Staphylococcus aureus is a leading cause of skin and soft tissue infections (SSTIs), which can escalate into systemic disease. While innate immune responses play a critical role in bacterial clearance, the bacterial components themselves can exacerbate inflammation. Here, we demonstrate that S. aureus lipoproteins (Lpp) and polymeric peptidoglycan (PG) synergistically induce skin abscesses in mice, in a process that requires both the lipid moiety of Lpp and the intact polymeric structure of PG. This synergy is mediated by Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and depends on infiltrating neutrophils and monocytes. Co-administration of Lpl1 and PG results in a 5-fold to 10-fold increase in macrophage inflammatory protein-2 (MIP-2) levels in the skin compared to either ligand alone, indicating a clear synergistic effect. Furthermore, we show that local alteration in coagulation and fibrinolysis contributes to the inflammatory response, as fibrinogen depletion significantly reduced lesion size. To extend these findings to a clinically relevant model, we employed an S. aureus double mutant that lacked both lipidation (Δlgt) and peptidoglycan O-acetyltransferase (ΔoatA). This strain exhibited markedly attenuated virulence in a murine skin infection model. Importantly, this attenuation was fully reversed by neutrophil depletion, indicating that neutrophils are essential mediators of the host responses to these bacterial structures. Our findings reveal a cooperative mechanism through which S. aureus cell wall components drive skin lesion development, and we identify potential therapeutic targets for reducing the severity of SSTIs. IMPORTANCE:Staphylococcus aureus is a bacterium that often causes skin infections, including painful abscesses. We discovered that two components of S. aureus, lipoproteins on its surface and peptidoglycan in its cell wall, collaborate to drive the formation of skin abscesses. This combination triggers potent immune responses by activating the receptor Toll-like receptor 2 (TLR2) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) on host cells. As a consequence, high numbers of neutrophils and monocytes swarm the infection site. The resulting immune overreaction, together with activation of the coagulation system, produces intense inflammation. We confirmed the importance of these bacterial components using mutant S. aureus strains in a skin infection model. These mutants generated much smaller abscesses in our experiments. Our findings highlight a cooperative mechanism that exacerbates staphylococcal infections. Targeting this synergy could be a valuable strategy to reduce disease severity.

Staphylococcus aureus is a major human pathogen whose resilience is largely attributed to a robust and dynamically remodeled cell wall. CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain-containing proteins constitute a large family of peptidoglycan hydrolases implicated in cell wall turnover, but their precise physiological roles remain poorly defined due to functional redundancy and limitations of prior mutagenesis approaches. Here, we performed a systematic functional analysis of CHAP proteins in the epidemic methicillin-resistant S. aureus USA300 JE2 by constructing clean, markerless deletions of 13 CHAP-encoding genes and selected double mutants. Phenotypic profiling revealed that CHAP proteins are not essential for growth but significantly influence β-lactam susceptibility, autolysis, biofilm formation, and virulence. Notably, deletion of sle1 (SAUSA300_0438) and SsaA-family genes (SAUSA300_2249, SAUSA300_2503) led to distinct alterations in penicillin-binding protein (PBP) expression, increased β-lactam sensitivity, and impaired biofilm development. Transmission electron microscopy demonstrated that these mutants exhibited significant changes in cell wall thickness, cell size, and septation dynamics, linking structural perturbations to observed functional defects. In a Galleria mellonella infection model, several CHAP mutants showed attenuated virulence. Collectively, our study establishes CHAP hydrolases as specialized regulators of cell envelope homeostasis and virulence in S. aureus, providing a foundation for their exploitation as targets for novel anti-staphylococcal strategies.

