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.

Hydroxytakakiamide and Other Constituents from a Marine Sponge-Associated Fungus Aspergillus fischeri MMERU23, and Antinociceptive Activity of Ergosterol Acetate, Acetylaszonalenin and Helvolic Acid.

An unreported prenylated indole derivative hydroxytakakiamide (4) was isolated, together with the previously described ergosterol (1), ergosterol acetate (2), and (3R)-3-(1H-indol-3-ylmethyl)-3, 4-dihydro-1H-1,4-benzodiazepine-2,5-dione (3), from the column fractions of the crude ethyl acetate extract of the culture of a marine sponge-associated fungus, Aspergillus fischeri MMERU 23. The structure of 4 was elucidated by the interpretation of 1D and 2D NMR spectral data and high-resolution mass spectrum. The absolute configuration of the stereogenic carbon in 3 was proposed to be the same as those of the co-occurring congeners on the basis of their biogenetic consideration and was supported by the comparison of its sign of optical rotation with those of its steroisomers. The crude ethyl acetate extract and 2 were evaluated, together with acetylaszonalenin (5) and helvolic acid (6), which were previously isolated from the same extract, for the in vivo antinociceptive activity in the mice model. The crude ethyl acetate extract exhibited antinociceptive activity in the acetic acid-induced writhing and formalin tests, while 2, 5, and 6 displayed the effects in the late phase of the formalin test. On the other hand, neither the crude ethyl acetate extract nor 2, 5, and 6 affected the motor performance of mice in both open-field and rotarod tests. Additionally, docking studies of 2, 5, and 6 were performed with 5-lipoxygenase (5-LOX) and phosphodiesterase (PDE) enzymes, PDE4 and PDE7, which are directly related to pain and inflammatory processes. Molecular docking showed that 6 has low affinity energy to PDE4 and PDE7 targets while retaining high affinity to 5-LOX. On the other hand, while 2 did not display any hydrogen bond interactions in any of its complexes, it achieved overall better energy values than 6 on the three antinociceptive targets. On the other hand, 5 has the best energy profile of all the docked compounds and was able to reproduce the crystallographic interactions of the 5-LOX complex.

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.

A Nanocomposite Dynamic Covalent Cross-Linked Hydrogel Loaded with Fusidic Acid for Treating Antibiotic-Resistant Infected Wounds.

Methicillin-resistant Staphylococcus aureus (MRSA) is associated with high levels of morbidity and is considered a difficult-to-treat infection, often requiring nonstandard treatment regimens and antibiotics. Since over 40% of the emerging antibiotic compounds have insufficient solubility that limits their bioavailability and thus efficacy through oral or intravenous administration, it is crucial that alternative drug delivery products be developed for wound care applications. Existing effective treatments for soft tissue MRSA infections, such as fusidic acid (FA), which is typically administered orally, could also benefit from alternative routes of administration to improve local efficacy and bioavailability while reducing the required therapeutic dose. Herein, we report an antimicrobial poly(oligoethylene glycol methacrylate) (POEGMA)-based composite hydrogel loaded with fusidic acid-encapsulating self-assembled polylactic acid-b-poly(oligo(ethylene glycol) methyl ether methacrylate) (PLA-POEGMA) nanoparticles for the treatment of MRSA-infected skin wounds. The inclusion of the self-assembled nanoparticles (380 nm diameter when loaded with fusidic acid) does not alter the favorable mechanical properties and stability of the hydrogel in the context of its use as a wound dressing, while fusidic acid (FA) can be released from the hydrogel over ∼10 h via a diffusion-controlled mechanism. The antimicrobial studies demonstrate a clear zone of inhibition in vitro and a 1-2 order of magnitude inhibition of bacterial growth in vivo in an MRSA-infected full-thickness excisional murine wound model even at very low antibiotic doses. Our approach thus can both circumvent challenges in the local delivery of hydrophobic antimicrobial compounds and directly deliver antimicrobials into the wound to effectively combat methicillin-resistant infections using a fraction of the drug dose required using other clinically relevant strategies.

