Structures of the Staphylococcus aureus ribosome inhibited by fusidic acid and fusidic acid cyclopentane.

The antibiotic fusidic acid (FA) is used to treat Staphylococcus aureus infections. It inhibits protein synthesis by binding to elongation factor G (EF-G) and preventing its release from the ribosome after translocation. While FA, due to permeability issues, is only effective against gram-positive bacteria, the available structures of FA-inhibited complexes are from gram-negative model organisms. To fill this knowledge gap, we solved cryo-EM structures of the S. aureus ribosome in complex with mRNA, tRNA, EF-G and FA to 2.5 Å resolution and the corresponding complex structures with the recently developed FA derivative FA-cyclopentane (FA-CP) to 2.0 Å resolution. With both FA variants, the majority of the ribosomal particles are observed in chimeric state and only a minor population in post-translocational state. As expected, FA binds in a pocket between domains I, II and III of EF-G and the sarcin-ricin loop of 23S rRNA. FA-CP binds in an identical position, but its cyclopentane moiety provides additional contacts to EF-G and 23S rRNA, suggesting that its improved resistance profile towards mutations in EF-G is due to higher-affinity binding. These high-resolution structures reveal new details about the S. aureus ribosome, including confirmation of many rRNA modifications, and provide an optimal starting point for future structure-based drug discovery on an important clinical drug target.

Dual Drug-Loaded Coaxial Nanofiber Dressings for the Treatment of Diabetic Foot Ulcer.

Introduction: Diabetes mellitus is frequently associated with foot ulcers, which pose significant health risks and complications. Impaired wound healing in diabetic patients is attributed to multiple factors, including hyperglycemia, neuropathy, chronic inflammation, oxidative damage, and decreased vascularization. Rationale: To address these challenges, this project aims to develop bioactive, fast-dissolving nanofiber dressings composed of polyvinylpyrrolidone loaded with a combination of an antibiotic (moxifloxacin or fusidic acid) and anti-inflammatory drug (pirfenidone) using electrospinning technique to prevent the bacterial growth, reduce inflammation, and expedite wound healing in diabetic wounds. Results: The fabricated drug-loaded fibers exhibited diameters of 443 ± 67 nm for moxifloxacin/pirfenidone nanofibers and 488 ± 92 nm for fusidic acid/pirfenidone nanofibers. The encapsulation efficiency, drug loading and drug release studies for the moxifloxacin/pirfenidone nanofibers were found to be 70 ± 3% and 20 ± 1 µg/mg, respectively, for moxifloxacin, and 96 ± 6% and 28 ± 2 µg/mg, respectively, for pirfenidone, with a complete release of both drugs within 24 hours, whereas the fusidic acid/pirfenidone nanofibers were found to be 95 ± 6% and 28 ± 2 µg/mg, respectively, for fusidic acid and 102 ± 5% and 30 ± 2 µg/mg, respectively, for pirfenidone, with a release rate of 66% for fusidic acid and 80%, for pirfenidone after 24 hours. The efficacy of the prepared nanofiber formulations in accelerating wound healing was evaluated using an induced diabetic rat model. All tested formulations showed an earlier complete closure of the wound compared to the controls, which was also supported by the histopathological assessment. Notably, the combination of fusidic acid and pirfenidone nanofibers demonstrated wound healing acceleration on day 8, earlier than all tested groups. Conclusion: These findings highlight the potential of the drug-loaded nanofibrous system as a promising medicated wound dressing for diabetic foot applications.

Emergence and spread of a mupirocin-resistant variant of the European epidemic fusidic acid-resistant impetigo clone of Staphylococcus aureus, Belgium, 2013 to 2023.