The rapid emergence of antibiotic-resistant bacteria has become a global concern. In particular, the overuse of antibiotics in the breeding industry has accelerated the emergence of antibiotic-resistant bacteria, but the mechanisms driving this phenomenon are not fully understood. Here, using the multidrug-resistant bacterium R. anatipestifer as a proof of concept, we show that the chaperone DnaK promoted the emergence and accumulation of antibiotic-resistant clones. Compared to the ∆dnaK, the wild type produced more resistant clones under antibiotic stress and accelerated the accumulation of resistance mutations under the second antibiotic stress. Importantly, it was indicated that the broad DnaK molecular chaperone inhibitor, telaprevir (TP), effectively decreases the frequency of antibiotics resistant (FOR) in R. anatipestifer through inhibiting the ATPase activation of the DnaK molecular chaperone. Taken together, this study suggested that the chaperone DnaK can be used as a target for drug design to control the emergence and accumulation of antibiotic-resistant clones in R. anatipestifer and reduce the risk of antibiotic resistance in clinical treatment.

Staphylococcus aureus has emerged as an important model organism in bacterial cell biology and pathogenesis due to its clinical relevance, genetic versatility, and adaptability. This review explores how S. aureus has contributed to advances in the fields of bacterial cell wall synthesis and cell division, particularly due to its minimal cell wall synthesis machinery and simple spherical shape. S. aureus has also been fundamental in antimicrobial resistance studies, particularly due to the increasing threat of antibiotic-resistant S. aureus strains. Furthermore, S. aureus' dual lifestyle as both a commensal and a pathogen has provided key insights into host-microbe interactions, biofilm formation, and immune evasion strategies. This review underscores the importance of continued research on S. aureus as a basis for the development of novel antimicrobial strategies and vaccine approaches.

Secondary bile acids (SBAs), which are metabolites produced by gut microbiota, have been implicated in both carcinogenic and anticancer processes. This review explores the dual role of SBAs, focusing on their molecular mechanisms and biological effects. The carcinogenic activities of SBAs include DNA damage, promotion of oxidative stress, and modulation of signaling pathways that drive tumorigenesis. Conversely, some SBAs exhibit anticancer properties by inducing apoptosis, inhibiting cell proliferation, and modulating immune responses. The article also discusses the complex interplay between SBAs and the host’s genetic and environmental factors, highlighting potential therapeutic implications and the need for targeted strategies to mitigate risks while harnessing beneficial effects. A comprehensive understanding of the delicate equilibrium between the deleterious and salutary impacts of SBAs has the potential to facilitate the development of innovative cancer prevention and treatment methodologies.

The human microbiota produces a diverse array of bioactive molecules, including classic neurotransmitters (dopamine and serotonin) and trace amines (tryptamine, tyramine, and phenylethylamine). Although long considered products of host metabolism, these aromatic monoamines are now also known to originate in part from the microbiota, where they are synthesized by bacterial aromatic L-amino acid decarboxylases (AADCs). This review explores the distribution, biochemical diversity, and host interactions of microbiota-encoded AADCs, highlighting their roles in gut and skin ecosystems. Bacterial AADCs vary in gene organization, substrate range, and expression patterns across taxa like Ruminococcus gnavus, Clostridium sporogenes, Enterococcus spp., and Staphylococcus spp. These enzymes contribute to microbial fitness through acid stress resistance, energy generation via proton motive force, epithelial adherence and internalization, and niche dominance. Critically, their products modulate host physiology via trace amine-associated receptors (TAARs) and other signaling pathways, influencing neurotransmission, immune response, barrier integrity, and metabolism. Microbiota-derived monoamines can enter systemic circulation and cross the blood–brain barrier, implicating them in disorders ranging from irritable bowel syndrome to neurodegeneration. Emerging data also reveal their impact on wound healing and drug efficacy, notably in Parkinson’s disease. By positioning microbial AADCs as key players in host-microbe chemical communication, this review underscores their relevance for health and disease and highlights them as potential therapeutic targets.