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.

New fusidane-type nortriterpenoids from the Arctic marine-derived fungus Simplicillium lamellicola culture medium with their inhibitory effect on benign prostatic hyperplasia.

Three new fusidane-type nortriterpenoids, simplifusinolide A, 24-epi simplifusinolide A, and simplifusidic acid L (1-3), were isolated from the EtOAc extract of the Arctic marine-derived fungus Simplicillium lamellicola culture medium, together with fusidic acid (4) and 16-O-deacetylfusicid acid (5). The structures of the isolated compounds were elucidated by NMR and MS analyses. The absolute configurations of compounds 1-3 were established by the quantum mechanical calculations of electronic circular dichroism and gauge-including atomic orbital NMR chemical shifts, followed by DP4 + analysis. Benign prostatic hyperplasia (BPH) is a major urological disorder in men worldwide. The anti-BPH potentials of the isolated compounds were evaluated using BPH-1 and WPMY-1 cells. Treatment with simplifusidic acid L (3) and fusidic acid (4) significantly downregulated the mRNA levels of the androgen receptor (AR) and its downstream effectors, inhibiting the proliferation of BPH-1 cells. Specifically, treatment with 24-epi simplifusinolide A (2) significantly suppressed the cell proliferation of both BPH-1 and DHT-stimulated WPMY-1 cells by inhibiting AR signaling. These results suggest the potential of 24-epi simplifusinolide A (2), simplifusidic acid L (3) and fusidic acid (4) as alternative agents for BPH treatment by targeting AR signaling.

Neutrophil-targeted combinatorial nanosystems for suppressing bacteremia-associated hyperinflammation and MRSA infection to improve survival rates.

There is currently no specific and effective treatment for bacteremia-mediated sepsis. Hence, this study engineered a combinatorial nanosystem containing neutrophil-targeted roflumilast-loaded nanocarriers and non-targeted fusidic acid-loaded nanoparticles to enable the dual mitigation of bacteremia-associated inflammation and methicillin-resistant Staphylococcus aureus (MRSA) infection. The targeted nanoparticles were developed by conjugating anti-lymphocyte antigen 6 complex locus G6D (Ly6G) antibody fragment on the nanoparticulate surface. The particle size and zeta potential of the as-prepared nanosystem were about 200 nm and -25 mV, respectively. The antibody-conjugated nanoparticles showed a three-fold increase in neutrophil internalization compared to the unfunctionalized nanoparticles. As a selective phosphodiesterase (PDE) 4 inhibitor, the roflumilast in the nanocarriers largely inhibited cytokine/chemokine release from the activated neutrophils. The fusidic acid-loaded nanocarriers were vital to eliminate biofilm MRSA colony by 3 log units. The nanoparticles drastically decreased the intracellular bacterial count compared to the free antibiotic. The in vivo mouse bioimaging demonstrated prolonged retention of the nanosystem in the circulation with limited organ distribution and liver metabolism. In the mouse bacteremia model, the multifunctional nanosystem produced a 1‒2 log reduction of MRSA burden in peripheral organs and blood. The functionalized nanosystem arrested the cytokine/chemokine overexpression greater than the unfunctionalized nanocarriers and free drugs. The combinatory nanosystem also extended the median survival time from 50 to 103 h. No toxicity from the nanoformulation was found based on histology and serum biochemistry. Furthermore, our data proved that the active neutrophil targeting by the versatile nanosystem efficiently alleviated MRSA infection and organ dysfunction caused by bacteremia. STATEMENT OF SIGNIFICANCE: Bacteremia-mediated sepsis poses a significant challenge in clinical practice, as there is currently no specific and effective treatment available. In our study, we have developed a novel combinatorial nanosystem to address this issue. Our nanosystem consists of neutrophil-targeted roflumilast-loaded nanocarriers and non-targeted fusidic acid-loaded nanoparticles, enabling the simultaneous mitigation of bacteremia-associated inflammation and MRSA infection. Our nanosystem demonstrated the decreased neutrophil activation, effective inhibition of cytokine release, elimination of MRSA biofilm colonies, and reduced intracellular bacterial counts. In vivo experiments showed prolonged circulation, limited organ distribution, and increased survival rates in a mouse bacteremia model. Importantly, our nanosystem exhibited no toxicity based on comprehensive assessments.

