Enterococcus faecium is a commensal but also a bacteremia causing pathogen, which is inherently resistant to several antimicrobials and has a great ability to acquire new traits. Bacterial membrane vesicles (MVs) are increasingly recognized as a mode of cell-free communication and a way to deliver virulence factors and/or antimicrobial resistance determinants. These features make MVs interesting research targets in research on critical hospital pathogens. This study describes for the first time that E. faecium strains produce MVs. It presents a morphological as well as a proteomic analysis of MVs isolated from four different, clinically relevant E. faecium strains grown under two different conditions and identifies MV-associated proteins in all of them. Interestingly, 11 virulence factors are found among the MV-associated proteins, including biofilm-promoting proteins and extracellular matrix-binding proteins, which may aid in enterococcal colonization. Additionally, 11 antimicrobial resistance-related proteins were MV-associated. Among those, all proteins encoded by the vanA-cluster of a vancomycin resistant strain were found to be MV-associated. This implies that E. faecium MVs may be utilized by the bacterium to release proteins promoting virulence, pathogenicity and antimicrobial resistance. Enterococcal infections, especially bacteremia and endocarditis, are challenging to treat because E. faecium have acquired resistance to multiple classes of antimicrobials, including ampicillin, aminoglycosides, and glycopeptides. Thus, research on different modes of enterococcal pathogenicity is warranted. This study utilized a proteomic approach to identify MV-associated proteins of different nosocomial E. faecium strains representing four clinically relevant sequence types (STs), namely ST17, ST18, ST78, and ST192. The presented data suggest that E. faecium MVs are involved in virulence and antimicrobial resistance.

Background. Infections by microbes (viruses, bacteria, and fungi) and parasites can cause serious diseases in both humans and animals. Heavy use of antimicrobials has created selective pressure and caused resistance to currently available antibiotics, hence the need for finding new and better antibiotics. Natural products, especially from plants, are known for their medicinal properties, including antimicrobial and anthelmintic activities. Geoclimatic variation, together with diversity in ethnomedicinal traditions, has made the Himalayas of Nepal an invaluable repository of traditional medicinal plants. We studied antiviral, antibacterial, antifungal, and anthelmintic activities of medicinal plants, selected based upon ethnobotanical evidence. Methods. Ethanolic and methanolic extracts were tested (1) on a panel of microbes: two Gram‐positive bacteria (Staphylococcus aureus and Listeria innocua), four Gram‐negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, and Shigella sonnei), and one fungal species: Candida albicans; (2) against three different viruses: yellow fever, chikungunya, and enterovirus; and (3) on the nematode Caenorhabditis elegans. Also, cytotoxicity was assessed on human hepatoma (Huh), rhabdosarcoma (RD), and Vero (VC) cell lines. Results. Of 18 plants studied, Ampelocissus tomentosa and Aleuritopteris anceps inhibited S. aureus (MIC 35 μg/mL and 649 μg/mL, respectively) and Pseudomonas aeruginosa (MIC 15 μg/mL and 38 μg/mL, respectively). Rhododendron arboreum and Adhatoda vasica inhibited S. enterica (MIC 285 μg/mL and 326 μg/mL, respectively). Kalanchoe pinnata, Ampelocissus tomentosa, and Paris polyphylla were active against chikungunya virus, and Clerodendrum serratum was active against yellow fever virus (EC₅₀ 15.9 μg/mL); Terminalia chebula was active against enterovirus (EC₅₀ 10.6 μg/mL). Ampelocissus tomentosa, Boenninghausenia albiflora, Dichrocephala integrifolia, and Kalanchoe pinnata significantly reduced C. elegans motility, comparable to levamisole. Conclusions. In countries like Nepal, with a high burden of infectious and parasitic diseases, and a current health system unable to combat the burden of diseases, evaluation of local plants as a treatment or potential source of drugs can help expand treatment options. Screening plants against a broad range of pathogens (bacteria, viruses, fungi, and parasites) will support bioprospecting in Nepal, which may eventually lead to new drug development.