Antibiotic resistance has become a global public health problem, which is closely related to humans, animals and the environment. Riemerella anatipestifer (R. anatipestifer, RA) is a Gram-negative, multi-resistant bacterium that infects ducks and other birds. However, the mechanisms underlying R. anatipestifer's resistance to antibiotics aren't fully to be understood. Here, we show that the R. anatipestifer dnaK-deficient strain (∆dnaK) is more sensitive than the wild type to various tested antibacterial agents. DnaK is important for alleviating oxidative stress damage, which has been shown to be necessary for efficient scavenging of reactive oxygen species (ROS) induced by bactericidal antibiotics. Furthermore, it is also essential for maintaining normal cell morphology and membrane permeability. Finally, the chaperone DnaK is also critical for the pathogenicity of R. anatipestifer, since it is required for the heat stress resistance inside ducks. Taken together, this study is important to provide strategies against the emergence of antimicrobial resistance and R. anatipestifer infection.

Staphylococcus chromogenes (S. chromogenes) is a predominant non-aureus staphylococcal species colonizing the teat skin and mammary gland of dairy ruminants. Although often linked to mild or subclinical mastitis, specific strains may also play protective roles against major udder pathogens. In this study, we characterized two S. chromogenes isolates (4S77 and 4S90) that displayed antimicrobial activity against Gram-positive bacteria. Complete genome sequencing revealed a conserved, plasmid-encoded biosynthetic gene cluster for the thiopeptide bacteriocin micrococcin P1 (MP1). All genes necessary for MP1 biosynthesis, modification, export, and immunity were identified, and compound production was confirmed by high-performance liquid chromatography and liquid chromatography-mass spectrometry. Comparative analysis with publicly available S. chromogenes genomes revealed that the MP1 cluster appears unique to these isolates. Both strains showed full phenotypic susceptibility to tested antibiotics, despite 4S90 carrying the lnuA gene, which did not confer detectable resistance under standard conditions. Classical staphylococcal toxin genes were also absent. Virulence gene profiling revealed a conserved repertoire of colonization- and persistence-associated genes, including factors involved in adhesion, capsule formation, and iron acquisition, but no markers of aggressive pathogenicity. Mobile genetic elements, including prophages and genomic islands, were common but did not carry antimicrobial resistance or virulence genes, suggesting a low risk of transmission of new pathogenic traits to the endogenous microbiome, including opportunistic bacteria. These findings suggest that MP1-producing S. chromogenes strains combine antimicrobial functionality with low virulence potential, highlighting their potential ecological role as protective commensals on the teat skin and in the broader mammary ecosystem of dairy ruminants. IMPORTANCE:Staphylococcus chromogenes is one of the most prevalent bacteria isolated from the mammary glands of dairy animals and is primarily considered a causative agent of subclinical mastitis. However, certain strains may also act as microbial competitors that inhibit more harmful pathogens. In this study, we identified two goat-derived S. chromogenes strains that produce micrococcin P1 (MP1), a potent antimicrobial compound effective against Gram-positive bacteria, including major mastitis pathogens. Genomic and phenotypic analyses revealed that these strains possess low virulence potential and retain antibiotic susceptibility, suggesting a possible protective role within the mammary microbiome. This is the first report of MP1 production in this species. Our findings highlight the functional diversity within S. chromogenes and suggest its potential application in microbiota-based strategies for mastitis prevention and antimicrobial stewardship in livestock.

Septic arthritis, primarily caused by Staphylococcus aureus, poses a significant risk of both mortality and morbidity due to its aggressive nature. The nuc1-encoded thermonuclease NucA of S. aureus degrades extracellular DNA/RNA, allowing the pathogen to escape neutrophil extracellular traps (NETs) and maintain the infection unabated. Here we show that in the mouse model for hematogenous septic arthritis, the Δnuc1 mutant is much less pathogenic and the severity of clinical septic arthritis is markedly reduced, including decreased weight loss, lower kidney bacterial load, reduced bone erosion, and much less IL-6 production. In vitro, S. aureus genomic DNA induces a robust TNF-α response in macrophage-like RAW 264.7 cells abrogated when the DNA is degraded by NucA. Moreover, the wild type induces high levels of TNF-α, IL-10, and IL-6 in neutrophils and osteoblast-like SAOS-2 cells, respectively. NucA exacerbates septic arthritis by increasing extracellular and intracellular survival of bacteria.

