Methicillin-resistant Staphylococcus Aureus Biofilm Formation Research Articles

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, andthe 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 2h. 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 61.5µg/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.

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.

Identification and Antibiotic Resistance Profile of Biofilm-forming Methicillin Resistant Staphylococcus aureus (MRSA) Causing Infection among Orthopedic Wound Patients

Background and Objectives: The biofilm-forming ability of Methicillin-Resistant Staphylococcus aureus(MRSA) strains have demonstrated the involvement of MRSA biofilm in antibiotic resistance, recalcitrant and persistent infections in humans. Despite a deeper understanding of the biofilm-forming ability of MRSAstrain, it is still essential to extend the research on the identification and antibiotic resistance profile of biofilm-forming MRSA causing infection among orthopedic wound patients.&#x0D; Methodology: A total of three hundred and thirty (303) patient-isolate of non-repeatable Staphylococcus aureus strains were obtained during the period of 2021 until 2022 from fracture and post-surgical orthopedic wound patients with wound duration &gt;2months at the National Orthopedic Hospital, Enugu (NOHE). S. aureus were identified using conventional microbiological cultures Technique followed by confirmation of MRSA strain through Brilliance MRSA 2 Agar. Antibiotic Susceptibility testing (AST) of biofilm-forming MRSA was performed using the Kirby–Bauer disk diffusion method and the results were interpreted using the Clinical Laboratory Standard Institute (CLSI) zone diameter breakpoints. Multidrug Resistance (MDR) was determined for biofilm-forming MRSA.&#x0D; Result:Of the 303 isolate of S. aureus, MRSA strain accounted 86(28.4 %) and 78(25.7 %) from post-surgical wound and fracture wound respectively while biofilm forming MRSA was identified in 101(33.4%) MRSA strain consisting of high proportion 66(21.8 %) fromPost-surgical wound followed by fracture wound samples recording 35(11.6 %). Association between MRSA production and biofilm formation was considered statistically significant at P&lt; .05. The proportion of biofilm-forming MRSA resistance to β-lactam accounted 71.4-100%, macrolide resistance recorded 65.7-92.4 %, lincosamideresistance 74.3-100 %, glycopeptide resistance proportion ranged from 62.8-100 % while low level of resistance to fluoroquinolones 19.7-42.9 % and Aminoglycoside 8.6-10.6 % was observed. Biofilm-forming MRSA isolate were MDR to one or more antibiotic antimicrobial agents in at least three categories withMDRIndex range ≥ 0.3 but majority of the isolate were 91.4% and 100% susceptible to Gentamicin and Imipenem.&#x0D; Conclusion: The invitro expression of biofilm formation among MRSA strain and their antibiotic resistance profile in this study makes them a potential threat and challenging pathogens with the ability to cause persistent infections in humans, especially among orthopedic wound patients. Thus the development of an antimicrobial stewardship program and regular detection of biofilm production is needed for timely intervention while judicious use of Imipenem and Gentamicin as a drug of choice for effective treatment of infection caused by biofilm-forming MRSA among orthopedic patients will avert the severity of infection. Further research of these sort should investigate the genotyping expression of a biofilm gene variant in other human diseases, different bacteria species, and orthopedic medical implant devices.

Dispersal of pathogen-associated multispecies biofilm by novel probiotic Bacillus subtilis in a contact-dependent manner.

Biofilms are involved in pathogenesis of various bacterial infections. Treatment of biofilm-related bacterial infection remains a major challenge due to the reduced efficacy of antibiotics and associated antibiotic resistance. Given the high prevalence of Enterotoxigenic Escherichia coli (ETEC), Salmonella Typhimurium (S. Typhimurium) and methicillin-resistant Staphylococcus aureus (MRSA)-related infections and associated drug resistance, it is imperative to develop alternative strategies for treatment and prevention. The current study investigated antibiofilm activity of a recently isolated Bacillus subtilis (B. subtilis-9) against these pathogens. Crystal violet staining showed that treatment with B. subtilis-9 significantly reduced biofilm biomass of ETEC (60%-80%), S. Typhimurium (68%-73%) and MRSA (66%-82%). In addition, B. subtilis-9 significantly reduced pre-formed biofilm biomass of ETEC (59%), S. Typhimurium (62%), MRSA (65%) and multispecies (58%). Fluorescence microscopy revealed that B. subtilis-9 treatment significantly reduced the thickness of biofilm and viability of the embedded bacteria. Additionally, B. subtilis-9 significantly reduced planktonic cell growth of ETEC (92%), S. Typhimurium (94%) and MRSA (93%). Interestingly, transwell assay showed that B. subtilis-9 exhibited antibiofilm properties in a cell-to-cell contact-dependent manner and significantly reduced mRNA expression of biofilm-related genes, bssS, luxS and ihfB in ETEC. Novel B. subtilis-9 exhibits a strong inhibitory activity against ETEC, S. Typhimurium and MRSA biofilm formation and adhesion to abiotic surfaces. With further investigations, our study could bring forward a novel Bacillus-based probiotic intervention strategy to combat pathogenic biofilms, in clinical and agricultural settings. Probiotic bacteria propose a potential alternative in combating biofilm-related infections, however, data on the efficacy and strain selection are limited. Data from this study are critical in further developing Bacillus-based novel probiotic applications that may reduce the use of antibiotics in biofilm-related infections in humans and animals.

