Escherichia coli atcc 25922

The bacterial strains used in the test were Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 700603 (Gram-positive), and Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Klebsiella pneumoniae ATCC 27853 (Gram-negative) were purchased from Thermoscientific.

Reference strains used for the tests were Escherichia coli ATCC 25,922 (Thermo Scientific) and Phocaeicola vulgatus ATCC 8482 (ATCC). Before assembling the co-culture system, bacteria were kept at -80ºC in Brain Heart Infusion Broth with glycerol (30%). At first, bacteria were inoculated on agar. Specifically, Trypton Soy Agar (TSA) (Sigma-Aldrich) was used for Escherichia coli, while Trypton Soy Agar supplemented with Defibrinated Sheep blood (5%) (Liofilchem) was used for Phocaeicola vulgatus. Both strains were cultivated for 24 h at 37ºC; Escherichia coli were cultured under aerobic conditions, while anaerobic conditions were used for Phocaeicola vulgatus. Bacterial suspensions were prepared using phosphate-buffered saline solution (Invitrogen).

Phenolic and carotenoid components’ standards were supplied by Sigma-Aldrich (Darmstadt, Germany). Plant Flavonoids Colorimetric Assay Kit was obtained from Elabscience Biotechnology Inc. (Houston, TX, USA). Prior to HPLC screening, each of the analyzed samples underwent filtration using a 0.45 µm MF-Millipore™ Membrane Filter acquired from Merck (Darmstadt, Germany). The bacterial reference strains: Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 700699, Bacillus cereus ATCC 14579, Enterococcus faecalis ATCC 29219, Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were purchased from Oxoid Ltd. (Hampshire, UK). Culture mediums for bacteria were purchased from Merck (Darmstadt, Germany). Sigma-Aldrich (St. Louis, MO, USA) provided the enzymatic mixtures for cell cultures, while Gibco Life Technologies (Paisley, UK) provided the culture medium constituents. Thermo Fisher Scientific Inc. (Waltham, MA, USA) provided the Annexin V-FITC with propidium iodide (PI) flow-cytometry Kit.

Susceptibility to antibiotics was carried out using the disk-diffusion method on Muller Hinton Agar at 37 °C for 18 h. Results were interpreted according to CLSI breakpoint criteria (2014). Three reference isolates (Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853) were used for quality control of the purchased disks (Oxoid, Basingstoke, Hampshire, UK). S. aureus isolates were tested on a total of 10 different antibiotic disks: oxacillin (OX5), cefoxitin (FOX30), penicillin G (P10), spiramycin (SP100), clindamycin (DA2), lincomycin (L15), erythromycin (E15 (RD5), vancomycin (VA30), amikacin (AK30), and ofloxacin (OFX5), whereas Enterobacteriaceae members were tested for amoxicillin (AMX25), amoxicillin/clavulanate (AMC10), cefotaxime (CTX30), ceftriaxone (CRO 30), ampicillin (AMP10), cefazolin (CF30), imipenem (IMP10), ofloxacin (OFX5), amikacin (AK10), and trimethoprim/sulfamethoxazole (SXT1.25/23.75). Non-fermentative bacilli (P. aeruginosa and A. baumannii) were tested for the following antibiotics: piperacillin (PIP100), ticarcillin (TIC75), ticarcillin–clavulanate (TCC75), aztreonam (ATM30), ceftazidime (CAZ30), piperacillin (PRL100), levofloxacin (LEV5), amikacin (AK30), colistin (CT10), and trimethoprim/sulfamethoxazole (SXT1.25/23.75).

The standard strains of three Gram-positive bacteria Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 29213, and S. epidermidis ATCC 12228, three Gram-negative bacteria Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 700603, and Pseudomonas aeruginosa ATCC 27853, and one yeast Candida albicans ATCC 10231 were obtained from Oxoid (Basingstoke, United Kingdom). Cation adjusted Mueller-Hinton broth (MHB) and Sabouraud dextrose broth (SDB) were used as cultivation media for antibacterial and antifungal microdilution assay, respectively, and were equilibrated with Tris-buffered saline (Sigma-Aldrich, Prague, Czech Republic). Mueller-Hinton agar (MHA) and Sabouraud dextrose agar (SDA) were used for subsequent determination of bactericidal and fungicidal concentrations, respectively. All media were purchased from Oxoid (Basingstoke, United Kingdom).

The S. aureus isolates were subjected to the Kirby-Bauer disc diffusion susceptibility tests following the clinical and laboratory standard institute (CLSI) guidelines [55 ]. Briefly, 24 antibiotics (Oxoid, Thermo Fischer Scientific, Canada) relevant to human and veterinary health from the classes of ß-lactams, aminoglycosides, cephalosporins, quinolones, macrolides, lincosamide, tetracycline, chloramphenicol, and sulphonamide were included in the study. The list of antibiotics and their corresponding antibiotic concentration breakpoints suggested by CLSI are provided in Table S2 [55 ]. Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Pseudomonas aeruginosa ATCC 27853 (Oxoid company, Canada) were used as quality control (QC) strains.

Various intestinal pathogens were used to evaluate the antagonistic effect of lactobacilli isolated from llama milk. The human intestinal pathogens Salmonella enterica ATCC 13076 and Escherichia coli ATCC 25922 were obtained from Oxoid (Argentina). The porcine pathogens enterotoxigenic E. coli TUCO-I5, enterohemorragic E. coli TUCO-I6, and Salmonella Typhimurium TUCO-I7 were obtained from the pathogen culture collection of the Laboratory of Food Microbiology (Faculty of Veterinary Sciences, University of Concepción). Escherichia coli and Salmonella strains were grown in BHI broth under the standard conditions (Quilodrán-Vega et al., 2016 (link)).
The study of inhibition of bacterial pathogens was performed as described previously (Quilodrán-Vega et al., 2016 (link)). Briefly, a 10 μl loop of a 2-day culture on MRS broth of the LAB strains was streaked on a line on MRS agar and incubated at 37°C at 5% CO₂ for 24 h. After incubation, a 10 μl loop of overnight cultures from the pathogen strains was streaked perpendicular to the lactobacilli strains lines. The plates were incubated at 37°C for 48 h under aerobic conditions. The inhibition zones were measured in cm.