CXCR2 perturbation promotes Staphylococcus aureus implant-associated infection.

Introduction. Staphylococcus aureus is the leading cause of acute medical implant infections, representing a significant modern medical concern. The success of S. aureus as a pathogen in these cases resides in its arsenal of virulence factors, resistance to multiple antimicrobials, mechanisms of immune modulation, and ability to rapidly form biofilms associated with implant surfaces. S. aureus device-associated, biofilm-mediated infections are often persistent and notoriously difficult to treat, skewing innate immune responses to promote chronic reoccurring infections. While relatively little is known of the role neutrophils play in response to acute S. aureus biofilm infections, these effector cells must be efficiently recruited to sites of infection via directed chemotaxis. Here we investigate the effects of modulating CXC chemokine receptor 2 (CXCR2) activity, predominantly expressed on neutrophils, during S. aureus implant-associated infection.Hypothesis. We hypothesize that modulation of CXCR2 expression and/or signalling activities during S. aureus infection, and thus neutrophil recruitment, extravasation and antimicrobial activity, will affect infection control and bacterial burdens in a mouse model of implant-associated infection.Aim. This investigation aims to elucidate the impact of altered CXCR2 activity during S. aureus biofilm-mediated infection that may help develop a framework for an effective novel strategy to prevent morbidity and mortality associated with implant infections.Methodology. To examine the role of CXCR2 during S. aureus implant infection, we employed a mouse model of indwelling subcutaneous catheter infection using a community-associated methicillin-resistant S. aureus (MRSA) strain. To assess the role of CXCR2 induction or inhibition during infection, treatment groups received daily intraperitoneal doses of either Lipocalin-2 (Lcn2) or AZD5069, respectively. At the end of the study, catheters and surrounding soft tissues were analysed for bacterial burdens and dissemination, and Cxcr2 transcription within the implant-associated tissues was quantified.Results. Mice treated with Lcn2 developed higher bacterial burdens within the soft tissue surrounding the implant site, which was associated with increased Cxcr2 expression. AZD5069 treatment also resulted in increased implant- and tissues-associated bacterial titres, as well as enhanced Cxcr2 expression.Conclusion. Our results demonstrate that CXCR2 plays an essential role in regulating the severity of S. aureus implant-associated infections. Interestingly, however, perturbation of CXCR2 expression or signalling both resulted in enhanced Cxcr2 transcription and elevated implant-associated bacterial burdens. Thus, CXCR2 appears finely tuned to efficiently recruit effector cells and mediate control of S. aureus biofilm-mediated infection.

Exploring secretory proteome and cytokine kinetic of human peripheral blood mononuclear cells exposed to methicillin-resistant Staphylococcus aureus biofilms and planktonic bacteria.

Staphylococcus aureus is a highly successful pathogen infecting various body parts and forming biofilms on natural and artificial surfaces resulting in difficult-to-treat and chronic infections. We investigated the secreted cytokines and proteomes of isolated peripheral blood mononuclear cells (PBMCs) from healthy volunteers exposed to methicillin-resistant S. aureus (MRSA) biofilms or planktonic bacteria. Additionally, the cytokine profiles in sera from patients with community-acquired pneumonia (CAP) caused by S. aureus were investigated. The aim was to gain insights into the immune response involved and differentiate between the planktonic and sessile MRSA forms. We identified 321 and 298 targets that were significantly differently expressed in PBMCs when exposed to planktonic or biofilm-embedded bacteria, respectively. PBMCs exposed to planktonic MRSA cells secreted increased levels of TNF-α, while IL-18 was elevated when exposed to the biofilm. The machine-learning analyses of the cytokine profiles obtained for the in vitro PBMCs and CAP sera distinguished between the two types of bacteria forms based on cytokines IL-18, IL12, and IL-17, and with a lower importance IL-6. Particularly, IL-18 which has not been correlated with S. aureus biofilms so far might represent a suitable marker for monitoring chronification during MRSA infection to individualize the therapy, but this hypothesis must be proved in clinical trials.

Deciphering the dynamics of methicillin-resistant Staphylococcus aureus biofilm formation: from molecular signaling to nanotherapeutic advances.