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.

Porin-independent accumulation in Pseudomonas enables antibiotic discovery.

Gram-negative antibiotic development has been hindered by a poor understanding of the types of compounds that can accumulate within these bacteria1,2. The presence of efflux pumps and substrate-specific outer-membrane porins in Pseudomonas aeruginosa renders this pathogen particularly challenging3. As a result, there are few antibiotic options for P. aeruginosa infections4 and its many porins have made the prospect of discovering general accumulation guidelines seem unlikely5. Here we assess the whole-cell accumulation of 345 diverse compounds in P. aeruginosa and Escherichia coli. Although certain positively charged compounds permeate both bacterial species, P. aeruginosa is more restrictive compared to E. coli. Computational analysis identified distinct physicochemical properties of small molecules that specifically correlate with P. aeruginosa accumulation, such as formal charge, positive polar surface area and hydrogen bond donor surface area. Mode of uptake studies revealed that most small molecules permeate P. aeruginosa using a porin-independent pathway, thus enabling discovery of general P. aeruginosa accumulation trends with important implications for future antibiotic development. Retrospective antibiotic examples confirmed these trends and these discoveries were then applied to expand the spectrum of activity of a gram-positive-only antibiotic, fusidic acid, into a version that demonstrates a dramatic improvement in antibacterial activity against P. aeruginosa. We anticipate that these discoveries will facilitate the design and development of high-permeating antipseudomonals.

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.

Biochemical and Cellular Characterization of the Function of Fluorophosphonate-Binding Hydrolase H (FphH) in Staphylococcus aureus Support a Role in Bacterial Stress Response.

The development of new treatment options for bacterial infections requires access to new targets for antibiotics and antivirulence strategies. Chemoproteomic approaches are powerful tools for profiling and identifying novel druggable target candidates, but their functions often remain uncharacterized. Previously, we used activity-based protein profiling in the opportunistic pathogen Staphylococcus aureus to identify active serine hydrolases termed fluorophosphonate-binding hydrolases (Fph). Here, we provide the first characterization of S. aureus FphH, a conserved, putative carboxylesterase (referred to as yvaK in Bacillus subtilis) at the molecular and cellular level. First, phenotypic characterization of fphH-deficient transposon mutants revealed phenotypes during growth under nutrient deprivation, biofilm formation, and intracellular survival. Biochemical and structural investigations revealed that FphH acts as an esterase and lipase based on a fold well suited to act on a small to long hydrophobic unbranched lipid group within its substrate and can be inhibited by active site-targeting oxadiazoles. Prompted by a previous observation that fphH expression was upregulated in response to fusidic acid, we found that FphH can deacetylate this ribosome-targeting antibiotic, but the lack of FphH function did not infer major changes in antibiotic susceptibility. In conclusion, our results indicate a functional role of this hydrolase in S. aureus stress responses, and hypothetical functions connecting FphH with components of the ribosome rescue system that are conserved in the same gene cluster across Bacillales are discussed. Our atomic characterization of FphH will facilitate the development of specific FphH inhibitors and probes to elucidate its physiological role and validity as a drug target.

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.

Identification and validation of fusidic acid and flufenamic acid as inhibitors of SARS-CoV-2 replication using DrugSolver CavitomiX.