BackgroundAntimicrobial resistance to mupirocin and fusidic acid, which are used for treatment of skin infections caused by Staphylococcus aureus, is of concern.AimTo investigate resistance to fusidic acid and mupirocin in meticillin-susceptible S. aureus (MSSA) from community-acquired skin and soft tissue infections (SSTIs) in Belgium.MethodsWe collected 2013-2023 data on fusidic acid and mupirocin resistance in SSTI-associated MSSA from two large Belgian laboratories. Resistant MSSA isolates sent to the Belgian Staphylococci Reference Centre were spa-typed and analysed for the presence of the eta and etb virulence genes and the mupA resistance gene. In addition, we whole genome sequenced MSSA isolates collected between October 2021 and September 2023.ResultsMupirocin resistance increased between 2013 and 2023 from 0.5-1.5% to 1.7-5.6%. Between 2018 and 2023, 91.4% (64/70) of mupirocin-resistant isolates were co-resistant to fusidic acid. By September 2023, between 8.9% (15/168) and 10.1% (11/109) of children isolates from the two laboratories were co-resistant. Of the 33 sequenced isolates, 29 were sequence type 121, clonal and more distantly related to the European epidemic fusidic acid-resistant impetigo clone (EEFIC) observed in Belgium in 2020. These isolates carried the mupA and fusB genes conferring resistance to mupirocin and fusidic acid, respectively, and the eta and etb virulence genes.ConclusionWe highlight the spread of a mupirocin-resistant EEFIC in children, with a seasonal trend for the third quarter of the year. This is of concern because this variant is resistant to the two main topical antibiotics used to treat impetigo in Belgium.

Enhancing the Topical Antibacterial Activity of Fusidic Acid via Embedding into Cinnamon Oil Nano-Lipid Carrier.

Presently, antimicrobial resistance is of great risk to remarkable improvements in health conditions and infection management. Resistance to various antibiotics has been considered a great obstacle in their usage, necessitating alternative strategies for enhancing the antibacterial effect. Combination therapy has been recognized as a considerable strategy that could improve the therapeutic influence of antibacterial agents. Therefore, the aim of this study was to combine the antibacterial action of compounds of natural origin like fusidic acid (FA) and cinnamon essential oil (CEO) for synergistic effects. A distinctive nanoemulsion (NE) was developed using cinnamon oil loaded with FA. Applying the Box-Behnken design (BBD) approach, one optimized formula was selected and integrated into a gel base to provide an FA-NE-hydrogel for optimal topical application. The FA-NE-hydrogel was examined physically, studied for in vitro release, and investigated for stability upon storage at different conditions, at room (25 °C) and refrigerator (4 °C) temperatures, for up to 3 months. Ultimately, the NE-hydrogel preparation was inspected for its antibacterial behavior using multidrug-resistant bacteria and checked by scanning electron microscopy. The FA-NE-hydrogel formulation demonstrated a pH (6.32), viscosity (12,680 cP), and spreadability (56.7 mm) that are acceptable for topical application. The in vitro release could be extended for 6 h, providing 52.0%. The formulation was stable under both test conditions for up to 3 months of storage. Finally, the FA-NE-hydrogel was found to inhibit the bacterial growth of not only Gram-positive but also Gram-negative bacteria. The inhibition was further elucidated by a scanning electron micrograph, indicating the efficiency of CEO in enhancing the antibacterial influence of FA when combined in an NE system.

Oral fusidic acid for the treatment of mild-to-moderate hidradenitis suppurativa.

BACKGROUND: Hidradenitis suppurativa (HS) is a debilitating inflammatory skin disease. Tetracyclines are one of the few therapeutic options recommended for mild-to-moderate disease. This study aimed to investigate the efficacy of systemic fusidic acid's (FA) effectiveness in treating HS. METHODS: This retrospective study analyzed 55 FA therapy cycles (TC, average weekly dose: 6409 mg; range: 5250-9800 mg; 2-12 weeks) in 49 patients. The outcome was evaluated using the Physician's Global Assessment (PGA) scale. Therapy response was defined as any reduction of inflammatory activity without the occurrence of flares. We also characterized adverse events and investigated predictors for treatment success. Results were compared to a matched control group receiving doxycycline. RESULTS: FA treatment (55 treatment cycles (TC); male: 45.5%; female: 54.5%) showed an overall response rate of 70.9% (39 TC). No worsening was observed. Significantly higher response rates were observed in females (83.3%, P = 0.026) and Hurley I (90.9%, P = 0.008). After multivariate adjustment, higher response rates were associated with the Hurley grade (P = 0.046) but not with gender (P = 0.0174). Adverse reactions (21.8% gastrointestinal symptoms) occurred in 27.3% (15 TC) and 46.7% within the first 4 weeks. Similar results were observed in the doxycycline control group (overall response rate: 76.4%). CONCLUSION: Oral FA is safe and improves symptoms in most patients. HS patients could benefit from oral FA treatment, especially in case of contraindications or resistance to tetracyclines.

Repurposing fusidic acid as an antimicrobial against enterococci with a low probability of resistance development.