Methicillin-resistant Staphylococcus aureus (MRSA) represents a global threat, necessitating the development of effective solutions to combat this emerging superbug. In response to selective pressures within healthcare, community, and livestock settings, MRSA has evolved increased biofilm formation as a multifaceted virulence and defensive mechanism, enabling the bacterium to thrive in harsh conditions. This review discusses the molecular mechanisms contributing to biofilm formation across its developmental stages, hence representing a step forward in developing promising strategies for impeding or eradicating biofilms. During staphylococcal biofilm development, cell wall-anchored proteins attach bacterial cells to biotic or abiotic surfaces; extracellular polymeric substances build scaffolds for biofilm formation; the cidABC operon controls cell lysis within the biofilm, and proteases facilitate dispersal. Beside the three main sequential stages of biofilm formation (attachment, maturation, and dispersal), this review unveils two unique developmental stages in the biofilm formation process for MRSA; multiplication and exodus. We also highlighted the quorum sensing as a cell-to-cell communication process, allowing distant bacterial cells to adapt to the conditions surrounding the bacterial biofilm. In S. aureus, the quorum sensing process is mediated by autoinducing peptides (AIPs) as signaling molecules, with the accessory gene regulator system playing a pivotal role in orchestrating the production of AIPs and various virulence factors. Several quorum inhibitors showed promising anti-virulence and antibiofilm effects that vary in type and function according to the targeted molecule. Disrupting the biofilm architecture and eradicating sessile bacterial cells are crucial steps to prevent colonization on other surfaces or organs. In this context, nanoparticles emerge as efficient carriers for delivering antimicrobial and antibiofilm agents throughout the biofilm architecture. Although metal-based nanoparticles have been previously used in combatting biofilms, its non-degradability and toxicity within the human body presents a real challenge. Therefore, organic nanoparticles in conjunction with quorum inhibitors have been proposed as a promising strategy against biofilms. As nanotherapeutics continue to gain recognition as an antibiofilm strategy, the development of more antibiofilm nanotherapeutics could offer a promising solution to combat biofilm-mediated resistance.

γδ T cell-intrinsic IL-1R promotes survival during Staphylococcus aureus bacteremia.

Staphylococcus aureus is a leading cause of bacteremia, further complicated by the emergence of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA). A better understanding of host defense mechanisms is needed for the development of host-directed therapies as an alternative approach to antibiotics. The levels of IL-1, IL-17, and TNF-α cytokines in circulation have been associated with predictive outcomes in patients with S. aureus bacteremia. However, their causative role in survival and the cell types involved in these responses during bacteremia is not entirely clear. Using a mouse model of S. aureus bacteremia, we demonstrated that IL-17A/F and TNF-α had no significant impact on survival, whereas IL-1R signaling was critical for survival during S. aureus bacteremia. Furthermore, we identified that T cells, but not neutrophils, monocytes/macrophages, or endothelial cells were the crucial cell type for IL-1R-mediated survival against S. aureus bacteremia. Finally, we determined that the expression of IL-1R on γδ T cell, but not CD4+ or CD8+ T cells was responsible for survival against the S. aureus bacteremia. Taken together, we uncovered a role for IL-1R, but not IL-17A/F and TNF-α in protection against S. aureus bacteremia. Importantly, γδ T cell-intrinsic expression of IL-1R was crucial for survival, but not on other immune cells or endothelial cells. These findings reveal potential cellular and immunological targets for host-directed therapies for improved outcomes against S. aureus bacteremia.

Current therapies and challenges for the treatment of Staphylococcus aureus biofilm-related infections.

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections and contributes to significant increase in morbidity and mortality especially when associated with medical devices and in biofilm form. Biofilm structure provides a pathway for the enrichment of resistant and persistent phenotypes of S. aureus leading to relapse and recurrence of infection. Minimal diffusion of antibiotics inside biofilm structure leads to heterogeneity and distinct physiological activity. Additionally, horizontal gene transfer between cells in proximity adds to the challenges associated with eradication of biofilms. This narrative review focuses on biofilm-associated infections caused by S. aureus, the impact of environmental conditions on biofilm formation, interactions inside biofilm communities, and the clinical challenges that they present. Conclusively, potential solutions, novel treatment strategies, combination therapies, and reported alternatives are discussed.