At present, few organisms are known to and capable of naturally producing laccases and white rot fungi are one such group. In the present study, three fungal species, namely,Ganoderma lucidum-CDBT1, Ganoderma japonicum,andLentinula edodes, isolated from their native habitat in Nepal were screened for laccase production, andG. lucidum-CDBT1 was found to express highest levels of enzyme (day 10 culture media showed 0.92 IU/mg total protein or 92 IU/mL laccase activity with ABTS as substrate). Lignin extracted from rice straw was used in Olga medium for laccase production and isolation fromG. lucidum-CDBT1. Presence of lignin (5 g/L) and copper sulfate (30 μM) in the media increased the extracellular laccase content by 111% and 114%, respectively. The laccase enzyme produced byG. lucidum-CDBT1 was fractionated by ammonium sulfate and purified by DEAE Sepharose anion exchange chromatography. The purified enzyme was found to have a molecular mass of 43 kDa and exhibits optimal activity at pH 5.0 and 30°C. The isolated laccase was thermally stable for up to 70°C for 1 h and exhibited broad pH stability. The kinetic constants,Km,Vmax, andKcat, determined using 2,2′-azinobis-(-3-ethylbenzothiazoline-6-sulfonic acid) as substrate were found to be 110 μM, 36 μmol/min/mg, and 246 min⁻¹, respectively. The isolated thermostable laccase will be used in future experiments for delignification process.

Nepal is very rich in biodiversity, and no extensive effort has yet been carried out to screen plants that are used by traditional healers against parasitic diseases. The aim of this study was to evaluate the in vitro antileishmanial and antimalarial activity of crude methanolic or ethanolic extracts of 29 plant species that are currently used by local people of Nepal for treating different ailments. Crude extracts of leaves, twigs, aerial parts, and/or roots of the selected plants were evaluated for in vitro inhibitory activity against intracellular amastigotes of Leishmania infantum and against erythrocytic stages of Plasmodium falciparum. To determine the selectivity index (SI), cytotoxicity was assessed on MRC-5 cells in parallel. Three plant species, namely Phragmites vallatoria and Ampelocissus tomentosa, for which no antiprotozoal activity has previously been reported, and Terminalia chebula revealed antiprotozoal activity. The extract of A. tomentosa exhibited moderate activity against L. infantum with an inhibitory concentration 50% (IC₅₀) of 13.2 ± 4.3 µg/ml and SI >3, while T. chebula exhibited fairly good antiplasmodial activity with IC₅₀ values of 4.5 ± 2.4 µg/ml and SI values >5. In countries like Nepal, where the current health system is unable to combat the burden of endemic parasitic diseases, evaluation of local plants as a potential source of the drug can help in expanding the treatment options. The extent of untapped resources available in these countries provides an opportunity for future bioprospecting.

Methicillin-resistant Staphylococcus aureus (MRSA) has evolved numerous antimicrobial resistance mechanisms and is identified as a serious public health threat by the World Health Organization and U.S. Centers for Disease Control and Prevention. The glycopeptide vancomycin (VAN) remains a cornerstone of therapy for severe MRSA infections despite increasing reports of therapeutic failure in hospitalized patients with bacteremia or pneumonia. Recently, the role of released bacterial-derived membrane vesicles (MVs) in antibiotic resistance has garnered attention. Here we examined the effect of exogenous MRSA-derived MVs on VAN activity against MRSA in vitro, using minimum inhibitory concentration and checkerboard assays, and ex vivo, incorporating components of host innate immunity such as neutrophils and serum complement present in blood. Additionally, the proteome of MVs from VAN-exposed MRSA was characterized to determine if protein expression was altered. The presence of MVs increased the VAN MIC against MRSA to values where clinical failure is commonly observed. Furthermore, the presence of MVs increased survival of MRSA pre-treated with sub-MIC concentrations of VAN in whole blood and upon exposure to human neutrophils but not human serum. Unbiased proteomic analysis also showed an elevated expression of MV proteins associated with antibiotic resistance (e.g., marR) or proteins that are functionally linked to cell membrane/wall metabolism. Together, our findings indicate MRSA-derived MVs are capable of lowering susceptibility of the pathogen to VAN, whole-blood- and neutrophil-mediated killing, a new pharmacodynamic consideration for a drug increasingly linked to clinical treatment failures.