ABSTRACT Manganese (Mn) is an essential element for bacteria, but the overload of manganese is toxic. In a previous study, we showed that the cation diffusion facilitator protein MetA and the resistance-nodulation-division efflux pump MetB are responsible for Mn efflux in the bacterial pathogen Riemerella anatipestifer CH-1. However, whether this bacterium encodes additional manganese efflux proteins is unclear. In this study, we show that R. anatipestifer CH-1 encodes a tellurium resistance C (TerC) family protein with low similarity to other characterized TerC family proteins. Compared to the wild type (WT), the terC mutant of R. anatipestifer CH-1 ( ∆terC ) is sensitive to Mn(II) intoxication. The ability of TerC to export manganese is higher than that of MetB but lower than that of MetA. Consistently, terC deletion ( ∆terC ) led to intracellular accumulation of Mn 2+ under excess manganese conditions. Further study showed that ∆terC was more sensitive than the WT to the oxidant hypoclorite but not to hydrogen peroxide. Mutagenesis studies showed that the mutant at amino acid sites of Glu116 (E116), Asp122 (D122), Glu245 (E245) Asp248 (D248), and Asp254 (D254) may be involved in the ability of TerC to export manganese. The transcription of terC was upregulated under excess manganese and downregulated under iron-limited conditions. However, this was not dependent on the manganese metabolism regulator MetR. In contrast to a strain lacking the manganese efflux pump MetA or MetB, the terC mutant is attenuated in virulence in a duckling model of infection due to increased sensitivity to duck serum. Finally, comparative analysis showed that homologs of TerC are distributed across the bacterial kingdom, suggesting that TerC exerts a conserved manganese efflux function. IMPORTANCE Riemerella anatipestifer is a notorious bacterial pathogen of ducks and other birds. In R. anatipestifer , the genes involved in manganese efflux have not been completely identified, although MetA and MetB have been identified as two manganese exporters. Additionally, the function of TerC family proteins in manganese efflux is controversial. Here, we demonstrated that a TerC family protein helps prevent Mn(II) intoxication in R. anatipestifer and that the ability of TerC to export manganese is intermediate compared to that of MetA and MetB. Sequence analysis and mutagenesis studies showed that the conserved key amino sites of TerC are Glu116, Asp122, Glu245, Asp248, and Asp254. The transcription of terC was regulated by manganese excess and iron limitation. Finally, we show that TerC plays a role in the virulence of R. anatipestifer due to the increased sensitivity to duck serum, rather than the increased sensitivity to manganese. Taken together, these results expand our understanding of manganese efflux and the pathogenic mechanisms of R. anatipestifer .

Inflammation including immunothrombosis by neutrophil extracellular traps (NETs) has important implications in acute ischemic stroke and can affect reperfusion status, susceptibility to stroke associated infections (SAI) as well as functional clinical outcome. NETs were shown to be prevalent in stroke thrombi and NET associated markers were found in stroke patients’ blood. However, little is known whether blood derived NET markers reflect the amount of NETs in thrombi. Conclusions from blood derived markers to thrombus composition might open avenues for novel strategies in diagnostic and therapeutic approaches. We prospectively recruited 166 patients with acute ischemic stroke undergoing mechanical thrombectomy between March 2018 and May 2021. Available thrombi (n = 106) were stained for NET markers DNA-histone-1 complexes and myeloperoxidase (MPO). Cell free DNA (cfDNA), deoxyribonuclease (DNase) activity, MPO-histone complexes and a cytokine-panel were measured before thrombectomy and after seven days. Clinical data, including stroke etiology, reperfusion status, SAI and functional outcome after rehabilitation, were collected of all patients. NET markers were present in all thrombi. At onset the median concentration of cfDNA in blood was 0.19 µg/ml increasing to 0.30 µg/ml at 7 days. Median DNase activity at onset was 4.33 pmol/min/ml increasing to 4.96 pmol/min/ml at 7 days. Within thrombi DNA-histone-1 complexes and MPO correlated with each other (ρ = 0.792; p < 0.001). Moreover, our study provides evidence for an association between the amount of NETs and endogenous DNase activity in blood with amounts of NETs in cerebral thrombi. However, these associations need to be confirmed in larger cohorts, to investigate the potential clinical implications for individualized therapeutic and diagnostic approaches in acute ischemic stroke.