Implant-associated biofilm infection established in an experimental Galleria mellonella model.

The study reports in vivo biofilm infection implemented in an insect model. We mimicked implant-associated biofilm infections in Galleria mellonella larvae using toothbrush bristles and methicillin-resistant Staphylococcus aureus (MRSA). In vivo biofilm formation on bristle was achieved by sequentially injecting a bristle and MRSA into the larval hemocoel. It was found that biofilm formation was in progress without any external sign of infection in most of the bristle-bearing larvae for 12 h after MRSA inoculation. Whereas the activation of the prophenoloxidase system did not affect the preformed in vitro MRSA biofilms, an antimicrobial peptide interfered with in vivo biofilm formation when injected into bristle-bearing larvae infected with MRSA. Finally, our confocal laser scanning microscopic analysis revealed that the biomass of the in vivo biofilm is greater compared to that of the in vitro biofilm and harbors a distribution of dead cells, which might be bacteria and/or host cells.

Switching from membrane disrupting to membrane crossing, an effective strategy in designing antibacterial polypeptide.

Drug-resistant bacterial infections have caused serious threats to human health and call for effective antibacterial agents that have low propensity to induce antimicrobial resistance. Host defense peptide-mimicking peptides are actively explored, among which poly-ß-l-lysine displays potent antibacterial activity but high cytotoxicity due to the helical structure and strong membrane disruption effect. Here, we report an effective strategy to optimize antimicrobial peptides by switching membrane disrupting to membrane penetrating and intracellular targeting by breaking the helical structure using racemic residues. Introducing ß-homo-glycine into poly-ß-lysine effectively reduces the toxicity of resulting poly-ß-peptides and affords the optimal poly-ß-peptide, ßLys50HG50, which shows potent antibacterial activity against clinically isolated methicillin-resistant Staphylococcus aureus (MRSA) and MRSA persister cells, excellent biosafety, no antimicrobial resistance, and strong therapeutic potential in both local and systemic MRSA infections. The optimal poly-ß-peptide demonstrates strong therapeutic potential and implies the success of our approach as a generalizable strategy in designing promising antibacterial polypeptides.

Host Response of Human Epidermis to Methicillin-Resistant Staphylococcus aureus Biofilm Infection and Synthetic Antibiofilm Peptide Treatment.

Bacterial biofilm infections associated with wounded skin are prevalent, recalcitrant, and in urgent need of treatments. Additionally, host responses in the skin to biofilm infections are not well understood. Here we employed a human organoid skin model to explore the transcriptomic changes of thermally-injured epidermis to methicillin-resistant Staphylococcus aureus (MRSA) biofilm colonization. MRSA biofilm impaired skin barrier function, enhanced extracellular matrix remodelling, elicited inflammatory responses including IL-17, IL-12 family and IL-6 family interleukin signalling, and modulated skin metabolism. Synthetic antibiofilm peptide DJK-5 effectively diminished MRSA biofilm and associated skin inflammation in wounded human ex vivo skin. In the epidermis, DJK-5 shifted the overall skin transcriptome towards homeostasis including modulating the biofilm induced inflammatory response, promoting the skin DNA repair function, and downregulating MRSA invasion of thermally damaged skin. These data clarified the underlying immunopathogenesis of biofilm infections and revealed the intrinsic promise of synthetic peptides in reducing inflammation and biofilm infections.

Analysis of the Antimicrobial and Anti-Biofilm Activity of Natural Compounds and Their Analogues against Staphylococcus aureus Isolates.