In this work, we present DrugSolver CavitomiX, a novel computational pipeline for drug repurposing and identifying ligands and inhibitors of target enzymes. The pipeline is based on cavity point clouds representing physico-chemical properties of the cavity induced solely by the protein. To test the pipeline's ability to identify inhibitors, we chose enzymes essential for SARS-CoV-2 replication as a test system. The active-site cavities of the viral enzymes main protease (Mpro) and papain-like protease (Plpro), as well as of the human transmembrane serine protease 2 (TMPRSS2), were selected as target cavities. Using active-site point-cloud comparisons, it was possible to identify two compounds-flufenamic acid and fusidic acid-which show strong inhibition of viral replication. The complexes from which fusidic acid and flufenamic acid were derived would not have been identified using classical sequence- and structure-based methods as they show very little structural (TM-score: 0.1 and 0.09, respectively) and very low sequence (~ 5%) identity to Mpro and TMPRSS2, respectively. Furthermore, a cavity-based off-target screening was performed using acetylcholinesterase (AChE) as an example. Using cavity comparisons, the human carboxylesterase was successfully identified, which is a described off-target for AChE inhibitors.

Persistence of the Staphylococcus aureus epidemic European fusidic acid-resistant impetigo clone (EEFIC) in Belgium.

OBJECTIVES: In August 2018, a public health alert was issued in Belgium regarding clusters of impetigo cases caused by the epidemic European fusidic acid-resistant impetigo clone (EEFIC) of Staphylococcus aureus. As a result, the Belgian national reference centre (NRC) was commissioned to update the epidemiology of S. aureus causing community-onset skin and soft tissues infection (CO-SSTI) to assess the proportion of EEFIC among them. METHODS: For 1 year, Belgian clinical laboratories were asked to send their first three S. aureus isolated from CO-SSTI each month. Isolates were tested for antimicrobial susceptibility to oxacillin, mupirocin and fusidic acid. Resistant isolates were also spa typed and tested for the presence of the genes encoding the Panton-Valentine leucocidin, the toxic shock syndrome toxin and the exfoliatins A and B. MLST clonal complexes were deduced from the spa types. RESULTS: Among the 518 S. aureus strains analysed, 487 (94.0%) were susceptible to oxacillin. Of these, 79 (16.2%) were resistant to fusidic acid, of which 38 (48.1%) belonged to the EEFIC. EEFIC isolates were mostly isolated from young patients with impetigo and showed a seasonal late summer peak. CONCLUSIONS: These results suggest the persistence of EEFIC in Belgium. Furthermore, its prevalence may lead to reconsideration of the treatment guidelines for impetigo.

Clonal Spreading of ST42 Staphylococcus haemolyticus Strains Occurs Possibly Due to fusB and tetK Resistant Genes and Capsule-Related Genes.

Multi-drug resistant Staphylococcus haemolyticus is a frequent nosocomial invasive bacteremia pathogen in hospitals. Our previous analysis showed one of the predominant strains, ST42 originated from ST3, had only one multilocus sequence typing (MLST) variation among seven loci in SH1431; yet no significant differences in biofilm formation observed between ST42 and ST3, suggesting that other factors influence clonal lineage change. Whole genome sequencing was conducted on two isolates from ST42 and ST3 to find phenotypic and genotypic variations, and these variations were further validated in 140 clinical isolates. The fusidic acid- and tetracycline-resistant genes (fusB and tetK) were found only in CGMH-SH51 (ST42). Further investigation revealed consistent resistant genotypes in all isolates, with 46% and 70% of ST42 containing fusB and tetK, respectively. In contrast, only 23% and 4.2% ST3 contained these two genes, respectively. The phenotypic analysis also showed that ST42 isolates were highly resistant to fusidic acid (47%) and tetracycline (70%), compared with ST3 (23% and 4%, respectively). Along with drug-resistant genes, three capsule-related genes were found in higher percentage distributions in ST42 than in ST3 isolates. Our findings indicate that ST42 could become endemic in Taiwan, further constitutive surveillance is required to prevent the spread of this bacterium.

Novel recombinant endolysin ointment with broad antimicrobial activity against methicillin-resistant Staphylococcus aureus isolated from wounds and burns.

