Penicillin g

Virus stocks were prepared from a previously published SARS-CoV-2 isolate (BetaCoV/Germany/BavPat1/2020) [29 (link)], which was kindly provided by Drs. Daniela Niemeyer und Christian Drosten, Charité Berlin, Germany. The isolate, referred to as SARS-CoV-2 München (SARS-CoV-2M) [30 (link)], was handled under the appropriate safety precautions in a BSL-3 facility (Freie Universität Berlin, Institut für Virologie) and propagated on Vero E6 cells (ATCC CRL-1586) in minimal essential medium (MEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (PAN Biotech), 100 IU/mL penicillin G and 100 µg/mL streptomycin (Carl Roth, Karlsruhe, Germany).

All laboratory manipulations with Mycoplasma were carried out under BSL2 conditions (Mwirigi et al., 2016 (link)). Two Mycoplasma mycoides subsp. capri (211/94 and 95010), two Mycoplasma mycoides subsp. Mycoides (Afadé and Gladysdale), and one Mycoplasma capricolum subsp capricolum (6443-90) (Tables 2, 3) were cultured in Pleuropneumonia Like-Organism (PPLO) broth (Difco^TM PPLO Broth) media prepared as follows: 21 g of PPLO was dissolved in 700 ml of distilled water and autoclaved for 15 min at 121°C. The mixture was cooled in a water bath to 55°C and supplemented with phenol red (Carl Roth GmbH) to a final concentration of 3%, 200 ml horse serum (Sigma), 0.25% of glucose (Carl Roth GmbH), 0.15% of penicillin G (Carl Roth GmbH) and 0.25% of thallium acetate (Carl Roth GmbH). A 48 well-plate was used to grow Mycoplasma strains where they were incubated at 37°C for a minimum period of seven days. Growth of Mycoplasma cells was determined by color change from red to yellow (Stemke and Robertson, 1982 (link)), as a result of pH change due to the growth. Stock cultures of Mmc, Mmm, and Mcc were grown to a density of approximately 1–3 10⁶ cells per ml as measured by both the colony forming units (CFU) method (Stemke and Robertson, 1982 (link)) and the use of FCM (Assunção et al., 2006 (link)) after cryopreservation in a freezer at -80°C for further antimycoplasmal activity tests.

At FU Berlin, African green monkey kidney VeroE6 cells (ATCC CRL-1586) were maintained at 37 °C with 5% CO₂ in Minimum Essential Medium (MEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS) (PAN Biotech), 100 IU/mL penicillin G and 100 µg/mL streptomycin (Carl Roth, Karlsruhe, Germany).
At CO-HEP, African green monkey kidney VeroE6 cells (kind gift from Prof. Jean Dubuisson) as well as human hepatoma Huh7.5 cells^{30 (link)} were maintained at 37 °C with 5% CO₂ in Dulbecco’s Modified Medium (DMEM) (Invitrogen, Paisley, UK) containing 10% heat inactivated FBS (Sigma, Saint Louis, Missouri, USA) and 100 U/mL penicillin + 100 µg/mL streptomycin (Gibco/Invitrogen Corporation, Carlsbad, California, USA). A549-hACE2 cells (Invivogen, Toulouse, France) were maintained at 37 °C with 5% CO₂ in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (Gibco, Paisley, UK) containing 10% heat inactivated FBS (Sigma, Saint Louis, Missouri, USA), 100 U/mL penicillin + 100 μg/mL streptomycin (Gibco/Invitrogen Corporation, Carlsbad, California, USA) and 0.5 µg/mL puromycin (Invivogen, Toulouse, France). Cells were sub-cultured every 2–3 days using trypsin (Sigma, Saint Louis, Missouri, USA) to maintain a sub-confluent cell layer.

Vero E6 cells (American Type Culture Collection, CRL-1586) were grown in minimal essential medium (MEM) containing 10% fetal bovine serum (PAN Biotech), penicillin G (100 IU/ml), and streptomycin (100 μg/ml; Carl Roth) at 37°C and 5% CO₂. SARS-CoV-2 variants B.1 (BetaCoV/Munich/BavPat1/2020) (39 (link)), B.1.1.7 (BetaCoV/Germany/ChVir21652/2020), B.1.351 (hCoV-19/Netherlands/NoordHolland_20159/2021), B.1.1.28.1 (hCoV-19/Netherlands/NoordHolland_10915/2021), and B.1.617.2 [SARS-CoV-2, Human, 2021, Germany ex India, 20A/452R (B.1.617)] were grown in Vero E6 cells. Virus stocks were stored at −80°C before experimental infections.

Single native polyps were placed in 48 well multiwell plates in 1 mL ASW supplemented with an antibiotic mixture (Provasoli’s antibiotic mixture with final concentrations of 360,000 U/L penicillin G, 1.5 mg/L chloramphenicol, 1.8 mg/L neomycin, and 9,000 U/L polymyxin B; all components from Carl Roth, Karlsruhe, Germany). No food was provided during the antibiotic treatment. The reduction and consequent change of the microbiota were tested by plating a single homogenized polyp (10 replicates) on Marine Bouillon agar plates (Carl Roth, Karlsruhe, Germany). Plates were incubated for 5 days at 20°C. Colony forming units (cfu) were calculated, and an 87 ± 9% reduction per polyp was determined.

African green monkey kidney VeroE6 cells (ATCC CRL-1586) and Crandell-Rees Feline Kidney (CRFK, ATCC CCL-94) cells were maintained at 37 °C with 5% CO₂ in Minimum Essential Medium (MEM; PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (PAN Biotech), 100 IU/mL penicillin G and 100 µg/mL streptomycin (Carl Roth, Karlsruhe, Germany).