(1) Background: Staphylococcus aureus (S. aureus) is one of the most frequent causes of biofilm-associated infections. With the emergence of antibiotic-resistant, especially methicillin-resistant S. aureus (MRSA), there is an urgent need to discover novel inhibitory compounds against this clinically important pathogen. In this study, we evaluated the antimicrobial and anti-biofilm activity of 11 compounds, including phenyl propenes and phenolic aldehydes, eugenol, ferulic acid, sinapic acid, salicylaldehyde, vanillin, cinnamoyl acid, and aldehydes, against drug-resistant S. aureus isolates. (2) Methods: Thirty-two clinical S. aureus isolates were obtained from Alkhidmat Diagnostic Center and Blood Bank, Karachi, Pakistan, and screened for biofilm-forming potential, and susceptibility/resistance against ciprofloxacin, chloramphenicol, ampicillin, amikacin, cephalothin, clindamycin, streptomycin, and gentamicin using the Kirby-Bauer disk diffusion method. Subsequently, 5 representative clinical isolates were selected and used to test the antimicrobial and anti-biofilm potential of 11 compounds using both qualitative and quantitative assays, followed by qPCR analysis to examine the differences in the expression levels of biofilm-forming genes (ica-A, fnb-B, clf-A and cna) in treated (with natural compounds and their derivatives) and untreated isolates. (3) Results: All isolates were found to be multi-drug resistant and dominant biofilm formers. The individual Minimum Inhibitory Concentration (MIC) of natural compounds and their analogues ranged from 0.75−160 mg/mL. Furthermore, the compounds, Salicylaldehyde (SALI), Vanillin (VAN), α-methyl-trans-cinnamaldehyde (A-MT), and trans-4-nitrocinnamic acid (T4N) exhibited significant (15−92%) biofilm inhibition/reduction percentage capacity at the concentration of 1−10 mg/mL. Gene expression analysis showed that salicylaldehyde, α-methyl-trans-cinnamaldehyde, and α-bromo-trans-cinnamaldehyde resulted in a significant (p < 0.05) downregulation of the expression of ica-A, clf-A, and fnb-A genes compared to the untreated resistant isolate. (4) Conclusions: The natural compounds and their analogues used in this study exhibited significant antimicrobial and anti-biofilm activity against S. aureus. Biofilms persist as the main concern in clinical settings. These compounds may serve as potential candidate drug molecules against biofilm forming S. aureus.

Ferrous sulfate-loaded hydrogel cures Staphylococcus aureus infection via facilitating a ferroptosis-like bacterial cell death in a mouse keratitis model.

Hydrogels loaded with ampicillin, vancomycin or other antibiotics are one of the most widely used therapeutic agents for keratitis caused by Staphylococcus aureus. However, emergence of methicillin-resistant S. aureus (MRSA) makes infections harder to be treated by antibiotic-based hydrogels, urging the development of novel antibacterial materials. Inspired by mammalian ferroptosis, we determined the bactericidal effects of ferrous sulfate (FeSO4) on S. aureus, and evaluated the therapeutic potential of FeSO4-loaded hydrogel in a mouse keratitis model. The results showed that FeSO4 facilitated ferroptosis-like cell death in S. aureus with the key characteristics of reactive oxygen species (ROS) generation and lipid peroxidation. Notably, FeSO4 also efficiently killed persisters and MRSA, and eliminated biofilms of S. aureus. RNA profiles demonstrated that ferroptosis-related genes were significantly up-regulated, and the genes responsible for cell wall and cell membrane biosynthesis were down-regulated after exposure to Fe2+, supporting the occurrence of ferroptosis and cell lysis. We further prepared a FeSO4-loaded hydrogel by using hyaluronic acid (HA) and ascorbate. The FeSO4 hydrogel has the characteristics of injectability, self-healing, uniform distribution of Fe2+ in the three-dimensional gel structure, appropriate fluidity, high-water retention, high efficacy to kill MRSA, and excellent biocompatibility. In a mouse keratitis model, we showed that treatment of animals with FeSO4 hydrogel led to a rapid recovery of from keratitis, prevented the dissimilation of MRSA to the lung, and alleviated systemic inflammation, demonstrating the therapeutic potential of FeSO4 hydrogel. Taken together, our results indicated that FeSO4 hydrogel is a promising alternative to current antibiotics-dependent therapeutic materials for the treatment of infections by MRSA.