Drug repurposing constitutes a strategy to combat antimicrobial resistance, by using agents with known safety, pharmacokinetics, and pharmacodynamics. Previous studies have implemented new fusidic acid (FA) front-loading-dose regimens, allowing higher serum levels than those achievable with ordinary doses. As susceptibility breakpoints are affected by serum level, we evaluated the repurposing of FA as an antimicrobial product against enterococci. FA minimum inhibitory concentrations (MICs) against standard enterococci strains; Enterococcus faecalis ATCC 29212 and Enterococcus faecium ATCC 27270 were 2 and 4 µg/mL, respectively. The MIC against 98 enterococcal clinical isolates was ≤ 8 µg/mL; all would be susceptible if categorized according to recalculated breakpoints (≥ 16 µg/mL), based on the serum level achieved using the front-loading regimen. FA administration in vivo, using the BALB/c mouse infection model, significantly reduced bacterial burden by two to three log10 units in the liver and spleen of mice infected with vancomycin-susceptible and -resistant strains. Exposure of the standard enterococcal strains to increasing, but not fixed, FA concentrations resulted in resistant strains (MIC = 128 µg/mL), with thicker cell walls and slower growth rates. Only one mutation (M651I) was detected in the fusA gene of the resistant strain derived from serial passage of E. faecium ATCC 27270, which was retained in the revertant strain after passage in the FA-free medium. In conclusion, FA can be repurposed as an antimicrobial drug against enterococci with a low probability of mutational resistance development, and can be employed for treatment of infections attributable to vancomycin-resistant enterococci.

Novel Microemulsion Containing Benzocaine and Fusidic Acid Simultaneously: Formulation, Characterization, and In Vitro Evaluation for Wound Healing.

Modern drug carrier technologies, such as microemulsions with small droplet sizes and high surface areas, improve the ability of low water solubility active ingredients to permeate and localize. The goal of this study was to create microemulsion formulations for wound healing that contained both fusidic acid (FA), an antibacterial agent, and benzocaine (BNZ), a local anesthetic. Studies on characterization were carried out, including viscosity, droplet size, and zeta potential. The drug-loaded microemulsion had a stable structure with -3.014 ± 1.265 mV of zeta potential and 19.388 ± 0.480 nm of droplet size. In both in vitro release and ex vivo permeability studies, the microemulsion was compared with Fucidin cream and oily BNZ solution. According to the drug release studies, BNZ release from the microemulsion and the BNZ solution showed a similar profile (p > 0.05), while FA release from the microemulsion had a higher drug release compared to Fucidin cream (p < 0.001). The microemulsion presented lower drug permeation (p > 0.05) for both active ingredients, on the other hand, provided higher drug accumulation compared to the control preparations. Moreover, according to the results of in vitro wound healing activity, the microemulsion indicated a dose-dependent wound healing potential with the highest wound healing activity at the highest concentrations. To the best of our knowledge, this developed BNZ- and FA-loaded microemulsion would be a promising candidate to create new opportunities for wound healing thanks to present the active ingredients, which have low water solubility, in a single formulation and achieved higher accumulation than control preparations.

The anti-staphylococcal fusidic acid as an efflux pump inhibitor combined with fluconazole against vaginal candidiasis in mouse model.