Background: Antibiotic resistance is a global public health concern that has been exacerbated by the overuse and misuse of antibiotics, leading to the emergence of resistant bacteria. The gut microbiota, often influenced by antibiotic usage, plays a crucial role in overall health. Therefore, this study aimed to investigate the prevalence of antibiotic resistant genes in the gut microbiota of Indonesian coastal and highland populations, as well as to identify vancomycin-resistant bacteria and their resistant genes. Methods: Stool samples were collected from 22 individuals residing in Pacet, Mojokerto, and Kenjeran, Surabaya Indonesia in 2022. The read count of antibiotic resistant genes was analyzed in the collected samples, and the bacterium concentration was counted by plating on the antibiotic-containing agar plate. Vancomycin-resistant strains were further isolated, and the presence of vancomycin-resistant genes was detected using a multiplex polymerase chain reaction (PCR).Results: The antibiotic resistant genes for tetracycline, aminoglycosides, macrolides, beta-lactams, and vancomycin were found in high frequency in all stool samples (100%) of the gut microbiota. Meanwhile, those meant for chloramphenicol and sulfonamides were found in 86% and 16% of the samples, respectively. Notably, vancomycin-resistant genes were found in 16 intrinsically resistant Gram-negative bacterial strains. Among the detected vancomycin-resistant genes, vanG was the most prevalent (27.3%), while vanA was the least prevalent (4.5%). Conclusion: The presence of multiple vancomycin resistance genes in intrinsically resistant Gram-negative bacterial strains demonstrated the importance of the gut microbiota as a reservoir and hub for the horizontal transfer of antibiotic resistant genes.

Polycyclic polyprenylated acylphloroglucinols (PPAPs) comprise a large group of compounds of mostly plant origin. The best-known compound is hyperforin from St. John’s wort with its antidepressant, antitumor and antimicrobial properties. The chemical synthesis of PPAP variants allows the generation of compounds with improved activity and compatibility. Here, we studied the antimicrobial activity of two synthetic PPAP-derivatives, the water-insoluble PPAP23 and the water-soluble sodium salt PPAP53. In vitro, both compounds exhibited good activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium. Both compounds had no adverse effects on Galleria mellonella wax moth larvae. However, they were unable to protect the larvae from infection with S. aureus because components of the larval coelom neutralized the antimicrobial activity; a similar effect was also seen with serum albumin. In silico docking studies with PPAP53 revealed that it binds to the F1 pocket of human serum albumin with a binding energy of −7.5 kcal/mol. In an infection model of septic arthritis, PPAP23 decreased the formation of abscesses and S. aureus load in kidneys; in a mouse skin abscess model, topical treatment with PPAP53 reduced S. aureus counts. Both PPAPs were active against anaerobic Gram-positive gut bacteria such as neurotransmitter-producing Clostridium, Enterococcus or Ruminococcus species. Based on these results, we foresee possible applications in the decolonization of pathogens.