Nanoapatites Doped and Co-Doped with Noble Metal Ions as Modern Antibiofilm Materials for Biomedical Applications against Drug-Resistant Clinical Strains of Enterococcus faecalis VRE and Staphylococcus aureus MRSA.

The main aim of our research was to investigate antiadhesive and antibiofilm properties of nanocrystalline apatites doped and co-doped with noble metal ions (Ag+, Au+, and Pd2+) against selected drug-resistant strains of Enterococcus faecalis and Staphylococcus aureus. The materials with the structure of apatite (hydroxyapatite, nHAp; hydroxy-chlor-apatites, OH-Cl-Ap) containing 1 mol% and 2 mol% of dopants and co-dopants were successfully obtained by the wet chemistry method. The majority of them contained an additional phase of metallic nanoparticles, in particular, AuNPs and PdNPs, which was confirmed by the XRPD, FTIR, UV-Vis, and SEM-EDS techniques. Extensive microbiological tests of the nanoapatites were carried out determining their MIC, MBC value, and FICI. The antiadhesive and antibiofilm properties of the tested nanoapatites were determined in detail with the use of fluorescence microscopy and computer image analysis. The results showed that almost all tested nanoapatites strongly inhibit adhesion and biofilm production of the tested bacterial strains. Biomaterials have not shown any significant cytotoxic effect on fibroblasts and even increased their survival when co-incubated with bacterial biofilms. Performed analyses confirmed that the nanoapatites doped and co-doped with noble metal ions are safe and excellent antiadhesive and antibiofilm biomaterials with potential use in the future in medical sectors.

Real time monitoring of Staphylococcus aureus biofilm sensitivity towards antibiotics with isothermal microcalorimetry.

Biofilm-associated infections with Staphylococcus aureus are difficult to treat even after administration of antibiotics that according to the standard susceptibility assays are effective. Currently, the assays used in the clinical laboratories to determine the sensitivity of S. aureus towards antibiotics are not representing the behaviour of biofilm-associated S. aureus, since these assays are performed on planktonic bacteria. In research settings, microcalorimetry has been used for antibiotic susceptibility studies. Therefore, in this study we investigated if we can use isothermal microcalorimetry to monitor the response of biofilm towards antibiotic treatment in real-time. We developed a reproducible method to generate biofilm in an isothermal microcalorimeter setup. Using this system, the sensitivity of 5 methicillin-sensitive S. aureus (MSSA) and 5 methicillin-resistant S. aureus (MRSA) strains from different genetic lineages were determined towards: flucloxacillin, cefuroxime, cefotaxime, gentamicin, rifampicin, vancomycin, levofloxacin, clindamycin, erythromycin, linezolid, fusidic acid, co-trimoxazole, and doxycycline. In contrast to conventional assays, our calorimetry-based biofilm susceptibility assay showed that S. aureus biofilms, regardless MSSA or MRSA, can survive the exposure to the maximum serum concentration of all tested antibiotics. The only treatment with a single antibiotic showing a significant reduction in biofilm survival was rifampicin, yet in 20% of the strains, emerging antibiotic resistance was observed. Furthermore, the combination of rifampicin with flucloxacillin, vancomycin or levofloxacin was able to prevent S. aureus biofilm from becoming resistant to rifampicin. Isothermal microcalorimetry allows real-time monitoring of the sensitivity of S. aureus biofilms towards antibiotics in a fast and reliable way.

Elasnin Effectively Eradicates Daptomycin-Resistant Methicillin-Resistant Staphylococcus aureus Biofilms.