BACKGROUND: Candida albicans is the most common fungus that causes vaginal candidiasis in immunocompetent women and catastrophic infections in immunocompromised patients. The treatment of such infections is hindered due to the increasing emergence of resistance to azoles in C. albicans. New treatment approaches are needed to combat candidiasis especially in the dwindled supply of new effective and safe antifungals. The resistance to azoles is mainly attributed to export of azoles outside the cells by means of the efflux pump that confers cross resistance to all azoles including fluconazole (FLC). OBJECTIVES: This study aimed to investigate the possible efflux pump inhibiting activity of fusidic acid (FA) in C. albicans resistant isolates and the potential use of Fusidic acid in combination with fluconazole to potentiate the antifungal activity of fluconazole to restore its activity in the resistant C. albicans isolates. METHODS: The resistance of C. albicans isolates was assessed by determination of minimum inhibitory concentration. The effect of Fusidic acid at sub-inhibitory concentration on efflux activity was assayed by rhodamine 6G efflux assay and intracellular accumulation. Mice model studies were conducted to evaluate the anti-efflux activity of Fusidic acid and its synergistic effects in combination with fluconazole. Impact of Fusidic acid on ergosterol biosynthesis was quantified. The synergy of fluconazole when combined with Fusidic acid was investigated by determination of minimum inhibitory concentration. The cytotoxicity of Fusidic acid was tested against erythrocytes. The effect of Fusidic acid on efflux pumps was tested at the molecular level by real-time PCR and in silico study. In vivo vulvovaginitis mice model was used to confirm the activity of the combination in treating vulvovaginal candidiasis. RESULTS: Fusidic acid showed efflux inhibiting activity as it increased the accumulation of rhodamine 6G, a substrate for ABC-efflux transporter, and decreased its efflux in C. albicans cells. The antifungal activity of fluconazole was synergized when combined with Fusidic acid. Fusidic acid exerted only minimal cytotoxicity on human erythrocytes indicating its safety. The FA efflux inhibitory activity could be owed to its ability to interfere with efflux protein transporters as revealed by docking studies and downregulation of the efflux-encoding genes of both ABC transporters and MFS superfamily. Moreover, in vivo mice model showed that using fluconazole-fusidic acid combination by vaginal route enhanced fluconazole antifungal activity as shown by lowered fungal burden and a negligible histopathological change in vaginal tissue. CONCLUSION: The current findings highlight FA's potential as a potential adjuvant to FLC in the treatment of vulvovaginal candidiasis.

The origin of sequence type 72 community-associated methicillin-resistant Staphylococcus aureus (MRSA) and fusidic acid (FA) resistant sequence type 5 MRSA: Analysis of FA resistance and spa type in a single center in South Korea.

INTRODUCTION: We investigated the prevalence of fusidic acid (FA) resistance in MSSA and MRSA stratified by sequence (ST) and spa types, and determined the prevalence of FA resistance mechanisms. METHODS: From August 2014 to April 2020, S. aureus blood isolates were collected in Asan Medical Center, Seoul, South Korea. Antimicrobial susceptibility tests were performed using broth microdilution and interpreted according to EUCAST's FA criteria. We performed spa typing for fusA mutation presence and acquired FA resistance determinants (fusB, fusC, and fusD) by PCR. RESULTS: Of the 590 MRSA isolates, 372 were FA resistant, and among 425 MSSA isolates, 136 were resistant. Of the 380 ST5-MRSA isolates, 350 were FA resistant, whereas only 1 of 14 ST5-MSSA isolates was FA resistant. Conversely, of the 163 ST72-MRSA isolates, only 8 were resistant, whereas 37 of 42 ST72-MSSA were resistant. The fusA mutation (80%) was the most common determinant. The one FA resistant ST5-MSSA isolate belonged to the t2460 spa type, the most common spa type (24 of 35 isolates) of FA resistant ST5-MRSA. In addition, t324 and t148, which are minor spa types of ST72-MSSA, were susceptible to FA, in contrast to other ST72-MSSA spa types, and the major spa type of ST72-MRSA (110 of 163 isolates). CONCLUSIONS: FA resistance was common in ST5-MRSA and ST72-MSSA, and rare in ST5-MSSA and ST72-MRSA. Our findings suggest that minor clones of ST5-MSSA isolates, with the fusA mutation and minor clones of ST72-MSSA susceptible to FA, may have evolved to harbor the mecA gene.

Effect of Fusidic Acid and Some Nitrogen-Containing Derivatives on Liposomal and Mitochondrial Membranes.

The paper assesses the membranotropic action of the natural antibiotic fusidic acid (FA) and its derivatives. It was found that a FA analogue with ethylenediamine moiety (derivative 2), in contrast to native FA and 3,11-dioxime analogue (derivative 1), is able to increase the mobility of the lipid bilayer in the zone of lipid headgroups, as well as to induce permeabilization of lecithin liposome membranes. A similar effect of derivative 2 is also observed in the case of rat liver mitochondrial membranes. We noted a decrease in the microviscosity of the mitochondrial membrane and nonspecific permeabilization of organelle membranes in the presence of this agent, which was accompanied by a decrease in mitochondrial Δψ and OXPHOS efficiency. This led to a reduction in mitochondrial calcium retention capacity. The derivatives also reduced the production of H2O2 by mitochondria. The paper considers the relationship between the structure of the tested compounds and the observed effects.

Synthesis and structure-activity relationship studies of fusidic acid derivatives as anti-inflammatory agents for acute lung injury.

