Salmonella enterica subsp enterica serovar Typhimurium (ATCC 35986) was grown under aerobic conditions at 37°C for 1–2 d on LB agar plates (Sigma-Aldrich). Growth of anaerobic bacteria was performed using a BD GasPak EZ Anaerobe Pouch System (BD Diagnostics). Prevotella stercorea (DSM No. 18206) was grown for 5–7 d under anaerobic conditions in liquid-chopped meat broth (Hardy Diagnostics) supplemented with 1% Trace Minerals (ATCC), 1% Vitamin Supplements (ATCC), 0.05% Tween 80, 29.7mM acetic acid, 8.1 mM propionic acid and 4.4 mM butyric acid (Sigma-Aldrich). Ruminococcus bromii (ATCC# 27255) was grown under anaerobic conditions at 37°C for 1–2 d in liquid-chopped meat broth (Hardy Diagnostics). The long-term stock of Bifidobacterium longum subsp infantis (ATCC 15697) was grown under anaerobic conditions at 37°C for 2–3 d in liquid-chopped meat broth (Hardy Diagnostics) and the working stock was grown on Brucella plates (Teknova) under anaerobic conditions at 37°C for 2–3 d. Acinetobacter junii (ATCC 17908) was grown under aerobic conditions at 26°C for 1–2 d using Nutrient Agar plates (Edge Biologicals). Single-use working stocks of all bacteria were prepared using DPBS and long-term stocks were prepared using 10% glycerol. All bacterial stocks were enumerated using the BD Cell Viability Kit (BD Bioscience) and stored at −80°C until use.
Lb agar plate
Manufactured by Merck Group
Sourced in United States
LB agar plates are a type of laboratory equipment used for the cultivation and growth of bacterial cultures. They consist of a petri dish filled with a solid culture medium made from Lysogeny Broth (LB) and agar. The LB agar provides essential nutrients and a stable growth environment for bacteria to proliferate and form visible colonies.
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Lab products found in correlation
Lb broth, by Merck Group (4 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (4 mentions)
Cell viability kit, by BD (2 mentions)
Gaspak ez anaerobe pouch system, by BD (2 mentions)
Liquid chopped meat broth, by Hardy Diagnostics (2 mentions)
Gentamicin, by Merck Group (2 mentions)
Lb medium, by Merck Group (2 mentions)
Butyric acid, by Merck Group (1 mentions)
Ruminococcus bromii, by American Type Culture Collection (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
Kanamycin, by Transgene (1 mentions)
Pet28b, by Thermo Fisher Scientific (1 mentions)
E coli strain bl21, by Merck Group (1 mentions)
Pet28b, by Merck Group (1 mentions)
Sheep blood agar plates, by Thermo Fisher Scientific (1 mentions)
Tetracycline, by Merck Group (1 mentions)
Tryptic soy agar tsa, by Thermo Fisher Scientific (1 mentions)
Tryptic soy broth (tsb), by Thermo Fisher Scientific (1 mentions)
Erythromycin, by Merck Group (1 mentions)
Type 45 ti rotor, by Beckman Coulter (1 mentions)
30 protocols using lb agar plate
1
Cultivation of Bacterial Strains
2
Quantifying Intracellular Salmonella Growth
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To measure Salmonella proliferation, cells were washed three times with PBS (to eliminate gentamycin) and lysed with 0.5% Sodium Deoxycholate (Sigma-Aldrich) at indicated time points. The lysate containing recovered intracellular bacteria was plated in appropriate dilutions on LB-Agar plates (Sigma-Aldrich) and incubated for another 12 h at 37 °C. CFUs were counted manually the day after.
3
Evaluating Antimicrobial Resistance Genes
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To evaluate the impact of resistance-determinant genes on MICs, blaOXA–1045 and blaOXA–213 were cloned into the vector pET28b (MiaoLingBio, Wuhan, China). To make pET28b-OXA1045 and pET28b-OXA213, PCR amplification and vector pET-28b were digested with BamHI and XhoI and then ligated to the pET-28b vector (Invitrogen, Carlsbad, California, United States). As previously described, the generated plasmid was chemically converted into E. coli strain BL21 (Sigma-Aldrich, St. Louis, MO, United States) (Liu et al., 2021 (link)). Potential transformants containing pET28b-OXA1045 were identified on LB agar plates (Sigma-Aldrich, St. Louis, MO, United States) containing 20 mg/L of kanamycin (TransGen, Beijing, China). PCR primers PET28AVF2/PET-VF were used to screen colonies on plates, followed by Sanger sequencing (Supplementary Datasheet 1 ). The empty vector pET-28b was turned into BL21 for use as a control.
