Tixagevimab/Cilgavimab: Still a Valid Prophylaxis against COVID-19 New Variants?

BACKGROUND: this study aims to evaluate the efficacy of tixagevimab/cilgavimab (Evusheld™) against various SARS-CoV-2 variants, including newer Omicron sublineages, in an immunocompromised cohort and in vitro. STUDY DESIGN: Conducted in Italy, this research involves immunocompromised patients who received Evusheld. It evaluates serum neutralization activity against different SARS-CoV-2 strains (20A.EU1, BA.5, BQ.1, XBB.1.5, XBB.1.16, and EG.5) before (T0), after 14 (T1), and after 30 (T2) days from the tixagevimab/cilgavimab injection. Furthermore, the in vitro activity of Evusheld against SARS-CoV-2 VOCs was evaluated. RESULTS: The cohort was composed of 72 immunocompromised patients. The serum neutralizing activity of tixagevimab/cilgavimab-treated patients was notably lower against newer variants such as BQ.1, XBB.1.5, XBB.1.16, and EG.5. Then, the in vitro study detailed specific EC50 values to quantify the activity of tixagevimab/cilgavimab against various SARS-CoV-2 VOCs. Newer variants like BQ.1 and XBB.1.5 exhibited notably lower neutralization, underscoring the challenges in effectively countering the evolving virus. Interestingly, tixagevimab/cilgavimab maintained reduced but still valid activity against EG.5 with an EC50 of 189 ng/mL and Cmax/EC90 of 110.7. CONCLUSIONS: Tixagevimab/cilgavimab efficacy wanes against novel subvariants. This underscores the critical need for ongoing adaptation and vigilance in prophylactic strategies to effectively counter the dynamic and unpredictable nature of the COVID-19 pandemic.

The hope and hype of ellagic acid and urolithins as ligands of SARS-CoV-2 Nsp5 and inhibitors of viral replication.

Non-structural protein 5 (Nsp5) is a cysteine protease that plays a key role in SARS-CoV-2 replication, suppressing host protein synthesis and promoting immune evasion. The investigation of natural products as a potential strategy for Nsp5 inhibition is gaining attention as a means of developing antiviral agents. In this work, we have investigated the physicochemical properties and structure-activity relationships of ellagic acid and its gut metabolites, urolithins A-D, as ligands of Nsp5. Results allow us to identify urolithin D as promising ligand of Nsp5, with a dissociation constant in the nanomolar range of potency. Although urolithin D is able to bind to the catalytic cleft of Nsp5, the appraisal of its viral replication inhibition against SARS-CoV-2 in Vero E6 assay highlights a lack of activity. While these results are discussed in the framework of the available literature reporting conflicting data on polyphenol antiviral activity, they provide new clues for natural products as potential viral protease inhibitors.

Synergistic Activity of Remdesivir-Nirmatrelvir Combination on a SARS-CoV-2 In Vitro Model and a Case Report.

BACKGROUND: This study aims to investigate the activity of the remdesivir-nirmatrelvir combination against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and to report a case of Coronavirus Disease 2019 (COVID-19) cured with this combination. METHODS: A Vero E6 cell-based infection assay was used to investigate the in vitro activity of the remdesivir-nirmatrelvir combination. The SARS-CoV-2 strains tested were 20A.EU1, BA.1 and BA.5. After incubation, a viability assay was performed. The supernatants were collected and used for viral titration. The Highest Single Agent (HSA) reference model was calculated. An HSA score >10 is considered synergic. RESULTS: Remdesivir and nirmatrelvir showed synergistic activity at 48 and 72 h, with an HSA score of 52.8 and 28.6, respectively (p < 0.0001). These data were confirmed by performing supernatant titration and against the omicron variants: the combination reduced the viral titer better than the more active compound alone. An immunocompromised patient with prolonged and critical COVID-19 was successfully treated with remdesivir, nirmatrelvir/ritonavir, tixagevimab/cilgavimab and dexamethasone, with an excellent clinical-radiological response. However, she required further off-label prolonged therapy with nirmatrelvir/ritonavir until she tested negative. CONCLUSIONS: Remdesivir-nirmatrelvir combination has synergic activity in vitro. This combination may have a role in immunosuppressed patients with severe COVID-19 and prolonged viral shedding.

The Interplay between Candida albicans, Vaginal Mucosa, Host Immunity and Resident Microbiota in Health and Disease: An Overview and Future Perspectives.

