (DFO), an iron chelator and natural siderophore, on antibiotic susceptibility in clinical methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Acinetobacter baumannii (CRAB) isolates. The in vitro antibacterial activity of DFO alone and in combination with vancomycin [VAN (30 μg)], amoxicillin (25 μg), colistin (10 μg), and imipenem (10 μg), was investigated against MRSA and CRAB isolates using the disk diffusion method. The spectrophotometric microplate method was used to detect the in vitro antibiofilm effect of DFO. DFO exhibited a synergistic effect with VAN, amoxicillin, and colistin and significantly disrupted mature biofilm formation in MRSA and CRAB isolates. Notably, the antibiofilm effect of DFO was more pronounced in CRAB strains. These findings highlight the potential of DFO as an antibiofilm agent candidate and suggest that it can enhance the antibiotic susceptibility of certain microorganism species.

Experimental Study on the Inhibition of Methicillin-Resistant Staphylococcus aureus Growth and Biofilm Formation by Berberine Hydrochloride Combined with Vancomycin

Our study aimed to investigate the combined effect of berberine hydrochloride and vancomycin on Methicillin-resistant Staphylococcus aureus (MRSA) planktonic bacteria. MRSA strains were isolated from patients with periprosthetic infections. In vitro experiments were conducted to examine changes in the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the bacteria when treated with the combination. The study found that berberine hydrochloride enhanced the antibacterial effect of vancomycin against MRSA. Interestingly, sub-inhibitory concentrations of vancomycin led to increased biofilm formation of MRSA, with a more pronounced effect as the concentration decreased. Berberine hydrochloride partly increased MRSA biofilm formation when combined with sub-inhibitory concentrations of vancomycin, but it reduced the promotion of MRSA biofilm formation at MIC concentrations. Notably, the combination of berberine hydrochloride and vancomycin at MIC concentrations decreased MRSA’s adhesion ability, possibly linked to the down-regulation of biofilm formation-related genes (icaA, sarA, and cidA). Overall, these findings suggest that berberine hydrochloride, in conjunction with vancomycin, can exert an inhibitory effect against MRSA to some extent. This combination has the potential to enhance the antimicrobial activity of vancomycin and may hold promise in combating MRSA infections.

Spinach protein-capped silver nanoparticles with low hemolytic activity inhibit methicillin-resistant Staphylococcus aureus biofilm formation as a new therapeutic approach

BackgroundThe methicillin-resistant Staphylococcus aureus (MRSA) was designated as a high-priority pathogen by the World Health Organization. Cardiovascular and thoracic surgery complications frequently involve MRSA infection. Treatment of newly emerging resistant S. aureus strains is clinically challenging due to the natural tendency of MRSA survival through biofilm formation. The failure of traditional antibiotic therapy necessitates development of next-generation therapeutic molecules. ObjectivesSilver nanoparticles were investigated to treat MRSA in order to overcome the challenges of antibiotic therapy. MethodsSpinach protein functionalized silver nanoparticles (SPAgNPs) were synthesized by reducing with sodium borohydride. The resazurin microtiter assay was used to determine the minimum inhibitory concentration (MIC) of SPAgNPs against MRSA. The antibiofilm activity was investigated using a crystal violet assay at sub-MIC levels and validated using scanning electron microscopy. To demonstrate the mechanism of action of SPAgNPs, the virulence factors involved in MRSA biofilm formation were examined. SEM was used to evaluate the hemocompatibility of human red blood cells. Results and conclusionThe synthesized SPAgNPs showed characteristic yellow colour with surface Plasmon resonance maximum at 418 nm. Powder X-ray diffraction and transmission electron microscopy analysis confirmed that the particles are crystalline with a size range from 14 to 40 nm. SPAgNPs have antibacterial activity at a minimum inhibitory concentration (MIC) of 1.25 mM, killing MRSA via disrupting membrane integrity and producing excess reactive oxygen species. Strikingly, SPAgNPs inhibit MRSA biofilm formation at sub-MIC (0.625 mM). Scanning electron microscopy study reveals that SPAgNPs trigger biofilm inhibition without causing cell damage. Hence the NPs are very effective in preventing pathogens from triggering stress responses. SPAgNPs suppress exopolysaccharide production, cell surface hydrophobicity, and staphyloxanthin biosynthesis. Testing SPAgNPs against human red blood cells reveals that NPs do not harm human cells, suggesting that the SPAgNPs are biocompatible. The findings show that SPAgNPs could be explored as next-generation materials for treating MDR pathogenic infection linked with biofilms.