MICs of ampicillin, ampicillin-sulbactam, piperacillin, piperacillin-tazobactam, oxacillin, cefazolin, cefoxitin, cefuroxime, ceftazidime, cefotaxime, imipenem, and meropenem for the transformants containing pET28b-OXA1045 (BL21:pET28b-OXA1045) and pET28b-OXA213 (BL21:pET28b-OXA213) were determined by the broth microdilution method. MICs for ampicillin in the presence of 4 mg/L of sulbactam were also determined based on the methods used to establish MICs for piperacillin-tazobactam.
MICs of ampicillin, ampicillin-sulbactam, piperacillin, piperacillin-tazobactam, oxacillin, cefazolin, cefoxitin, cefuroxime, ceftazidime, cefotaxime, imipenem, and meropenem for the transformants containing pET28b-OXA1045 (BL21:pET28b-OXA1045) and pET28b-OXA213 (BL21:pET28b-OXA213) were determined by the broth microdilution method. MICs for ampicillin in the presence of 4 mg/L of sulbactam were also determined based on the methods used to establish MICs for piperacillin-tazobactam.
4
Cultivation of Livestock-Associated MRSA ST398
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The whole genome sequenced wild type (WT) livestock-associated methicillin-resistant S. aureus ST398 (Genbank accession AM990992) [21] (link) and S. aureus RN4220 were grown in Brain Heart Infusion (BHI) (Oxoid, Difco) broth at 37°C with aeration. S. aureus SH1000 pMARGH2b, S. aureus SH1000 pFA545 and S. aureus RN4220 pFA545gen were grown in BHI or Tryptic Soy Broth (TSB) (Oxoid) with 5 mg/l erythromycin (Sigma), 5 mg/l tetracycline (Sigma) and 16 mg/l gentamicin (Sigma) respectively, at 30°C with aeration. For solid growth BHI agar, sheep blood agar plates (Oxoid) or Tryptic Soy Agar (TSA) (Oxoid) were applied and supplemented with the appropriate antibiotic if needed. Escherichia coli DH10 was cultured in Luria Broth (LB) at 37°C with aeration or on LB agar plates (Sigma).
5
Isolation of Outer Membrane Vesicles from S. proteamaculans
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S. proteamaculans 94 colonies were grown in LB medium (Sigma Aldrich, Darmstadt, Germany) at 30 °C in aerated flasks for 41 h. Outer membrane vesicle (OMV) formation was induced by adding hydrogen peroxide to the medium until a final concentration of 250 μM (Biolot, St. Petersburg, Russia) for 1 h; after this, a OMV isolation was performed. The bacteria were centrifuged at 3160× g for 25 min, the supernatant was additionally clarified with two repeated centrifugation cycles and the resulting supernatant was filtered through a membrane with a pore diameter of 0.22 μm (Millipore, Watford, UK). The supernatant was subjected to ultracentrifugation at 40,000× g for 1 h at 4 °C using a Type 45 Ti rotor (Beckman Instruments, Indianapolis, IN, USA). Sediments containing OMVs were suspended in PBS (Biolot, St. Petersburg, Russia). The OMV suspension was tested for sterility by plating on LB agar plates (Sigma Aldrich, Darmstadt, Germany).
6
Ionizing Radiation and Nitric Oxide Effects
7
Phagocytosis in Salmonella Infection
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We collected peritoneal lavage fluids at indicated time-points after S. typhimurium infection. Total and differential cell counts were determined with the XE 2100TM cell counter (Sysmex, Vienna, Austria). Additionally, differential cell counts were carried out in smears stained with May-Gruenwald-Giemsa and using FACS analysis as described below. To determine in vivo phagocytosis in lavage fluids, leukocytes were collected by centrifugation, lyzed in 0.5% sodium deoxycholic acid (Sigma-Aldrich, St. Louis, MO) as previously described, and plated on LB agar plates (Sigma-Aldrich, St. Louis, MO) under sterile conditions in serial dilutions for 24 h (Nairz et al, 2011 (link)).
8
Engineered E. coli Cell Growth
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Plasmids containing engineered genetic circuits were transformed into Nissle 1917 E. coli (Mutaflor®). Nissle cells were cultured in LB broth (Sigma) and grown on LB agar plates (Sigma) containing appropriate antibiotics. Singular colonies were picked into LB broth and grown overnight in a shaking incubator (30 °C, 250 rpm). The next day, optical density measurements (OD600) were taken, and the saturated cultures were diluted to 0.1 OD600. Diluted cultures were then allowed to grow to exponential phase until they reached 0.6 OD600 before starting assays. Optical density measurements were taken using a Nanodrop 2000c (Thermo Scientific) in cuvette mode.
9
Bacterial Growth Curve and Viability
10
Cultivation of Anaerobic and Aerobic Bacteria
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