Vulvovaginal candidiasis (VVC), which is primarily caused by Candida albicans, is an infection that affects up to 75% of all reproductive-age women worldwide. Recurrent VVC (RVVC) is defined as >3 episodes per year and affects nearly 8% of women globally. At mucosal sites of the vagina, a delicate and complex balance exists between Candida spp., host immunity and local microbial communities. In fact, both immune response and microbiota composition play a central role in counteracting overgrowth of the fungus and maintaining homeostasis in the host. If this balance is perturbed, the conditions may favor C. albicans overgrowth and the yeast-to-hyphal transition, predisposing the host to VVC. To date, the factors that affect the equilibrium between Candida spp. and the host and drive the transition from C. albicans commensalism to pathogenicity are not yet fully understood. Understanding the host- and fungus-related factors that drive VVC pathogenesis is of paramount importance for the development of adequate therapeutic interventions to combat this common genital infection. This review focuses on the latest advances in the pathogenic mechanisms implicated in the onset of VVC and also discusses novel potential strategies, with a special focus on the use of probiotics and vaginal microbiota transplantation in the treatment and/or prevention of recurrent VVC.

Stabilization of the Dimeric State of SARS-CoV-2 Main Protease by GC376 and Nirmatrelvir.

The main protease (Mpro or 3CLpro) is an enzyme that is evolutionarily conserved among different genera of coronaviruses. As it is essential for processing and maturing viral polyproteins, Mpro has been identified as a promising target for the development of broad-spectrum drugs against coronaviruses. Like SARS-CoV and MERS-CoV, the mature and active form of SARS-CoV-2 Mpro is a dimer composed of identical subunits, each with a single active site. Individual monomers, however, have very low or no catalytic activity. As such, inhibition of Mpro can be achieved by molecules that target the substrate binding pocket to block catalytic activity or target the dimerization process. In this study, we investigated GC376, a transition-state analog inhibitor of the main protease of feline infectious peritonitis coronavirus, and Nirmatrelvir (NMV), an oral, bioavailable SARS-CoV-2 Mpro inhibitor with pan-human coronavirus antiviral activity. Our results show that both GC376 and NMV are capable of strongly binding to SARS-CoV-2 Mpro and altering the monomer-dimer equilibrium by stabilizing the dimeric state. This behavior is proposed to be related to a structured hydrogen-bond network established at the Mpro active site, where hydrogen bonds between Ser1' and Glu166/Phe140 are formed in addition to those achieved by the latter residues with GC376 or NMV.

Development of a new indole derivative dry powder for inhalation for the treatment of biofilm-associated lung infections.

The aim of this work was to produce an inhalable dry powder formulation of a new anti-biofilm compound (SC38). For this purpose, chitosan was used as a polymeric carrier and l-leucine as a dispersibility enhancer. SC38 was entrapped by spray-drying into previously optimized chitosan microparticles. The final formulation was fully characterized in vitro in terms of particle morphology, particle size and distribution, flowability, aerodynamic properties, anti-biofilm activity and effects on lung cell viability. The SC38-loaded chitosan microparticles exhibited favorable aerodynamic properties with emitted and respirable fractions higher than 80 % and 45 % respectively. The optimized formulation successfully inhibited biofilm formation at microparticle concentrations starting from 20 µg/mL for methicillin-sensitive and 100 µg/mL for methicillin-resistant Staphylococcus aureus and showed a relatively safe profile in lung cells after 72 h exposure. Future in vivo tolerability and efficacy studies are needed to unravel the potential of this novel formulation for the treatment of difficult-to-treat biofilm-mediated lung infections.

Nelfinavir: An Old Ally in the COVID-19 Fight?

After almost three years of the pandemic, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is still spreading around the world, causing notable sanitary and social issues. New antiviral therapies are constantly under investigation. However, few options have been approved for the treatment of COVID-19. Clinical trials are currently ongoing to evaluate the efficacy of nelfinavir on mild−moderate COVID-19. This study aims to investigate the activity of this compound on SARS-CoV-2 "Variants of Concern" (VOCs), comparing its effectiveness with the approved drugs remdesivir and molnupiravir. The experiments were conducted in a biosafety level 3 facility. In this study, we used a Vero-E6-cell-based infection assay to investigate the in vitro activity of nelfinavir, molnupiravir, and remdesivir. Four strains of SARS-CoV-2 were tested: 20A.EU1, B.1.1.7, P.1, and B.1.617.2. All compounds reached micromolar/submicromolar EC50, EC90, and EC99. Furthermore, the Cmax/EC50 and Cmax/EC90 ratios were >1 for all compounds and all variants tested. Our study demonstrated that nelfinavir, as molnupiravir, and remdesivir are effective in vitro on SARS-CoV-2 variants.

