Kanamycin km

Bacterial strains used in this study are listed and described in S1 Table. For all experiments UPEC was grown overnight in Luria-Bertani (LB) medium at 37°C, shaking, in the presence of kanamycin (Km, 50 μg/ml, Sigma-Aldrich, Sweden) or ampicillin (Amp, 100 ug/ml, Sigma-Aldrich, Sweden) when indicated. On the day of the experiment a fresh culture was cultivated to a density of OD₆₀₀ = 0.5–0.6 (≈ 1–2 x 10⁸ CFU/mL). Bacteria were used directly for infection in the biomimetic model, but otherwise washed twice in PBS before use. ARD371 (LT002 pBAD-HlyA) and ARD372 (LT005 pBAD-HlyA) were constructed by cloning hlyA of CFT073 into the pBAD vector under the control of an arabinose-inducible promoter. Briefly, the open reading frame of hlyA was PCR amplified from the CFT073 gDNA using primers Sacl_hlyA_FW and BstBl_hlyA_RV (S1 Table) and subjected to double digestion (SacI-HF and BstBI enzymes, NEB) and purification (GE healthcare DNA purification kit), and was ligated to the pBAD plasmid, resulting in pBAD-HlyA. The pBAD-HlyA plasmid was electroporated into LT002 (CFT073 ΔhlyA) and LT005 (LT002 gfp+). Induction of hlyA was achieved by adding 0.2% arabinose to the media. All constructs were confirmed by genomic sequencing and the expression of hemolysin confirmed using a hemolytic assay (S2A Fig).

All bacterial strains used in this study are listed in Supplementary file 1. Escherichia coli strains were grown in Luria–Bertani broth (LB; Difco, France) or Luria–Bertani agar (LA; Difco) medium at 37°C with aeration. When needed, the antibiotics ampicillin (Amp; Sigma-Aldrich, Germany) and erythromycin (Ery; Sigma-Aldrich) were added at a final concentration of 100 µg/ml, and kanamycin (Km; Sigma-Aldrich) was added at a final concentration of 30 µg/ml. Lactococcus lactis LL108 strain was grown in M17 broth (Difco), supplemented with sucrose (0.5 M) and glucose (0.5% wt/vol) at 30°C without aeration. Erythromycin was used when required at 100 µg/ml. S. aureus strains were grown at 30°C with aeration in tryptic soy broth (TSB; Difco) or on tryptic soy agar (TSA; Difco). Medium was supplemented when required with Ery at 10 µg/ml and/or 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal; Apollo Scientific, UK) at 100 µg/ml. S. pneumoniae was grown in C + Y medium at 37°C, without aeration, or on TSA plates supplemented with 5% vol/vol sheep blood (Probiológica, Portugal). Tetracycline (Sigma-Aldrich) and Ery were used, when required, at 1 µg/ml and 0.25 µg/ml, respectively.

3-Oxo-hexanoyl-homoserine lactone (3OC6-HSL) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions (1–10 mM) were prepared by dissolving appropriate amounts of 3OC6-HSL in ethyl acetate (Nacalai Tesque, Kyoto, JP) acidified with glacial acetic acid (0.01% (v/v); Nacalai Tesque, Kyoto, JP) and stored at -20°C. 6-(β-D-2-Deoxyribofuranosyl)-3,4-dihydro-8H-pyrimido[4 (link),5 (link)-c][1 (link),2 (link)]oxazin-7-one (dP) was purchased from Berry and Associates (Dexter, MI, USA). Stock solutions (1–1,000 μM) were prepared by dissolving appropriate amounts of dP in DMSO as 1,000 × stock (stored at 4°C). The oligonucleotides used in this work were all synthesized by FASMAC Co., Ltd (Kanagawa, JP). All other chemicals and media were of the highest available grade. Antibiotics were added to the growth medium as required at the following concentrations: 50 μg/mL carbenicillin (Carb; Sigma-Aldrich, St. Louis, MO, USA), 30 μg/mL chloramphenicol (Cm; Nacalai Tesque, Kyoto, JP), and 50 μg/mL kanamycin (Km; Sigma-Aldrich, St. Louis, MO, USA).

L. biflexa sevorar Patoc strain Patoc (Paris) and L. interrogans serovar Manilae strain L495 were used in this study as described earlier [17] (link). Bacteria were grown in Ellinghausen-McCullough-Johnson-Harris (EMJH) medium (Bio-Rad) at 28°C without agitation. EMJH agar plates were obtained by solidification of EMJH medium with 1.2% noble agar (Difco). Leptospires were counted using a Petroff-Hauser chamber. Antibiotics were used in vitro at the following concentrations: 100 µg/mL ampicillin (Amp, MP Biomedical); 25 µg/ml kanamycin (Km, Sigma-Aldrich); 50 µg/ml spectinomycin (Spc, Sigma-Aldrich). Thymidine (dT, Sigma-Aldrich) and diaminopimelate (DAP, Sigma-Aldrich) were added when necessary at the final concentration of 0.3 mM.

Streptococcusagalactiae GD201008-001 was isolated in 2010 from tilapia with meningoencephalitis in Guangdong Province, China [21 (link)]. GD201008-001 wild-type strain (WT) and its derived luxS mutant strain (ΔluxS) and the luxS complemented strain (CΔluxS) [13 (link)] were maintained in Todd-Hewitt broth (THB) or in chemically defined medium (CDM) [22 (link)]. Escherichia coli was cultured in Luria–Bertani (LB) medium. For plasmids screening required, media were supplemented with antibiotics using the concentration below: 100 μg/mL spectinomycin (Sp, Sigma, St. Louis, MO, USA), 10 μg/mL erythromycin (Em, Sigma), 100 μg/mL kanamycin (Km, Sigma) or 100 μg/mL ampicillin (Ap, Sigma). The details of bacterial strains and plasmids are listed in Additional file 1. Macrophage cell line RAW 264.7 were maintained in DMEM (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco).

A genetically engineered E. coli MG1655 substrain was initially cultured overnight from single colony at 37 °C and 200 rpm in tryptone broth (TB) medium [10 g tryptone (Sigma-Aldrich^®, St. Louis, MO) and 5 g NaCl (Sigma-Aldrich, St. Louis, MO) in 1 l ddH₂O, pH 7] containing 50 μg/ml kanamycin (KM) (Sigma-Aldrich, St. Louis, MO) and 100 μg/ml ampicillin (AMP) (Sigma-Aldrich, St. Louis, MO). Then, 100 μl of the overnight bacterial culture was transferred into fresh 10 ml TB medium which consisted of 1 μM biotin (Sigma-Aldrich, St. Louis, MO), 50 μg/ml KM, and 100 μg/ml AMP and further cultured at 34 °C and 270 rpm for 2h. Subsequently, 100 μM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma-Aldrich, St. Louis, MO) was added into the medium and the bacteria were further cultured at 34 °C and 270 rpm until their optical density at 600 nm (OD₆₀₀) reached 0.6, which was measured with a Synergy HTX multi-mode plate reader (BioTek Instruments, Winooski, VT).