E faecalis

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Enterococcus faecalis (E. faecalis) is a Gram-positive, facultatively anaerobic bacterium. It is a commonly used laboratory strain for various microbiological applications.

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E. faecalis ATCC 29212 (15 citations) Enterococcus faecalis (E. faecalis) (2 citations) E. faecalis ATCC 51299 (2 citations)

53 protocols using e faecalis

Bacterial Strain Reference Panel

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Reference strains for the two groups included in this study; vancomycin-resistant Enterococcus (VRE) strains and vancomycin-sensitive Enterococcus (VSE) strains, and the standard strain of Bacteroides fragilis were purchased from Pro-Lab (Neston, South Wirral, Cheshire, UK), NCIMB Limited or the National Collection of Type Cultures (NCTC; Health Protection Agency, Porton Down, UK). These referenced bacterial strains were as follow: Bacteroides fragilis ATCC 25285, VSE strains (E. faecalis NCTC 77, E faecium NCIMB 2699) and VRE strains (E faecium ATCC 19434, E faecalis ATCC 29212, E faecalis ATCC 51299).

, & Ozbak H.A. (2018). A novel high-resolution melting analysis approach for rapid detection of vancomycin-resistant enterococci. Annals of Saudi Medicine, 38(3), 200-207.

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Antibiotic Susceptibility Profiling

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Minimum inhibitory concentrations (MICs) were determined in duplicate by a standard double dilution method according to CSLI guidelines [52 ] using 96-well microtiter cell-culture plates, as previously described [49 (link)]. All reference strain bacteria, including S. aureus (ATCC 12600), E. faecium (ATCC 19434), E. faecalis (ATCC 51299), E. coli (ATCC 35218), K. pneumoniae (ATCC 49472), P. aeruginosa (ATCC 27853), and S. typhimurium (ATCC 14028), as well as S. aureus (ATCC BAA-2312) and antibiotic-resistant K. pneumoniae (ATCC BAA-2814), were obtained from the Microbiology Research Group at the Department of Life Sciences (DLS), Faculty of Science and Technology (FST), University of the West Indies. Control incubations were carried out in parallel with increasing concentrations of antibiotics (ampicillin for S. aureus, E. faecalis, and E. coli; vancomycin for S. aureus (ATCC BAA-2312); and ciprofloxacin for the sensitive K. pneumoniae strain, P. aeruginosa, and S. typhimurium), in order to monitor the validity and reproducibility of the assays. The published antibiotic sensitivity/resistance profiles for all bacterial strains were confirmed in the authors’ laboratory prior to setting up the MIC experiments.

Barran G., Kolodziejek J., Coquet L., Leprince J., Jouenne T., Nowotny N., Conlon J.M, & Mechkarska M. (2020). Peptidomic Analysis of Skin Secretions of the Caribbean Frogs Leptodactylus insularum and Leptodactylus nesiotus (Leptodactylidae) Identifies an Ocellatin with Broad Spectrum Antimicrobial Activity. Antibiotics, 9(10), 718.

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Antibacterial and Antiviral Efficacy Determination

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Antibacterial assays were carried out by Micromyx, LLC in accordance with methods from the Clinical and Laboratory Standards Institute. Minimal inhibitory concentrations were determined with the following strains (listed in Table 1 and Supplementary Table 14): S. aureus ATCC 29213, S. aureus MMX 2011, S. pneumoniae ATCC 49619, S. pyogenes MMX 6253, S. agalactiae MMX 6189, E. faecalis ATCC 29212, E. faecalis MMX 486, B. subtilis ATCC 6633, E. coli ATCC 25922, K. pneumoniae MMX 214, P. aeruginosa ATCC 27853, A. baumannii ATCC 19606, V. cholerae BAA-2163, C. difficile ATCC 700057, and B. fragilis ATCC 25285.
Antiviral assays were performed by Virapur in accordance with methods from the Clinical and Laboratory Standards Institute. Minimal inhibitory concentrations were determined with the following viruses and host cells (listed in Table 1 and Supplementary Table 14): Influenza A/Perth/16/2009 in MDCK cells, Influenza B/Wisconsin/1/2010 in MDCK cells, Herpes Simplex 1 Strain MacIntyre in Vero cells, Herpex Simplex 2 Strain G in Vero cells, Vaccinia virus WR in Vero cells, Rhinovirus 8 in HeLa cells and Respiratory Syncytial Virus in Hep2 cells.
All assays (antibacterial and antiviral) were carried out in triplicates and yielded identical MIC values for all replicates (Table 1). As such a range or an error was not available to report.