BNT162b2 Elicited an Efficient Humoral Response Against Different Strains of SARS-CoV-2 in People Living with HIV.

BACKGROUND: Vaccines have had a fundamental impact in containing the ongoing Coronavirus Disease 2019 (COVID-19) pandemic. However, there are few efficacy data relating to frail patients, including the HIV-positive patient. OBJECTIVE: This study evaluated the Severe Acute Respiratory Syndrome Coronavirus 2 (SARSCoV- 2) serum neutralization in People Living with HIV (PLWH) compared to a cohort of healthy volunteers both vaccinated with BNT162b2. METHODS: A serum sample was then withdrawn 14-21 days after the second dose of the vaccine and a serum neutralization assay was performed on Vero E6 cells. The experiments were performed using two strains of SARS-CoV-2 as 20A.EU1 and B.1.617.2. RESULTS: PLWH on Antiretroviral Therapy (ART) showed a vaccine response comparable to the healthy subjects. No correlation between CD4 count or CD4/CD8 and neutralizing antibodies (NTAbs) has been found. No differences in NT-Abs between patients with CD4 nadir above or under 200 cells/µl have been found. In both cohorts, vaccine-elicited serum better neutralized 20A.EU1 than B.1.617.2 strain. CONCLUSION: PLWH in ART and with good immuno-virological recovery showed a vaccine response comparable to that of healthy subjects and regardless of their immunological status at HIV infection diagnosis. However, larger studies are needed to confirm our results and to evaluate the vaccine response even in patients with low CD4 counts.

The Combination of Molnupiravir with Nirmatrelvir or GC376 Has a Synergic Role in the Inhibition of SARS-CoV-2 Replication In Vitro.

Introduction: The development of effective vaccines has partially mitigated the trend of the SARS-CoV-2 pandemic; however, the need for orally administered antiviral drugs persists. This study aims to investigate the activity of molnupiravir in combination with nirmatrelvir or GC376 on SARS-CoV-2 to verify the synergistic effect. Methods: The SARS-CoV-2 strains 20A.EU, BA.1 and BA.2 were used to infect Vero E6 in presence of antiviral compounds alone or in combinations using five two-fold serial dilution of compound concentrations ≤EC90. After 48 and 72 h post-infection, viability was performed using MTT reduction assay. Supernatants were collected for plaque-assay titration. All experiments were performed in triplicate, each being repeated at least three times. The synergistic score was calculated using Synergy Finder version 2. Results: All compounds reached micromolar EC90. Molnupiravir and GC376 showed a synergistic activity at 48 h with an HSA score of 19.33 (p < 0.0001) and an additive activity at 72 h with an HSA score of 8.61 (p < 0.0001). Molnupiravir and nirmatrelvir showed a synergistic activity both at 48 h and 72 h with an HSA score of 14.2 (p = 0.01) and 13.08 (p < 0.0001), respectively. Conclusion: Molnupiravir associated with one of the two protease-inhibitors nirmatrelvir and GC376 showed good additive-synergic activity in vitro.

Lactobacillus iners Cell-Free Supernatant Enhances Biofilm Formation and Hyphal/Pseudohyphal Growth by Candida albicans Vaginal Isolates.

Candida albicans is a commensal fungus of the vaginal mucosa and the principal etiological agent of vaginal candidiasis. Vaginal dysbiosis has been reported during vulvovaginal candidiasis (VVC), with a progressive decrease in Lactobacillus crispatus population and an increase in L. iners population. To date, the role of L. iners in VVC pathogenesis remains scarcely explored. Herein we investigated the in vitro effect of L. iners cell-free supernatant (CFS) on the ability of C. albicans to form biofilms. Biomass and metabolic activity were measured by crystal violet and XTT assays. Further, light microscopy was performed to determine the effect of L. iners CFS on biofilm cellular morphology. We found that L. iners CFS induced a significant increase in biofilm formation by C. albicans clinical isolates which were categorized as moderate or weak biofilm producers. This effect was associated with an enhancement of hyphal/pseudohyphal growth, and the expression levels of HWP1 and ECE1, which are typical hyphae-associated genes, were upregulated. Overall, these results suggest that L. iners contributes to the pathogenesis of VVC and highlight the complexity of the interaction between C. albicans and vaginal lactobacilli. Understanding these interactions could prove essential for the development of new strategies for treating VVC.