Acute lung injury (ALI) are severe forms of diffuse lung disease that impose a substantial health burden all over the world. In the United States, approximately 190,000 cases per year of ALI each year, with an associated 74,500 deaths per year. Anti-inflammatory therapy has become a reasonable approach for the treatment of patients with ALI. In this study, fusidic acid derivatives were used to design new anti-inflammatory compounds with high pharmacological activity and low toxicity. A total of 30 new fusidic acid derivatives were discovered, synthesized, and screened for their anti-inflammatory activity against lipopolysaccharide (LPS)-treated RAW264.7 cells. Of them, b2 was found to be the most active, with a higher efficiency compared with fusidic acid and celecoxib in 10 µM. In vitro, we further measured b2 inhibited inflammatory factor NO (IC50 = 5.382 ± 0.655 µM), IL-6 (IC50 = 7.767 ± 0.871 µM), and TNF-α (IC50 = 7.089 ± 0.775 µM) and b2 inhibited inflammatory cytokines COX-2 and iNOS, ROS production, NF-κB/MAPK and Bax/Bcl-2 signaling pathway pathway. In vivo,b2 attenuated ALI pathological changes and inhibited inflammatory cytokines COX-2 and iNOS in lung tissue and NF-κB/MAPK and Bax/Bcl-2 signaling pathway. In conclusion, b2 may be a promising anti-inflammatory lead compound.

Polymyxin B and fusidic acid, a novel potent synergistic combination against Klebsiella pneumoniae and Escherichia coli isolates with polymyxin B resistance.

The increasing prevalence of multidrug-resistant (MDR) Gram-negative bacteria and comparatively limited options of antibiotics pose a major threat to public health worldwide. Polymyxin B is the last resort against extensively resistant Gram-negative bacterial infections. However, a large number of Gram-negative bacteria exhibited high-level resistance to Polymyxin B, bringing challenges for antimicrobial chemotherapy. Combination therapies using polymyxins and other antibiotics are recommended to treat multidrug-resistant pathogens. In this study, we selected Gram-negative bacterial strains, including Klebsiella pneumoniae and Escherichia coli, to explore whether fusidic acid and polymyxin B have a synergistic killing effect. Through broth microdilution, we observed that minimum inhibitory concentrations (MICs) against polymyxin B in the isolates tested were significantly reduced by the addition of fusidic acid. Notably, chequerboard analysis indicated a synergistic effect between polymyxin B and fusidic acid. In addition, subsequent time-kill experiments showed that the combination of polymyxin B and fusidic acid was more effective than a single drug in killing bacteria. Finally, our investigation utilizing the murine model revealed a higher survival rate in the combination therapy group compared to the monotherapy group. Our research findings provide evidence of the synergistic effect between polymyxin B and fusidic acid. Fusidic acid was shown to increase the sensitivity of multi-drug resistant E. coli and K. pneumoniae to polymyxin B, thereby enhancing its bactericidal activity. This study provides new insights into a potential strategy for overcoming polymyxin B resistance, however, further investigations are required to evaluate their feasibility in real clinical settings.

Curcumin/Fusidic Acid Bitherapy Loaded Mixed Micellar Nanogel for Acne Vulgaris Treatment: In Vitro and In Vivo Studies.

The combination of herbal drugs with a topical antibacterial for managing a chronic disease like acne vulgaris has emerged lately to settle side effects and bacterial multidrug resistance. Mixed micelles (MMs) incorporated into nanogel were explored for hybrid delivery of curcumin (Cur) and fusidic acid (FA) combination presenting a multi-strategic treatment. Curcumin-fusidic acid-loaded mixed micelles (Cur-FA-MMs) were assessed for size, surface charge, compatibility, in vitro release, and encapsulation. The selected formula was further loaded into nanogel and investigated for viscosity, ex vivo permeation, and in vivo potential. Cur-FA-MMs exhibited uniform nanosized spherical morphology, and negative surface charge affording high encapsulation for both drugs with a biphasic in vitro release over a period of 48h and good colloidal stability. The attained Cur-FA-MM-loaded nanogel had optimum viscosity with remarkable permeation coefficient values nearly 2-fold that related to plain nanogel. The pharmacodynamic effect of Cur on FA was pronounced by the significant improvement of the skin's degree of inflammation, epidermal hypertrophy, and congestion in animals treated with Cur-FA-MM-loaded nanogel. In conclusion, micellar nanogel could enable the progressive effect of Cur (an antioxidant with reported antibiotic activity) on FA (antibiotic) and decrease the risk of emerging antibiotic resistance by enhancing the solubility and permeation of Cur.