Elasnin is a recently reported antibiofilm agent that is effective against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). Remarkably, we observed that elasnin has a superior activity in eradicating daptomycin-resistant MRSA strain biofilm, with a lower minimum biofilm eradication concentration (MBEC) value of 0.625 µg/mL, compared to 2.5 µg/mL for the wild type. Confocal microscopy further confirmed the higher biofilm eradication on the daptomycin-resistant strain, displaying ∼53% decrease in cell density upon elasnin treatment, while the wild-type strain was only decreased by ∼15%. Quantitative proteomics revealed that the daptomycin-resistant strain has a lower expression of the membrane, cell wall, and extracellular proteins, and also proteins involved in the arginine biosynthesis, pathogenesis, and cell adhesion compared to the wild type, which may result in weaker biofilm development. This study highlights the potential clinical application of elasnin through its superior biofilm eradication activity against a daptomycin-resistant MRSA strain, and revealed the associated processes governing this superior activity through proteomics analysis. IMPORTANCE Due to the increased use of daptomycin for the treatment of MRSA infections, the emergence of daptomycin-resistant strains has become prevalent in recent years. In this study, we discovered that elasnin, a newly reported antibiofilm compound, has a superior activity in eradicating daptomycin-resistant MRSA strain biofilms compared to the wild type. Follow-up analysis revealed the reason behind this superior activity, which is the lower expression of key proteins that play a role in pathogenesis and cell adhesion in the daptomycin-resistant strain, leading to weaker biofilm development. This showcases the potential use of elasnin in clinical settings where daptomycin-resistant strains and biofilm formation are prevalent. Altogether, our study provides new insights into the mechanism of elasnin in MRSA biofilm cells and identified its superior biofilm eradicating activity in the daptomycin-resistant strain.

MRSA-induced endothelial permeability and acute lung injury are attenuated by FTY720 S-phosphonate.

Disruption of the lung endothelial barrier is a hallmark of acute respiratory distress syndrome (ARDS), for which no effective pharmacologic treatments exist. Prior work has demonstrated that FTY720 S-phosphonate (Tys), an analog of sphingosine-1-phosphate (S1P) and FTY720, exhibits potent endothelial cell (EC) barrier protective properties. In this study, we investigated the in vitro and in vivo efficacy of Tys against methicillin-resistant Staphylococcus aureus (MRSA), a frequent bacterial cause of ARDS. Tys-protected human lung EC from barrier disruption induced by heat-killed MRSA (HK-MRSA) or staphylococcal α-toxin and attenuated MRSA-induced cytoskeletal changes associated with barrier disruption, including actin stress fiber formation and loss of peripheral VE-cadherin and cortactin. Tys-inhibited Rho and myosin light chain (MLC) activation after MRSA and blocked MRSA-induced NF-κB activation and release of the proinflammatory cytokines, IL-6 and IL-8. In vivo, intratracheal administration of live MRSA in mice caused significant vascular leakage and leukocyte infiltration into the alveolar space. Pre- or posttreatment with Tys attenuated MRSA-induced lung permeability and levels of alveolar neutrophils. Posttreatment with Tys significantly reduced levels of bronchoalveolar lavage (BAL) VCAM-1 and plasma IL-6 and KC induced by MRSA. Dynamic intravital imaging of mouse lungs demonstrated Tys attenuation of HK-MRSA-induced interstitial edema and neutrophil infiltration into lung tissue. Tys did not directly inhibit MRSA growth or viability in vitro. In conclusion, Tys inhibits lung EC barrier disruption and proinflammatory signaling induced by MRSA in vitro and attenuates acute lung injury induced by MRSA in vivo. These results support the potential utility of Tys as a novel ARDS therapeutic strategy.

Hesperidin inhibits biofilm formation, virulence and staphyloxanthin synthesis in methicillin resistant Staphylococcus aureus by targeting SarA and CrtM: an in vitro and in silico approach.

Methicillin resistant Staphylococcus aureus is considered multidrug resistant bacterium due to developing biofilm formation associated with antimicrobial resistance mechanisms. Therefore, inhibition of biofilm formation is an alternative therapeutic action to control MRSA infections. The present study revealed the non-antibacterial biofilm inhibitory potential of hesperidin against ATCC strain and clinical isolates of S. aureus. Hesperidin is a flavanone glycoside commonly found in citrus fruit. Hesperidin has been shown to exhibits numerous pharmacological activities. The present study aimed to evaluate the antibiofilm and antivirulence potential of hesperidin against MRSA. Results showed that hesperidin treatment significantly impedes lipase, hemolysin, autolysin, autoaggregation and staphyloxanthin production. Reductions of staphyloxanthin production possibly increase the MRSA susceptibility rate to H2O2 oxidative stress condition. In gene expression study revealed that hesperidin treatment downregulated the biofilm-associated gene (sarA), polysaccharide intracellular adhesion gene (icaA and icaD), autolysin (altA), fibronectin-binding protein (fnbA and fnbB) and staphyloxanthin production (crtM). Molecular docking analysis predicted the ability of hesperidin to interact with SarA and CrtM proteins involved in biofilm formation and staphyloxanthin production in MRSA.