Xu F., Wu Y., Zhang C., Davis K.M., Moon K., Bushin L.B, & Seyedsayamdost M.R. (2019). A Genetics-Free Method for High-Throughput Discovery of Cryptic Microbial Metabolites. Nature chemical biology, 15(2), 161-168.

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Antibacterial and Antiviral Efficacy Determination

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Antibiotic Susceptibility Testing Protocols

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Control strains S. aureus ATCC29213, S. aureus ATCC12598, S. aureus ATCC BAA-1556 (USA300), S. aureus ATCC 700699 (Mu50-NRS1), E. faecalis ATCC29212, E. faecalis ATCC51299, A. baumannii ATCC17978; E. coli ATCC25922, P. aeruginosa ATCC27853, K. pneumoniae ATCC700603; and the MDR strain K. pneumoniae ATCC BAA-2814 were obtained from the American Type Culture Collection (Manassas, VA, USA).
S. aureus was grown on Mannitol Salt Agar (CM0085B, Thermo Scientific™ Oxoid™, Basingstoke, UK); E. faecalis and E. faecium on Bile Esculine Agar (CM0888, Thermo Scientific™ Oxoid™, Basingstoke, UK); and A. baumannii, E. coli, P. aeruginosa, and K. pneumoniae on MecConkey agar (CM0007, Thermo Scientific™ Oxoid™, Basingstoke, UK) at 37 °C for 24 h.

Bongiorno D., Musso N., Bonacci P.G., Bivona D.A., Massimino M., Stracquadanio S., Bonaccorso C., Fortuna C.G, & Stefani S. (2021). Heteroaryl-Ethylenes as New Lead Compounds in the Fight against High Priority Bacterial Strains. Antibiotics, 10(9), 1034.

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Bacterial Strains and C. elegans Infection Assay

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The bacterial strains used in this experiment were E. coli OP50, V. cholerae wild type C6706 (O1 El Tor isolated from Peru) [37] (link), JZV133 (a V. cholerae strain expressing green fluorescence protein (GFP)) [38] (link), Staphylococcus aureus (S. aureus, ATCC^#25923), Pseudomonas aeruginosa (P. aeruginosa, ATCC^#27853), Enterococcus faecalis (E. faecalis, ATCC^#47077), Salmonella typhimurium (S. typhimurium, ATCC^#14028), and E. coli O157:H7 (ATCC^#700927). The E. coli, V. cholerae, P. aeruginosa, and S. typhimurium strains were cultured in Luria-Bertani (LB) medium. The S. aureus and E. faecalis strains were cultured in brain-heart infusion (BHI) medium.
All C. elegans strains were maintained at 20°C on nematode growth medium (NGM) seeded with E. coli OP50 feeding strain. 100 µl of OP50 was dropped on the center of 60 mm NGM plates, which were allowed to dry overnight before the culture assays were carried out. Strains used in this study were: N2 Bristol (wild type), sek-1(ag1), pmk-1(km25), daf-2 (e1370), age-1(hx546), daf-16 (mgDf50), hsf-1(sy441), fmo-2(ok2147), dod-22(ok1918), and CL2070(hsp-16.2::GFP). All the strains were obtained from the Caenorhabditis Genetics Center (CGC), University of Minnesota, USA.

Dinh J., Angeloni J.T., Pederson D.B., Wang X., Cao M, & Dong Y. (2014). Cranberry Extract Standardized for Proanthocyanidins Promotes the Immune Response of Caenorhabditis elegans to Vibrio cholerae through the p38 MAPK Pathway and HSF-1. PLoS ONE, 9(7), e103290.

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Antimicrobial Susceptibility Testing Protocol

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The following bacterial strains were used in this study: M. tuberculosis H37Rv, M. abscessus (ATCC 19977), A. baumannii (strain 6M-1b, Clinical Microbiology, Johns Hopkins University), K. pneumoniae (ATCC 35657), E. cloacae (ATCC 13047), P. aeruginosa (PA14), E. faecalis (ATCC 19433) and methicillin-sensitive S. aureus (ATCC 29213). M. tuberculosis and M. abscessus were grown and assessed as previously described ^{7 (link)}. Cation-adjusted Mueller-Hinton broth was used to grow A. baumannii, K. pneumoniae, E. cloacae, P. aeruginosa, E. faecalis and S. aureus at 35 °C according to Clinical and Laboratory Standard Institute guidelines ³⁵. Rifampin, isoniazid and all β-lactams except evolved carbapenems were obtained from commercial vendors. Compounds were 95%–99% pure when random samples were analyzed using LC-MS.