Bicarbonate and CO2 are essential substrates for carboxylation reactions in bacterial central metabolism. In Staphylococcus aureus, the bicarbonate transporter, MpsABC (membrane potential-generating system) is the only carbon concentrating system. An mpsABC deletion mutant can hardly grow in ambient air. In this study, we investigated the changes that occur in S. aureus when it suffers from CO2/bicarbonate deficiency. Electron microscopy revealed that ΔmpsABC has a twofold thicker cell wall thickness compared to the parent strain. The mutant was also substantially inert to cell lysis induced by lysostaphin and the non-ionic surfactant Triton X-100. Mass spectrometry analysis of muropeptides revealed the incorporation of alanine into the pentaglycine interpeptide bridge, which explains the mutant’s lysostaphin resistance. Flow cytometry analysis of wall teichoic acid (WTA) glycosylation patterns revealed a significantly lower α-glycosylated and higher ß-glycosylated WTA, explaining the mutant’s increased resistance towards Triton X-100. Comparative transcriptome analysis showed altered gene expression profiles. Autolysin-encoding genes such as sceD, a lytic transglycosylase encoding gene, were upregulated, like in vancomycin-intermediate S. aureus mutants (VISA). Genes related to cell wall-anchored proteins, secreted proteins, transporters, and toxins were downregulated. Overall, we demonstrate that bicarbonate deficiency is a stress response that causes changes in cell wall composition and global gene expression resulting in increased resilience to cell wall lytic enzymes and detergents.

Recent findings indicate that human microbiota can excrete trace amines, dopamine, and serotonin. These neurotransmitters (NTs) can either affect classical neurotransmitter signaling or directly trigger trace amine-associated receptors (TAARs), with still unclear consequences for host physiology. Compared to gut microbiota, less information is available on the role of skin microbiota in NT production. To explore this, 1909 skin isolates, mainly from the genera Staphylococcus, Bacillus, and Corynebacterium, were tested for NT production. Only 6.7% of the isolates were capable of producing NTs, all of which belonged to the Staphylococcus genus. Based on substrate specificity, we identified two distinct profiles among the NT producers. One group primarily produced tryptamine (TRY) and phenylethylamine (PEA), while the other mainly produced tyramine (TYM) and dopamine (Dopa). These differing production profiles could be attributed to the activity of two distinct aromatic amino acid decarboxylase enzymes, SadA and TDC, responsible for generating the TRY/PEA and TYM/Dopa product spectra, respectively. SadA and TDC orthologues differ in structure and size; SadA has approximately 475 amino acids, whereas the TDC type consists of about 620 amino acids. The genomic localization of the respective genes also varies: tdc genes are typically found in small, conserved gene clusters, while sadA genes are not. The heterologous expression of sadA and tdc in Escherichia coli yielded the same product spectrum as the parent strains. The possible effects of skin microbiota-derived NTs on neuroreceptor signaling in the human host remain to be investigated.

Background: After two decades from its introduction in the lateral skull base paraganglioma surgery, the indications and results of preoperative internal carotid artery stenting should be critically assessed. Materials and Methods: Monocentric retrospective study on 26 patients affected by head and neck paragangliomas (19 tympanojugular paragangliomas, 4 carotid body paragangliomas, 3 vagal paragangliomas) preoperatively treated with internal carotid artery stents between 2008 and 2023. The preoperative findings, the intraoperative complications and the final surgical results were analyzed. Results: The stent complication rate was less than 3.1%. Self-expanding highly flexible intracranial nitinol stents were applied. In all cases, it was possible to completely mobilize the internal carotid artery and perform a vascular dissection of the tumor. Gross total tumor resection was possible in 85% of cases. The median follow up was 7.83 y (SD +/− 3.93 y). No local recurrence was observed. Conclusions: The preoperative vascular stent facilitates tumor dissection from the internal carotid artery without risk of vascular damage, helping the surgeon to achieve surgical radicality. The vascular stent is indicated in the case of revision surgeries, circumferential involvement of the vessel and in cases with non-insufficient intracerebral crossflow. Procedural complications, temporary antiplatelet therapy and delay of surgery are the limitations of the procedure.