Glucocorticoid-Induced Leucine Zipper-Mediated TLR2 Downregulation Accounts for Reduced Neutrophil Activity Following Acute DEX Treatment.

Glucocorticoids are the most powerful anti-inflammatory and immunosuppressive pharmacological drugs available, despite their adverse effects. Glucocorticoid-induced leucine zipper (GILZ) is a glucocorticoid-induced gene that shares several anti-inflammatory properties with glucocorticoids. Although immunosuppressive effects of glucocorticoids on neutrophils remain poorly understood, we previously demonstrated that GILZ suppresses neutrophil activation under glucocorticoid treatment. Here, we sought to explore the regulation of Toll-like receptor 2 (TLR2) by the synthetic glucocorticoid dexamethasone (DEX) on neutrophils and the associated GILZ involvement. Peripheral blood neutrophils were isolated from wild type and GILZ-knock-out (KO) mice. TLR2 was found to be downregulated by the in vivo administration of glucocorticoids in wild type but not in GILZ-KO neutrophils, suggesting the involvement of GILZ in TLR2 downregulation. Accordingly, the TLR2-associated anti-fungal activity of neutrophils was reduced by DEX treatment in wild type but not GILZ-KO neutrophils. Furthermore, GILZ did not interact with NF-κB but was found to bind with STAT5, a pivotal factor in the regulation of TLR2 expression. A similar modulation of TLR2 expression, impaired phagocytosis, and killing activity was observed in circulating human neutrophils treated in vitro with DEX. These results demonstrate that glucocorticoids reduce the ability of neutrophils to respond to infections by downregulating TLR2 via GILZ, thereby reducing critical functions.

Cross-neutralization of SARS-CoV-2 B.1.1.7 and P.1 variants in vaccinated, convalescent and P.1 infected.

OBJECTIVES: The emergence of new variants of concern (VOCs) of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) around the world significantly complicated the exit from Coronavirus disease 2019 (COVID-19) pandemic. The aim of this study was to evaluate the serum neutralizing activity of three cohorts. METHODS: BNT162b2-elicited serum (N = 103), candidates as hyper-immune plasma donors (N = 90) and patients infected with the SARS-CoV-2 P1 variant (N = 22) were enrolled. Three strains of SARS-CoV-2 have been tested: 20A.EU1, B.1.1.7 (alpha) and P.1 (gamma). Neutralizing antibodies (NT-Abs) titers against SARS-CoV-2 were evaluated. RESULTS: B.1.1.7 and P.1 are less efficiently neutralized by convalescent wild-type infected serums if compared to 20A.EU1 strain (mean titer 1.6 and 6.7-fold lower respectively). BNT162b2 vaccine-elicited human sera show an equivalent neutralization potency on the B.1.1.7 but it is significantly lower for the P.1 variant (mean titer 3.3-fold lower). Convalescent P.1 patients are less protected from other SARS-CoV-2 strains with an important reduction of neutralizing antibodies against 20A.EU1 and B.1.1.7, about 12.2 and 10.9-fold, respectively. CONCLUSIONS: BNT162b2 vaccine confers immunity against all the tested VOCs, while previous SARS-CoV-2 infection may be less protective.

SARS-CoV2 infection impairs the metabolism and redox function of cellular glutathione.

Viral infections sustain their replication cycle promoting a pro-oxidant environment in the host cell. In this context, specific alterations of the levels and homeostatic function of the tripeptide glutathione have been reported to play a causal role in the pro-oxidant and cytopathic effects (CPE) of the virus. In this study, these aspects were investigated for the first time in SARS-CoV2-infected Vero E6 cells, a reliable and well-characterized in vitro model of this infection. SARS-CoV2 markedly decreased the levels of cellular thiols, essentially lowering the reduced form of glutathione (GSH). Such an important defect occurred early in the CPE process (in the first 24 hpi). Thiol analysis in N-acetyl-Cys (NAC)-treated cells and membrane transporter expression data demonstrated that both a lowered uptake of the GSH biosynthesis precursor Cys and an increased efflux of cellular thiols, could play a role in this context. Increased levels of oxidized glutathione (GSSG) and protein glutathionylation were also observed along with upregulation of the ER stress marker PERK. The antiviral drugs Remdesivir (Rem) and Nelfinavir (Nel) influenced these changes at different levels, essentially confirming the importance or blocking viral replication to prevent GSH depletion in the host cell. Accordingly, Nel, the most potent antiviral in our in vitro study, produced a timely activation of Nrf2 transcription factor and a GSH enhancing response that synergized with NAC to restore GSH levels in the infected cells. Despite poor in vitro antiviral potency and GSH enhancing function, Rem treatment was found to prevent the SARS-CoV2-induced glutathionylation of cellular proteins. In conclusion, SARS-CoV2 infection impairs the metabolism of cellular glutathione. NAC and the antiviral Nel can prevent such defect in vitro.