The major problem in the management of burn wounds is infections. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of infection in burn wounds. Antibiotic-resistant bacteria around the world have become a major therapeutic challenge. Bacteriophages and their lysin are suggested as an antimicrobial alternative agent. The approach of this study was to evaluate the potential of recombinant phage lysin ointment efficacy in MRSA burn wound infection in vitro. Whole genome sequencing was performed to the three isolated bacteriophages by ABM, USA using Illumina next-generation sequencing (NGS) technology. De novo assembly and genetic analysis carried out. Expression of lysin genes was performed by cloning using Escherichia coli JM109. Lysin protein extraction and purification was performed before and after cloning using precipitation by ammonium sulfate, dialysis, and gel filtration chromatography. Dose-dependent assay and time-kill curve experiment was performed for 2 lysins showed that recombinant lysin 2 functions more than its non-recombinant lysins 2 with the same concentration of 0.5 µg/mL. Both lysins' ointment was prepared and compared with commercial ointments. 62 (78.4%) out of 79 wounds a burns swabs were detected as S. aureus and S methicillin-resistant S. aureus (MRSA) rate was determined to be 29 (46.8%) in total, while 33 isolates (53.2%) determined as methicillin-sensitive S. aureus (MSSA). According to the antibiotic susceptibility test results, all S. aureus isolates were identified as sensitive against vancomycin, ceftaroline, and linezolid. Results shows one lysogenic bacteriophage and three distinct lytic specific S. aureus bacteriophage were isolated from sewage. For each of the three samples, a single contig was possible to be obtained. Sample BP-SA2 had the best coverage, and the contig was slightly longer than the other bacteriophages. In addition, BLAST search identified Staphylococcus bacteriophage vB-SscM-1 (accession KX171212.1) as the closest match to the public database. Finally, the gene annotation was checked, and two potential lysin genes were identified. Besides the two ends, there are only 4 SNPs between the three genomes. It should be noted that the two lysin genes from the genomes have no SNPs, and are identical across the three genomes. It can be seen that the three bacteriophages (BP-SA1), (BP-SA 2), and (BP-SA3) form their own tight cluster. It can be seen that (BP-SA 2) is more closely related to Staphylococcus bacteriophage vB-SscM-1 genome and most noticeable 5' region of S5 and vB-SscM-1 are now located at 3' end of vB-Sau-Clo6. The investigation of the two lysin genes in (BP-SA 2) by whole genome sequencing showed that there is some homology with vB-SscM-1; although the first gene is annotated as hypothetical protein, the second gene is annotated as amidase. The same two lysin genes are identified in all three bacteriophage genomes by the RAST. The putative protein sequences of the discovered phage lysin was analyzed using protein search with UniProt/Swiss-Prot database, and all matches suggest that the putative protein of the discovered phage lysin is a real endolysin. The three samples of bacteriophage were harboring both (Lysin 1 and lysin 2) genes were amplified. Afterward, 2-lysin genes were cloned successfully; for the dose-dependent assay, the same incubation time of recombinant lysins and its two non-recombinant lysins with the bacteria for 30 min. It is found that the bactericidal activity of these groups increased in correlation with their concentrations. For the time-kill curve experiment, it showed that Recombinant lysin 2 functions more than its non-recombinant lysins 2 with the same concentration of 0.5 µg/mL. Both lysins' ointments have potential activity against S. aureus isolates more than mupirocin and have a similar activity with Fusidic acid through applying 10 µL from lysin 1 ointment, lysin 2 ointment, mupirocin ointment 2%, and Fusidic acid cream 2%. In vitro lytic spectrum analysis revealed that 100% (29/29) tested S. aureus were sensitive. One dose of lysin ointment resulted in a reduction of 3.3 log units in the number of bacteria (from an initial count of 2 × 105 CFU/mg) at 18 hours compared with one dose of mupirocin, PBS, or Aquaphor. Specifically, this study provides evidence that the application of lysin ointment has significant potential as an alternative strategy for MRSA infections.