A novel antibacterial compound decreases MRSA biofilm formation without the use of antibiotics in a murine model.

Despite significant advancements in material science, surgical site infection (SSI) rates remain high and prevention is key. This study aimed to demonstrate the in vivo safety and antibacterial efficacy of titanium implants treated with a novel broad-spectrum biocidal compound (DBG21) against methicillin-resistant Staphylococcus aureus (MRSA). Titanium (Ti) discs were covalently bound with DBG21. Untreated Ti discs were used as controls. All discs were implanted either untreated for 44 control mice or DBG21-treated for 44 treated mice. After implantation, 1 × 107 colony forming units (CFU) of MRSA were injected into the operating site. Mice were killed at 7 and 14 daysto determine the number of adherent bacteria (biofilm) on implants and in the peri-implant surrounding tissues. Systemic and local toxicity were assessed. At both 7 and 14 days, DBG21-treated implants yielded a significant decrease in MRSA biofilm (3.6 median log10 CFU [99.97%] reduction [p < 0.001] and 1.9 median log10 CFU [98.7%] reduction [p = 0.037], respectively) and peri-implant surrounding tissues (2.7 median log10 CFU/g [99.8%] reduction [p < 0.001] and 5.6 median log10 CFU/g [99.9997%] reduction [p < 0.001], respectively). There were no significant differences between control and treated mice in terms of systemic and local toxicity. DBG-21 demonstrated a significant decrease in the number of biofilm bacteria without associated toxicity in a small animal implant model of SSI. Preventing biofilm formation has been recognized as a key element of preventing implant-related infections.

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.

Isolation of lectin from Musa acuminata for its antibiofilm potential against Methicillin-resistant Staphylococcus aureus and its synergistic effect with Enterococcus species.

Methicillin resistant Staphylococcus aureus (MRSA) is an emerging pathogen posing a considerable burden on the healthcare system due to its involvement in skin and soft tissue infections (SSTIs). Lectins are carbohydrate binding proteins found ubiquitously in animals, plants and microorganisms. Extraction and isolation of proteins from Musa acuminata were performed by using Affinity chromatography with Sephadex G 75 to determine antibiofilm activity against MRSA. Enterococcus strains obtained from dairy products, beans and vegetables were also screened for its potential to inhibit growth and biofilm formation of MRSA by using 96 well microtiter plates. Synergistic effect of cell free supernatant of Enterococcus with proteins from ripe banana were also tested. BanLec was successfully isolated and appeared as 15 KDa band after SDS-PAGE (15%) while multiple bands of unbound protein fractions were observed. The unbound fractions showed inhibition of planktonic cells and biofilm but BanLec exhibited no significant effect. The CFS of Enterococcus faecium (LCM002), Enterococcus lactis (LCM003) and Enterococcus durans (LCM004 and LCM005) displayed antagonistic effects against pathogen. The synergistic effect of CFS from E. lactis (LCM003) and unbound proteins showed inhibition of biofilm and pathogenic growth. This study demonstrates the use of Enterococcus species and plant proteins against pathogens and results suggested that it can inhibit the growth of resistant strains of Staphylococcus aureus and their synergistic effect has opened new ways to tackle emerging resistance. Furthermore, after assessment of Enterococcus as probiotics, this could be used in food industries as well as in treatment of severe skin infections.