SARS-CoV-2 Survival on Surfaces and the Effect of UV-C Light.

The aim of this study was to establish the persistence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on inanimate surfaces such as plastic, stainless steel, and glass during UV-C irradiation which is a physical means commonly utilized in sanitization procedures. The viral inactivation rate, virus half-life, and percentage of titer reduction after UV-C irradiation were assessed. Infectivity was maintained on plastic and glass until 120 h and on stainless steel until 72 h. The virus half-life was 5.3, 4.4, and 4.2 h on plastic, stainless steel, and glass, respectively. In all cases, titer decay was >99% after drop drying. UV-C irradiation efficiently reduced virus titer (99.99%), with doses ranging from 10.25 to 23.71 mJ/cm2. Plastic and stainless steel needed higher doses to achieve target reduction. The total inactivation of SARS-CoV-2 on glass was obtained with the lower dose applied. SARS-CoV-2 survival can be long lasting on inanimate surfaces. It is worth recommending efficient disinfection protocols as a measure of prevention of viral spread. UV-C can provide rapid, efficient and sustainable sanitization procedures of different materials and surfaces. The dosages and mode of irradiation are important parameters to consider in their implementation as an important means to fight the SARS-CoV-2 pandemic.

Discovery of a AHR pelargonidin agonist that counter-regulates Ace2 expression and attenuates ACE2-SARS-CoV-2 interaction.

The severe acute respiratory syndrome (SARS)-CoV-2 is the pathogenetic agent of Corona Virus Induced Disease (COVID)19. The virus enters the human cells after binding to the angiotensin converting enzyme (ACE)2 receptor in target tissues. ACE2 expression is induced in response to inflammation. The colon expression of ACE2 is upregulated in patients with inflammatory bowel disease (IBD), highlighting a potential risk of intestinal inflammation in promoting viral entry in the human body. Because mechanisms that regulate ACE2 expression in the intestine are poorly understood and there is a need of anti-SARS-CoV-2 therapies, we have settled to investigate whether natural flavonoids might regulate the expression of Ace2 in intestinal models of inflammation. The results of these studies demonstrated that pelargonidin activates the Aryl hydrocarbon Receptor (AHR) in vitro and reverses intestinal inflammation caused by chronic exposure to high fat diet or to the intestinal braking-barrier agent TNBS in a AhR-dependent manner. In these two models, development of colon inflammation associated with upregulation of Ace2 mRNA expression. Colon levels of Ace2 mRNA were directly correlated with Tnf-α mRNA levels. Molecular docking studies suggested that pelargonidin binds a fatty acid binding pocket on the receptor binding domain of SARS-CoV-2 Spike protein. In vitro studies demonstrated that pelargonidin significantly reduces the binding of SARS-CoV-2 Spike protein to ACE2 and reduces the SARS-CoV-2 replication in a concentration-dependent manner. In summary, we have provided evidence that a natural flavonoid might hold potential in reducing intestinal inflammation and ACE2 induction in the inflamed colon in a AhR-dependent manner.

Initial In Vivo Evaluation of a Novel Amikacin-Deoxycholate Hydrophobic Salt Delivers New Insights on Amikacin Partition in Blood and Tissues.