Quorum sensing inhibitory potential of vaccenic acid against Chromobacterium violaceum and methicillin-resistant Staphylococcus aureus.

Quorum sensing (QS) is a potential target for inhibiting bacterial antibiotic resistance and associated pathogenicity. The present study aimed to investigate vaccenic acid anti-QS and antibiofilm potential against Chromobacterium violaceum and methicillin-resistant Staphylococcus aureus (MRSA). In the broth microdilution method, we determined the minimum inhibitory concentration (MIC) of vaccenic acid against C. violaceum and MRSA. Then, we determined the vaccenic acid anti-QS potential against C. violaceum via a violacein inhibition assay. Vaccenic acid at a sub-MIC concentration significantly inhibited violacein pigment production. Vaccenic acid also inhibits C. violaceum and MRSA biofilm formation at sub-MIC concentrations. The effect of vaccenic acid antivirulence potential was evaluated by phenotypic virulence assays. The results showed that vaccenic acid at a sub-MIC concentration significantly inhibited the virulence production of C. violaceum (chitinase and motility) and MRSA (hemolysin and staphyloxanthin production). Quantitative PCR analysis revealed the downregulation of QS associated genes upon vaccenic acid treatment. This resulted in the downregulation of genes involved in QS mechanisms such as cviI, cviR, and SarA and pigment production such as vioB and crtM. The results of the present study suggest that vaccenic acid is a promising agent to combat C. violaceum and MRSA.

Targeted inhibition of methicillin-resistant Staphylococcus aureus biofilm formation by a graphene oxide-loaded aptamer/berberine bifunctional complex

Biofilm formation is known to promote drug resistance in methicillin-resistant Staphylococcus aureus (MRSA), which is closely related to persistent infections in hospital settings. In this study, a DNA aptamer specific to penicillin-binding protein 2a (PBP2a) with a dissociation constant (K d) of 82.97 ± 8.86 nM was obtained after 14 cycles of systematic evolution of ligands by exponential enrichment (SELEX). Next, a bifunctional complex containing the aptamer intercalated by berberine into the double-strand region was prepared and adsorbed onto the surface of graphene oxide (GO) by π-stacking interactions. The GO-loaded aptamer/berberine bifunctional complex showed significantly higher inhibition of MRSA biofilm formation than the control. Furthermore, this study shows that the complex possesses anti-biofilm activity, which can be attributed to the ability of the aptamer to reduce cell-surface attachment by blocking the function of PBP2a and berberine to attenuate the level of the accessory gene regulator (agr) system, which plays an important role in mediating MRSA biofilm formation. Therefore, the simultaneous delivery of berberine and PBP2a-targted aptamer using GO may have potential for the treatment of chronic infections caused by MRSA biofilms. It also provides a new avenue for multitarget treatment of bacterial biofilms.

Mptx2 defends against peritoneal infection by methicillin-resistant staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) has an increasing prevalence of multi-drug resistance. There is an urgent need for developing novel approaches to combat MRSA infection. Mucosal pentraxin 2 (Mptx2) is predicted to be a member of the pentraxin family, but its biological function is still unknown. This study is aimed to explore the roles of Mptx2 in MRSA-associated peritoneal infection. The recombinant Mptx2 protein is used to evaluate its antibacterial activity. Biofilm formation assay and macrophage phagocytic experiment are performed to explore the involved mechanisms. The effects of Mptx2 on peritoneal infection are investigated in a MRSA-induced peritoneal infected model. We here show that addition of Mptx2 suppresses the growth and biofilm formation of MRSA in vitro. Enzyme-linked immunosorbent assay (ELISA) binding analysis shows that Mptx2 protein directly binds to the MRSA. Additionally, Mptx2 supplementation promotes macrophages to phagocytize and clear the MRSA. In the MRSA-infected peritonitis model, Mptx2 administration reduces MRSA loading in peritoneal organs and alleviates peritoneal damage. Mptx2 knockout aggravates MRSA infection-induced peritoneal injury. In conclusion, our findings reveal that Mptx2 has bactericidal activity against MRSA both in vitro and in vivo, which may shed light on the discovery and development of novel strategies for MRSA-infected peritonitis.