Natural biomass-derived carbon dots as potent antimicrobial agents against multidrug-resistant bacteria and their biofilms

Infections caused by multidrug-resistant (MDR) bacteria have become a major challenge for healthcare systems worldwide. Antibacterial carbon dots (CDs) have received increasing attention for fighting MDR bacteria. In this study, we synthesized nitrogen and sulfur self-doped garlic carbon dots (GCDs) via a one-step hydrothermal method. The prepared GCDs are sphere-like nanoparticles with graphite lattice structure and positive charge (+8.53 mV). The minimum inhibitory concentrations (MICs) of GCDs against methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Salmonella Typhimurium (MRST), and generic Escherichia coli were 25 μg/mL, 75 μg/mL, and 50 μg/mL, respectively. The MICs of kanamycin against MRSA and MRST were approximately 64 and 18 times higher than those of GCDs, respectively. GCDs at 200 μg/mL eliminated all three bacteria within 180 min. GCDs also effectively inhibited the biofilm formation of MRSA and MRST in a dose-dependent manner. The antimicrobial mechanisms of GCDs against MDR bacteria include: (1) electrostatic interaction facilitated penetration into MDR bacteria; (2) disruption of cell structure and subsequent leakage of intracellular components; (3) induction of oxidative stress and inhibition of the antioxidant enzymes. In addition, GCDs showed high biocompatibility in hemolysis assays and vitro cytotoxicity assays. In conclusion, this study developed a novel antimicrobial nanomaterial based on natural plants, providing an important idea for the rational use of natural biomass against MDR bacterial infections.

Effect of Ruta graveolens Extract on the Major Virulence Factors in Methicillin Resistant Staphylococcus aureus.

Rising Antibiotic Resistance has put the world in real threat. Methicillin resistant Staphylococcus aureus (MRSA), is a predominant cause of suppurative chronic skin and soft-tissue infections. Novel insights have focused the light on plant extracts. In this study, Ruta graveolens ethanolic active extract was tested for its potential anti-virulence activities in MRSA. A total of 100 MRSA strains causing skin and soft tissue infections were isolated and antibiotic susceptibility testing was done. Ability to form biofilm was tested phenotypically. Furthermore, the antimicrobial activity of Ruta graveolens was evaluated followed by detection of its Minimum inhibitory concentration (MIC). The inhibitory activity of this extract on biofilm formation was investigated. Afterwards, we investigated its effect on the transcription of biofilm-related genes and mecA gene. All tested isolates were sensitive to Vancomycin and Linezolid while high resistance was noted with both Fusidic acid (83%) and Gentamicin (68%). (83%) of the isolates were biofilm producers. Ruta graveolens extract showed strong antimicrobial activity against the MRSA strains with MIC 0.78 mg/mL. At subinhibitory concentration (1/2 MIC), the extract had high biofilm inhibitory effects with mean inhibition (70%). Moreover, transcriptional analysis results showed that the mean percentages of inhibition in expression of mecA, icaA and icaD genes were 52.3%, 34.8% and 33.7%, respectively, in which all showed statistically significant difference (p ≤ 0.05). The current study proposes the ability of Ruta graveolens extract to reduce the biofilm formation and antibiotic resistance of MRSA through downregulation of some biofilm forming genes and mecA gene which confers resistance to B-lactam antibiotics. This may decrease our reliance on antibiotics and improve our ability to effectively treat biofilm-related skin and soft-tissue infections caused by MRSA.

GltS regulates biofilm formation in methicillin-resistant Staphylococcus aureus

Biofilm-based infection is a major healthcare burden. Methicillin-resistant Staphylococcus aureus (MRSA) is one of major organisms responsible for biofilm infection. Although biofilm is induced by a number of environmental signals, the molecule responsible for environmental sensing is not well delineated. Here we examined the role of ion transporters in biofilm formation and found that the sodium-glutamate transporter gltS played an important role in biofilm formation in MRSA. This was shown by gltS transposon mutant as well as its complementation. The lack of exogenous glutamate also enhanced biofilm formation in JE2 strain. The deficiency of exogenous glutamate intake accelerated endogenous glutamate/glutamine production, which led to the activation of the urea cycle. We also showed that urea cycle activation was critical for biofilm formation. In conclusion, we showed that gltS was a critical regulator of biofilm formation by controlling the intake of exogenous glutamate. An intervention to target glutamate intake may be a potential useful approach against biofilm.