BackgroundUrinary tract infections (UTIs) represent one of the most prevalent bacterial infections, with Enterococcus species now recognized as the second leading cause of these infections. This study focused on symptomatic UTI cases to investigate the risk factors associated with Enterococcus faecalis clinical isolates in patients from Tehran, Iran.MethodsUrine samples were collected from patients presenting with symptomatic UTIs. The identification of E. faecalis isolates was performed using standard microbiological techniques, with confirmation via polymerase chain reaction (PCR). Antibiotic susceptibility testing was conducted using the Kirby–Bauer disc diffusion method. The presence of virulence genes was determined through PCR, and biofilm formation was assessed using the microtiter plate method. Additionally, multi-locus sequence typing (MLST) was utilized to genotype linezolid-resistant isolates.ResultsOut of 300 UTI cases, E. faecalis was identified as the causative agent in 160 instances. Notably, a high proportion of these isolates exhibited resistance to tetracycline (83.8%) and minocycline (82.5%). Linezolid resistance was observed in 1.3% (n = 2) of the isolates. Conversely, the highest susceptibility rates were observed for vancomycin, penicillin G, ampicillin, and nitrofurantoin, each demonstrating a 98.8% susceptibility rate. Biofilm formation was detected in 25% of the E. faecalis isolates. A significant majority (93.8%) of the isolates harbored the efbA and ace genes, with varying frequencies of esp (72.5%), asa1 (61.2%), cylA (52.5%), and gelE (88.8%) genes. MLST analysis demonstrated that both linezolid-resistant isolates, characterized by strong biofilm formation and the presence of virulence genes, were assigned to the ST150 lineage, which has not been previously documented in clinical settings.ConclusionThe emergence of the ST150 clonal lineage, underscores its clinical significance, particularly in relation to linezolid resistance in E. faecalis. This study adds to the growing body of evidence linking specific clonal lineages with antibiotic resistance, highlighting the critical need for ongoing surveillance and molecular characterization of resistant pathogens.

A new route to the macrolactone antibiotic berkeleylactone A was developed. As a key step, a ring-closing alkyne metathesis (RCAM) of an ester substrate featuring 1-propynyl termini was used. The carboxylic part of the substrate was easily assembled using alkyne chemistry, like carboxylation of a diyne followed by isomerization of the ynoate section to a dienoate and dihydroxylation of the 4,5-double bond. The synthesis of the alcohol part of the ester started with opening of (R)-propylene oxide with an acetylide and was followed by two triple bond migrations. After successful RCAM which formed the C8-C9 bond, the triple bond was selectively hydrogenated to the corresponding alkene before the 4,5-diol was oxidized to the 5-hydroxy-4-oxo derivative. At this stage, the thioether was formed and the 8,9-double bond reduced. We also prepared the 8,9-didehydro analog of berkeleylactone A. However, it turned out that its antimicrobial activity was slightly reduced.

This study aims to compare antibacterial effects of green‐synthesized silver nanoparticles (AgNPs) with silver nitrate (AgNO 3 ). AgNPs were successfully synthesized using Eucalyptus camaldulensis leaf extract as a reducing and stabilizing agent. Minimum inhibitory concentrations (MIC) of AgNPs and AgNO 3 against Staphylococcus aureus and Pseudomonas aeruginosa ranged between 4.8 and 6.75 µg mL −1 . Growth curves demonstrated that inhibition of P. aeruginosa occurred right after AgNPs were added and throughout the period of the study (72 h). Antibacterial effects of both AgNPs and AgNO 3 could be abrogated by cysteine and 2‐mercaptoethanol, thiol‐containing compounds. Galleria mellonella model revealed relatively low toxic effects of both AgNPs and AgNO 3 . At 20MIC of AgNPs (≈137.8 mg kg −1 ), more than 80% survival of G. mellonella was observed. Unexpectedly, silver‐containing agents could not rescue larvae after S. aureus infection. Further ex vivo experiments in the presence of coelomic larval fluid demonstrated the reduction of antibacterial activity of both AgNPs and AgNO 3 . It was speculated that anionic molecules present in the coelomic fluid might neutralize the action of Ag ions. Binding of AgNPs or AgNO 3 to albumin, a major protein in human blood which transport several endogenous compounds was not detected, indicating that the silver‐containing agents could be applied as an antimicrobial agent.