Biomineralized Cascade Enzyme-Encapsulated ZIF-8 Nanoparticles Combined with Antisense Oligonucleotides for Drug-Resistant Bacteria Treatment.

The unrestrained use of antibiotics accelerates the development of drug-resistant bacteria and leads to an increasing threat to human health. Therefore, there is an urgent need to explore novel and effective strategies for the treatment of bacterial infections. Herein, zeolite imidazole framework-8 (ZIF-8) material was utilized to construct biomineralized nanomaterial (GOx&HRP@ZIF-8/ASO) by encapsulating biological cascade enzymes and combining with antisense oligonucleotides (ASOs), which achieved effective and synergistic antidrug-resistant bacteria therapy. Various in vitro assays confirmed that GOx&HRP@ZIF-8/ASO exhibited excellent antibacterial properties against Escherichia coli, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA) during catalysis of glucose (Glu), especially the minimum inhibitory concentration (MIC) against MRSA was only 16 µg/mL. Compared with simple ZIF-8 (32.85%) and ftsZ ASO (58.65%), GOx&HRP@ZIF-8/ASO+Glu exhibited superb biofilm destruction ability, and the bacteria removal efficiency of the MRSA biofilm could be as high as 88.2%, indicating that the reactive oxygen species (ROS) produced by the cascade enzyme reaction imparted the main synergistic antibacterial capability, and simultaneously, ftsZ ASO significantly enhanced the antibacterial effect by inhibiting the expression of the ftsZ gene. In vivo anti-infection treatment experiments revealed that GOx&HRP@ZIF-8/ASO exhibited the best wound repairing performance and excellent biocompatibility in the presence of Glu. These findings suggested that GOx&HRP@ZIF-8/ASO has favorably realized high-efficiency treatment of MRSA infection and filled the gap in the antibacterial application of biological enzymes.

Dynamic changes of inflammatory response and oxidative stress induced by methicillin-resistant Staphylococcus aureus in mice.

This study is to analyze the dynamic changes of inflammation and oxidative stress in mice infected with MRSA and to provide experimental basis for clinically formulating reasonable treatment plans. We established a model of MRSA infection in mice, detected the fluctuations in the concentration of proinflammatory cytokines and oxidative stress factors with time, and combined with the results of microscopic examination of tissue sections to explain the infection in vivo caused by MRSA. The results showed that on the 1st, 3rd, and 7th day of MRSA infection, the number of leukocytes and eosinophils decreased at first and then increased, monocytes increased continuously, and neutrophils and basophils decreased. At the same time, the levels of proinflammatory cytokines IL-1ß, IL-6, and TNF-α increased. The concentration of glutathione peroxide decreased, and the oxidative metabolites increased. Tissue sections also showed that inflammation and oxidative stress occurred in mice. It is obvious that MRSA infection can lead to significant inflammation and oxidative stress. Therefore, while treating MRSA infection, attention should be paid to the levels of inflammation and oxidative stress in different periods to achieve better treatment effects.

Staphylococcal Phages Adapt to New Hosts by Extensive Attachment Site Variability.