Prevalence and genetic characteristics of fusidic acid resistant Staphylococcus aureus clinical isolates: Emergence of t030 strains carrying fusB in Tehran, Iran.

The literature on fusidic acid resistant Staphylococcus aureus strains is scarce in Iran, although the emergence of these strains in health care settings is increasing. This descriptive cross-sectional study was conducted on 68 fusidic acid resistant S. aureus strains to learn about the molecular characteristics and antimicrobial resistance of strains isolated from hospitalized patients. In the present study, the prevalence of resistance to fusidic acid in S. aureus isolates was 15.1%. Fusidic acid resistance determinative factors (fusB, fusC and fusD) were identified by multiplex PCR assay. To detect the existence of fusA and fusE determinants and their mutation status, amplifications and sequencing were performed. Molecular characterization of fusidic acid resistant isolates was investigated by SCCmec and spa typing methods. All strains were MRSA and multi drug resistant. Two (2.9%) and 31 (45.6%) isolates were resistant to vancomycin and mupirocin respectively. The SCCmec type IV was highly prevalent representing 50% followed by types III (51.5%), and SCCmec types II (13.2%). fusB, was the most predominant acquired gene (66.2%) followed by fusC (19.1%), and fusA (14.7%). The mutations in fusA were present in 10 isolates with 5 (50%) having L461K mutation showing fusidic acid MIC values of ≥256 µg ml-1 followed by H457Y (40%), and H457Q (10%) showing fusidic acid MIC values of 128 and 64 µg ml-1 respectively. Isolates were allocated to ten particular t030 (22.1%), t037 (14.6%), t408 (11.8%), t064 (11.8%), t008 (10.3%), t002 (8.8%), t005 (5.9%), t790 (5.9%), t318 (4.4%), and t018 (4.4%) spa types. fusA positive isolates were assigned to particular spa types t002 (60%), and t005 (40%). There may be be a spreading of fusidic acid resistance among MRSA, creating worrying public concern. This research notes the importance of adequate data of local prevalence of FA-resistant MRSA in Iran for taking appropriate measures to treat, control and reduce the incidence of these isolates.

Metabolic profiling for the discovery of two rare fusidane-type heterodimers from the fungal endophyte Acremonium pilosum F47.

In our process of studying fusidane-type antibiotics, metabolomics-guided chemical investigation on the endophytic Acremonium pilosum F47 led to the isolation of two unique heterodimers, acremonidiols B and C (1 and 2) consisting of a fusidane-type triterpenoid motif and a steroid unit. Four biosynthetically related known natural products including fusidic acid (FA, 3), as well as ergosterol derivatives (4-6) were also obtained. Their structures were determined by the analyses of ESI-HRMS and NMR data. Compounds 1 and 2, as hybrid molecules comprising the fusidane triterpenoid and steroid, are rare in nature. Compared with the clinically used antibiotic FA (3), new compounds 1 and 2 showed no obvious antibiotic activity, indicating the importance of free C-21 carboxyl group for antibacterial activity.

Synthesis of fusidane triterpenoid Mannich bases as potential antibacterial and antitumor agents.

Mannich bases (8 examples) were synthesized via aminomethylation of fusidane propargyl esters. In vitro antimicrobial screening against key ESKAPE pathogens showed that the fusidic acid based Mannich products exhibit a high antimicrobial effect against Gram-positive bacteria Staphylococcus aureus and the fungus Cryptococcus neoformans. Moreover, the cytotoxic effect of fusidic acid and its analogs, which showed high antibacterial activity, was determined by MTT assay on cancer HepG2, HCT-116, SH-SY5Y, MCF-7, A549 and conditionally normal cells HEK293. A remarkable cytotoxic activity of fusidic acid propargyl ester and its aminomethylene derivatives against cancer and nontumoral HEK293 cells with IC50 values within 4.2-25 µM was found.