Design, synthesis fusidic acid derivatives alleviate acute lung injury via inhibiting MAPK/NF-κB/NLRP3 pathway.

Acute lung injury (ALI) refers to a series of lung lesions resulting from multiple lung injuries, even leading to morbidity and death, abundant previous reports have showed that anti-inflammatory as a key to treatment of ALI. Fusidic acid (FA) as an antibiotic has significant anti-bacterial activity and anti-inflammatory effects. In this study, we designed and synthesized 34 FA derivatives to identify new anti-inflammatory drugs. The anti-inflammatory activities of the derivatives were screened using lipopolysaccharide (LPS)-induced RAW264.7 cells to evaluate the anti-inflammatory activity of the compounds, we measured nitric oxide (NO) and interleukin-6 (IL-6). Most of compounds showed inhibitory effects on inflammatory NO and IL-6 in LPS-induced RAW264.7 cells. Based on the screening results, compound a1 showed the strongest anti-inflammatory activity. Compared with FA, the inhibition rate NO and IL-6 of compound a1 increased 3.08 and 2.09 times at 10 µM, respectively. We further measured a1 inhibited inflammatory factor NO (IC50 = 3.26 ± 0.42 µM), IL-6 (IC50 = 1.85 ± 0.21 µM) and TNF-α (IC50 = 3.88 ± 0.55 µM). We also demonstrated that a1 markedly inhibits the expression of certain immune-related cytotoxic factors, including cyclooxygenase-2 (COX-2) and inducible nitric-oxide synthase (iNOS). In vivo results indicate that a1 can reduce lung inflammation and NO, IL-6, TNF-α, COX-2 and iNOS in LPS-induced ALI mice. On the one hand, we demonstrated a1 inhibits the mitogen-activated protein kinase (MAPK) signaling pathway by down-regulating the phosphorylation of p38 MAPK, c-Jun N-terminal kinase (c-JNK) and extracellular signal-regulated kinase (ERK). Moreover, a1 also suppressing the phosphorylation of inhibitory NF-κB inhibitor α (IκBα) inhibits the activation of the nuclear factor-κB (NF-κB) signaling pathway. On the other hand, we demonstrated a1 also role in anti-inflammatory by inhibits nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) inflammasome and further inhibits Caspase-1 and inflammatory factor interleukin-1ß (IL-1ß). In conclusion, our study demonstrates that a1 has an anti-inflammatory effect and alleviates ALI by regulating inflammatory mediators and suppressing the MAPK, NF-κB and NLRP3 inflammasome signaling pathways.

Enterococcus faecalis OG1RF Evolution at Low pH Selects Fusidate-Sensitive Mutants in Elongation Factor G and at High pH Selects Defects in Phosphate Transport.

Enterococcus bacteria inhabit human and soil environments that show a wide range of pH values. Strains include commensals as well as antibiotic-resistant pathogens. We investigated the adaptation to pH stress in E. faecalis OG1RF by conducting experimental evolution under acidic (pH 4.8), neutral pH (pH 7.0), and basic (pH 9.0) conditions. A serial planktonic culture was performed for 500 generations and in a high-pH biofilm culture for 4 serial bead transfers. Nearly all of the mutations led to nonsynonomous codons, indicating adaptive selection. All of the acid-adapted clones from the planktonic culture showed a mutation in fusA (encoding elongation factor G). The acid-adapted fusA mutants had a trade-off of decreased resistance to fusidic acid (fusidate). All of the base-adapted clones from the planktonic cultures as well as some from the biofilm-adapted cultures showed mutations that affected the Pst phosphate ABC transporter (pstA, pstB, pstB2, pstC) and pyrR (pyrimidine biosynthesis regulator/uracil phosphoribosyltransferase). The biofilm cultures produced small-size colonies on brain heart infusion agar. These variants each contained a single mutation in pstB2, pstC, or pyrR. The pst and pyrR mutants outgrew the ancestral strain at pH 9.2, with a trade-off of lower growth at pH 4.8. Additional genes that had a mutation in multiple clones that evolved at high pH (but not at low pH) include opp1BCDF (oligopeptide ABC transporter), ccpA (catabolite control protein A), and ftsZ (septation protein). Overall, the experimental evolution of E. faecalis showed a strong pH dependence, favoring the fusidate-sensitive elongation factor G modification at low pH and the loss of phosphate transport genes at high pH. IMPORTANCE E. faecalis bacteria are found in dental biofilms, where they experience low pH as a result of fermentative metabolism. Thus, the effect of pH on antibiotic resistance has clinical importance. The loss of fusidate resistance is notable for OG1RF strains in which fusidate resistance is assumed to be a stable genetic marker. In endodontal infections, enterococci can resist calcium hydroxide therapy that generates extremely high pH values. In other environments, such as the soil and plant rhizosphere, enterococci experience acidification that is associated with climate change. Thus, the pH modulation of natural selection in enterococci is important for human health as well as for understanding soil environments.