Bacterial pathogens commonly carry prophages that express virulence factors, and human strains of Staphylococcus aureus carry Sa3int phages, which promote immune evasion. Recently, however, these phages have been found in livestock-associated, methicillin-resistant S. aureus (LA-MRSA). This is surprising, as LA-MRSA strains contain a mutated primary bacterial integration site, which likely explains why the rare integration events that do occur mostly happen at alternative locations. Using deep sequencing, we show that after initial integration at secondary sites, Sa3int phages adapt through nucleotide changes in their attachment sequences to increase homology with alternative bacterial attachment sites. Importantly, this homology significantly enhances integrations in new rounds of infections. We propose that promiscuity of the phage-encoded tyrosine recombinase is responsible for establishment of Sa3int phages in LA-MRSA. Our results demonstrate that phages can adopt extensive population heterogeneity, leading to establishment in strains lacking bona fide integration sites. Ultimately, their presence may increase virulence and zoonotic potential of pathogens with major implications for human health. IMPORTANCE A growing number of humans are being infected by antibiotic resistant Staphylococcus aureus originating from livestock. The preference of S. aureus for humans or animals is in part determined by factors encoded by viruses (phages) that reside in the bacterial genome. Here, we reveal a process by which phages adapt to and become integrated in new strains of S. aureus lacking the preferred phage integration site. We propose that this is due to the relaxed specificity of a phage-encoded enzyme called recombinase. As this recombinase is used by many other phages, our results might have implications for a broader range of phages. Importantly, the adaptation described here enables S. aureus to jump between host organisms and increases its zoonotic threat.

Composition, Anti-MRSA Activity and Toxicity of Essential Oils from Cymbopogon Species.

Many of the essential oils obtained from medicinal plants possess proven antimicrobial activity and are suitable for medicinal purposes and applications in the food industry. The aim of the present work was the chemical analysis of 19 essential oils (EOs) from seven different Cymbopogon species (C. nardus, C. citratus, C winterianus, C. flexuosus, C. schoenanthus, C. martinii, C. giganteus). Five different chemotypes were established by GC/MS and TLC assay. The EOs, as well as some reference compounds, i.e., citronellol, geraniol and citral (neral + geranial), were also tested for their antimicrobial and antibiofilm activity against methicillin-resistant Staphylococcus aureus (MRSA) by the microdilution method and direct bioautography. The toxicity of EOs was evaluated by Danio rerio 'Zebrafish' model assay. All examined EOs showed moderate to high activity against MRSA, with the highest activity noted for C. flexuosus-lemongrass essential oil, both in microdilution and direct autobiography method. Significant difference in the toxicity of the examined EOs was also detected.

Investigation of novel putative immunogenic targets against Staphylococcus aureus using a reverse vaccinology strategy.

BACKGROUND: The emergence of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) strains is a significant public health concern. Considering the high morbidity and mortality of invasive S. aureus infections and multi-drug resistant strains, there is an urgent need for non-antibiotic immune-based approaches to cure these infections. Despite all efforts, vaccine candidates targeting S. aureus failed in human clinical trials, and no approved vaccine is available against this pathogen. Therefore, this study aimed to introduce suitable candidates for immunization against S. aureus using a comprehensive reverse vaccinology approach. METHODS: In this study, we retrieved putative immunogenic targets from three different levels (literature review, automated reverse vaccinology, and manual reverse vaccinology) and evaluated them using several immunoinformatics analyses including antigenicity, allergenicity, PSI-BLAST to human proteome, physiochemical properties, B-cell, and T-cell epitopes. In the next step, the quartile method scoring was used to the shortlisted proteins. Finally, the molecular docking and immune simulation of immunogenic targets were performed. RESULTS: This study presents 12 vaccine candidates, including three enzymatic proteins (WP_000222271.1, WP_001170274, and WP_000827736.1), three cell wall-associated proteins (WP_001125631.1, WP_000731642, and WP_000751265.1), two hemolysins (WP_000594517.1, and WP_000916697.1), one secretion involved protein (WP_000725226.1), one heme­iron binding protein (WP_001041573.1), one superantigen like protein (WP_000668994.1) and one hypothetical proteins (WP_000737711.1). CONCLUSION: Through quartile scoring method, immune simulation and molecular docking, four promising targets including lytic transglycosylase IsaA, HlgA, secretory antigen precursor SsaA, and heme uptake protein IsdB were selected as the shortlisted proteins. It seems that a polarized immunization (Th1/Th17) response is needed for protection against this bacterium. An optimized formulation based on these putative immunogenic proteins and a wisely adjuvant selection may drive the immune system toward a full protection.