Kumar P., Kaushik A., Lloyd E.P., Li S.G., Mattoo R., Ammerman N.C., Bell D.T., Perryman A.L., Zandi T.A., Ekins S., Ginell S.L., Townsend C.A., Freundlich J.S, & Lamichhane G. (2016). Non-classical transpeptidases yield insight into new antibacterials. Nature chemical biology, 13(1), 54-61.

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Antibacterial Evaluation of Plant Extracts

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Reference strains originated from the American Type Culture Collection (ATCC, Manassas, VA, USA), and the strain isolated from food originated from the Department of Biotechnology, Microbiology and Food Evaluation (SGGW, Poland). The study of aqueous extract (A) and ethanolic extract (E) used 5 strains of Gram-positive bacteria (B. cereus ATCC 11778, E. faecalis ATCC 29212, S. aureus ATCC 25923, S. epidermidis ATCC 12228, L. innocua SGGW) and 5 strains of Gram-negative bacteria (E. coli ATCC 25922, K. pneumoniae ATCC13883, P. mirabilis ATCC 35659, P. aeruginosa ATCC 27853 S. Enteritidis ATCC 13076). The study of other extracts (E1, S, P) used only 1 strain of Gram-positive bacteria (S. aureus ATCC 25932) and 1 strain of Gram-negative bacteria (S. Enteritidis ATCC 13076). The bacterial strains were cultured on Mueller-Hinton Agar (BTL, Poland) and incubated at 37 °C for 24 h. The bacterial inocula were prepared in sterile 0.85% NaCl (w/v) solution to reach a population of approximately 10⁸CFU × mL⁻¹.

Cendrowski A., Kraśniewska K., Przybył J.L., Zielińska A, & Kalisz S. (2020). Antibacterial and Antioxidant Activity of Extracts from Rose Fruits (Rosa rugosa). Molecules, 25(6), 1365.

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Antimicrobial Screening of Natural Extracts

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Antimicrobial screening of fractions obtained at each isolation step was performed by broth dilution protocol based on the NCCLS (National Committee for Clinical Laboratory Standards) procedures for antimicrobial vulnerability testing, 14th informational Suppl, NCCLS; M100-S14: 2002, Wayne, PA (Wayne 2002 ). The antimicrobial activity isolates were checked against 7 bacterial, and 4 fungal strains. Bacterial strains selected for bioactivity testing were S. aureus (ATCC 43300), E. faecalis (ATCC 51299), B. subtilis (ATCC 6633), E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), K. pneumoniae (ATCC 13883), A. baumannii (ATCC 19606) while selected strains from kingdom fungi were C. albicans (ATCC 10231), C. neoformans (ATCC 66031), F. solani (ATCC 0300) and A. niger (ATCC 0198).

Afsar T., Razak S., Almajwal A., Shabbir M., Khan K., Trembley J, & Alruwaili N.W. (2022). Bioassay-guided isolation and characterization of lead antimicrobial compounds from Acacia hydaspica plant extract. AMB Express, 12, 156.

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Antimicrobial Peptide Characterization

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Indolicidin (sequence: ILPWKWPWWPWRR-NH₂, molecular weight: 1,906.3 g/mol) and FMOC-WWRR-NH₂ (molecular weight: 924.1) were obtained from ChemPep (USA) as lyophilized powder and acetate salts with a purity >95%. Ethanol and Congo red were purchased from Sigma-Aldrich (Australia). Sodium chloride was purchased from Alfa Aesar (United Kingdom). The negative staining agent phosphotungstic acid was purchased from Proscitech (Australia). A Milipore filter system was utilized to obtain Milli-Q water. BD Difco Mueller Hinton Broth (MHB) and Sabouraud broth (SB) were purchased from Cell biosciences (Australia). Strains: E. coli NCTC 10418, P. aeruginosa ATCC 9721, E. faecalis ATCC 19433, S. aureus ATCC 25923, S. aureus ATCC BAA-1756 (methicillin resistant), Streptococcus pneumoniae ATCC 49619, Candida albicans ATCC 10231 and Candida auris DSM 21092 (Fluconazole resistant) were provided by the RMIT microbial culture collection. Bacterial strains were stored in 50% glycerol at −20 and −80°C.

Cardoso P., Appiah Danso S., Hung A., Dekiwadia C., Pradhan N., Strachan J., McDonald B., Firipis K., White J.F., Aburto-Medina A., Conn C.E, & Valéry C. (2023). Rational design of potent ultrashort antimicrobial peptides with programmable assembly into nanostructured hydrogels. Frontiers in Chemistry, 10, 1009468.

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