In this study, an initial in vivo evaluation of a new amikacin-deoxycholate hydrophobic salt aimed at potentiating amikacin action against hard-to-treat lung infections was undertaken by quantifying, for the first time, amikacin in whole blood. Pharmacokinetic evaluation after intranasal administration in a murine model showed higher drug retention in the lungs compared to blood, with no significant differences between the salt and the free drug. Upon repeated administrations, the two treatments resulted in nonsignificant tissue damage and mild higher inflammation for the hydrophobic salt. Whole-blood analysis highlighted an unreported high partition of amikacin in blood components up to 48 h, while significant lung levels were measured up to 72 h. Such a new observation was considered responsible for the nearly overlapping pharmacokinetic profiles of the two treatments. To overcome such an issue, a dry powder in an inhalable form may be best suited. Moreover, if confirmed in humans, and considering the current once-a-day regimen for amikacin aerosols, important yet-to-be-explored clinical implications may be postulated for such amikacin persistence in the organism.

Optimized Extraction of Amikacin from Murine Whole Blood.

Amikacin (Amk) analysis and quantitation, for pharmacokinetics studies and other types of investigations, is conventionally performed after extraction from plasma. No report exists so far regarding drug extraction from whole blood (WB). This can represent an issue since quantification in plasma does not account for drug partitioning to the blood cell compartment, significantly underrating the drug fraction reaching the blood circulation. In the present work, the optimization of an extraction method of Amk from murine WB has been described. The extraction yield was measured by RP-HPLC-UV after derivatization with 1-fluoro-2,4-dinitrobenzene, which produced an appreciably stable derivative with a favorable UV/vis absorption. Several extraction conditions were tested: spiked Amk disulfate solution/acetonitrile/WB ratio; presence of organic acids and/or ammonium hydroxide and/or ammonium acetate in the extraction mixture; re-dissolution of the supernatant in water after a drying process under vacuum; treatment of the supernatant with a solution of inorganic salts. The use of 5% (by volume) of ammonium hydroxide in a hydro-organic solution with acetonitrile, allowed the almost quantitative (95%) extraction of the drug from WB.

Is recurrence possible in coronavirus disease 2019 (COVID-19)? Case series and systematic review of literature.

Can a patient diagnosed with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) be infected again? This question is still unsolved. We tried to analyze local and literature cases with a positive respiratory swab after recovery. We collected data from symptomatic patients diagnosed with SARS-CoV-2 infection in the Italian Umbria Region that, after recovery, were again positive for SARS-CoV-2 in respiratory tract specimens. Samples were also assessed for infectivity in vitro. A systematic review of similar cases reported in the literature was performed. The study population was composed of 9 patients during a 4-month study period. Among the new positive samples, six were inoculated in Vero-E6 cells and showed no growth and negative molecular test in culture supernatants. All patients were positive for IgG against SARS-CoV-2 nucleoprotein and/or S protein. Conducting a review of the literature, 1350 similar cases have been found. The presumptive reactivation occurred in 34.5 days on average (standard deviation, SD, 18.7 days) after COVID-19 onset, when the 5.6% of patients presented fever and the 27.6% symptoms. The outcome was favorable in 96.7% of patients, while the 1.1% of them were still hospitalized at the time of data collection and the 2.1% died. Several hypotheses have been formulated to explain new positive respiratory samples after confirmed negativity. According to this study, the phenomenon seems to be due to the prolonged detection of SARS-CoV-2 RNA traces in respiratory samples of recovered patients. The failure of the virus to replicate in vitro suggests its inability to replicate in vivo.

Tedizolid-Rifampicin Combination Prevents Rifampicin-Resistance on in vitro Model of Staphylococcus aureus Mature Biofilm.

Staphylococcus aureus infections associated with implanted medical devices are difficult to treat and require long-lasting antibiotic therapies, especially when device removal is not possible or easy such as in the case of joint prostheses. Biofilm formation is a major cause of treatment failure and infection recurrence. This study aimed to test, for the first time, the in vitro combination of tedizolid plus rifampicin on methicillin-sensitive (MSSA ATCC 6538) and methicillin-resistant (MRSA ATCC 43300) S. aureus mature biofilm. Here, we demonstrated that the combination of tedizolid with rifampicin significantly disaggregated pre-formed biofilm of both strains, reduced their metabolic activity and exerted bactericidal activity at clinically meaningful concentrations. Notably, tedizolid was able to completely prevent the emergence of resistance to rifampicin. Moreover these effects were similar to those obtained with daptomycin plus rifampicin, a well-known and widely used combination. Preliminary results on some MRSA clinical isolates confirmed the efficacy of this combination in reducing biofilm biomass and preventing rifampicin resistance onset. Further in vivo studies are needed to confirm the validity of this promising therapeutic option that can be useful against biofilm-associated S. aureus infections.