Staphylococcus aureus

Five bacterial strains, Gram-positive and Gram-negative bacteria, were evaluated for their susceptibility to the test compounds. The selected strains, Streptococcus mutans ATCC 35668, Streptococcus pyogenes ATCC 19615, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa (ATCC 27853, Microbiologics, France) were reference strains specific to human bacteria infections. The strains were reconstituted by inoculation on Columbia agar +5% sheep blood (bioMérieux, France), then the plates were incubated for 24 h at 37 °C.

The microbial strains used to determine the TPEO antibacterial and antifungal activity were reference strains: Escherichia coli ATCC 25922, Shigella flexneri ATCC 12022, Streptococcus pyogenes ATCC 19615, Salmonella typhimurium ATCC 14028, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Candida albicans ATCC 10231 and Candida parapsilosis ATCC 22019 (Microbiologics, Molsheim, France). According to the EFSA One Health 2020 Zoonoses Report [36 (link)], these strains generated the majority of foodborne outbreaks across the EU member states.

Gram-negative strains Escherichia coli (ATCC 25922), Enterobacter aerogenes (ATCC 13048) and Pseudomonas aeruginosa (ATCC 27853), Gram-positive strains Staphylococcus aureus (ATCC 25923) and Enterococcus faecalis (ATCC 29212), and fungal strain Candida albicans (ATCC 90028) were obtained from Microbiologics Inc., and used for the antimicrobial screening. Strains were selected according to the guidelines set for clinical laboratories by Clinical and Laboratory Standards Institute [CLSI, formerly National Committee on Clinical Laboratory Standards [22] ] and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Bacterial strains were grown on Mueller Hinton II Agar (MHA) (BBL, BD) and Mueller Hinton II broth (MHB) (BBL, BD), whereas Candida was initiated on Sabouraud Dextrose Agar (SDA) (Difco, BD) plates. Media were prepared into MilliQ water according to manufacturer’s instruction and autoclaved at 121°C for 15 min. Bacteria were plated on MHA plates and incubated at 37°C for 16–18 h. Bacterial suspension for the assays was prepared by subculturing the bacteria into MHB and by incubating at 37°C for 16–20 h at 110 rpm prior to the assay. Candida strain was grown on SDA plates at 27°C for 16–18 h and suspended into sterile 0.9% saline for the assay.