3-Amino-Substituted Analogues of Fusidic Acid as Membrane-Active Antibacterial Compounds.

Fusidic acid (FA) is an antibiotic with high activity against Staphylococcus aureus; it has been used in clinical practice since the 1960s. However, the narrow antimicrobial spectrum of FA limits its application in the treatment of bacterial infections. In this regard, this work aims both at the study of the antimicrobial effect of a number of FA amines and at the identification of their potential biological targets. In this way, FA analogues containing aliphatic and aromatic amino groups and biogenic polyamine, spermine and spermidine, moieties at the C-3 atom, were synthesized (20 examples). Pyrazinecarboxamide-substituted analogues exhibit a high antibacterial activity against S. aureus (MRSA) with MIC ≤ 0.25 µg/mL. Spermine and spermidine derivatives, along with activity against S. aureus, also inhibit the growth and reproduction of Gram-negative bacteria Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa, and have a high fungicidal effect against Candida albicans and Cryptococcus neoformans. The study of the membrane activity demonstrated that the spermidine- and spermine-containing compounds are able to immerse into membranes and disorder the lipidsleading to a detergent effect. Moreover, spermine-based compounds are also able to form ion-permeable pores in the lipid bilayers mimicking the bacterial membranes. Using molecular docking, inhibition of the protein synthesis elongation factor EF-G was proposed, and polyamine substituents were shown to make the greatest contribution to the stability of the complexes of fusidic acid derivatives with biological targets. This suggests that the antibacterial effect of the obtained compounds may be associated with both membrane activity and inhibition of the elongation factor EF-G.

Purification of PaTx-II from the Venom of the Australian King Brown Snake and Characterization of Its Antimicrobial and Wound Healing Activities.

Infections caused by multi-drug-resistant (MDR) bacteria are a global threat to human health. As venoms are the source of biochemically diverse bioactive proteins and peptides, we investigated the antimicrobial activity and murine skin infection model-based wound healing efficacy of a 13 kDa protein. The active component PaTx-II was isolated from the venom of Pseudechis australis (Australian King Brown or Mulga Snake). PaTx-II inhibited the growth of Gram-positive bacteria in vitro, with moderate potency (MICs of 25 µM) observed against S. aureus, E. aerogenes, and P. vulgaris. The antibiotic activity of PaTx-II was associated with the disruption of membrane integrity, pore formation, and lysis of bacterial cells, as evidenced by scanning and transmission microscopy. However, these effects were not observed with mammalian cells, and PaTx-II exhibited minimal cytotoxicity (CC50 > 1000 µM) toward skin/lung cells. Antimicrobial efficacy was then determined using a murine model of S. aureus skin infection. Topical application of PaTx-II (0.5 mg/kg) cleared S. aureus with concomitant increased vascularization and re-epithelialization, promoting wound healing. As small proteins and peptides can possess immunomodulatory effects to enhance microbial clearance, cytokines and collagen from the wound tissue samples were analyzed by immunoblots and immunoassays. The amounts of type I collagen in PaTx-II-treated sites were elevated compared to the vehicle controls, suggesting a potential role for collagen in facilitating the maturation of the dermal matrix during wound healing. Levels of the proinflammatory cytokines interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2) and interleukin-10 (IL-10), factors known to promote neovascularization, were substantially reduced by PaTx-II treatment. Further studies that characterize the contributions towards efficacy imparted by in vitro antimicrobial and immunomodulatory activity with PaTx-II are warranted.

Characterisation of Methicillin-Resistant Staphylococcus aureus from Alexandria, Egypt.

The present study aims to characterise clinical MRSA isolates from a tertiary care centre in Egypt's second-largest city, Alexandria. Thirty isolates collected in 2020 were genotypically characterised by microarray to detect their resistance and virulence genes and assign them to clonal complexes (CC) and strains. Isolates belonged to 11 different CCs and 14 different strains. CC15-MRSA-[V+fus] (n = 6), CC1-MRSA-[V+fus+tir+ccrA/B-1] (PVL+) (n = 5) as well as CC1-MRSA-[V+fus+tir+ccrA/B-1] and CC1153-MRSA-[V+fus] (PVL+) (both with n = 3) were the most common strains. Most isolates (83%) harboured variant or composite SCCmec V or VI elements that included the fusidic acid resistance gene fusC. The SCCmec [V+fus+tir+ccrA/B-1] element of one of the CC1 isolates was sequenced, revealing a presence not only of fusC but also of blaZ, aacA-aphD and other resistance genes. PVL genes were also common (40%). The hospital-acquired MRSA CC239-III strain was only found twice. A comparison to data from a study on strains collected in 2015 (Montelongo et al., 2022) showed an increase in fusC and PVL carriage and a decreasing prevalence of the CC239 strain. These observations indicate a diffusion of community-acquired strains into hospital settings. The beta-lactam use in hospitals and the widespread fusidic acid consumption in the community might pose a selective pressure that favours MRSA strains with composite SCCmec elements comprising mecA and fusC. This is an unsettling trend, but more MRSA typing data from Egypt are required.

Enhancing Antibiotics Efficacy by Combination of Kuraridin and Epicatechin Gallate with Antimicrobials against Methicillin-Resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is an opportunistic pathogen and a major cause of nosocomial and community-acquired infections. The alarming rise in Methicillin-resistant S. aureus (MRSA) infection worldwide and the emergence of vancomycin-resistant MRSA strains have created an urgent need to identify new and alternative treatment options. Triple combinations of antimicrobials with different antimicrobial mechanisms may be a good choice to overcome antimicrobial resistance. METHODS: In this study, we combine two natural compounds: kuraridin from Sophora flavescens and epicatechin gallate (ECG) from Camellia sinensis (Green tea), which could provide the best synergy with antibiotics against a selected panel of laboratory MRSA with known resistant mechanisms and clinical community-associated (CA) and hospital-associated (HA) MRSA as well. RESULTS: The combined use of ECG and kuraridin was efficacious in inhibiting the growth of a panel of tested MRSA strains. The antibacterial activities of gentamicin, fusidic acid and vancomycin could be further enhanced by the addition of ECG and kuraridin. In time-kill study, when vancomycin (0.5 µg/mL) was combined with ECG (2 µg/mL) and kuraridin (2 µg/mL), a very strong bactericidal growth inhibition against 3 tested strains ATCC25923, MRSA ST30 and ST239 was observed from 2 to 24 h. ECG and kuraridin both possess anti-inflammatory activities in bacterial toxin-stimulated peripheral blood mononuclear cells by suppressing the production of inflammatory cytokines (IL-1ß, IL-6 and TNFα) and are non-cytotoxic. In a murine pneumonia model infected with ATCC25923, MRSA ST30 or ST239, the combined use of ECG and kuraridin with vancomycin could significantly reduce bacterial counts. CONCLUSIONS: The present findings reveal the potential of ECG and kuraridin combination as a non-toxic herbal and antibiotics combination for MRSA treatment with antibacterial and anti-inflammatory activities.

Fusidic Acid and Its Major Active Metabolite Penetration into Cerebrospinal Fluid for Assessing Treatment of Intracranial Infections.

Fusidic acid (FA) had excellent antimicrobial effects due to its unique mechanism of action. Since 1962, FA has been widely used in the systemic and topical treatment of staphylococcal infections and exhibits a well-characterized potency against methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and methicillin-resistant coagulase-negative Staphylococci. In view of the spectrum of activity, no cross-resistance with other clinically used antibiotics, and potential penetration into brain tissue, FA was used to treat possible gra-positive bacteria in 3 patients with intracranial infections in the present report. FA and its active metabolite (3-keto FA) were measured in plasma and cerebrospinal fluid (CSF) to assess the treatment of FA, and the results indicated that 1,500 mg per day of FA was sufficient to achieve therapeutic concentrations in both plasma and CSF in intracranial infection patients, while the dosage did not experience unexpected regimen-related toxicity.