Staphylococcus aureus (ATCC25923) was grown on blood agar plates (BioMerieux SA, Fr). Legionella pneumophila (ATCC33152) was grown on buffered charcoal yeast extract (BCYE) agar (Oxoid). Mycobacterium avium (BAA-535) was grown on 7H10 agar (Becton Dickinson).
Bcye agar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, France
BCYE (Buffered Charcoal Yeast Extract) agar is a microbiological growth medium used for the cultivation and isolation of Legionella species, the causative agent of Legionnaires' disease. It provides the essential nutrients and growth factors required by Legionella bacteria. The agar base, charcoal, and supplementary ingredients in the formulation support the specific growth requirements of Legionella.
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9 protocols using bcye agar
1
Bacterial Growth Conditions Protocol
2
Isolation and Identification of Legionella
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Two respiratory specimens were collected from each patient: one sample inoculated onto the culture media and the other stored at −70°C until molecular assay. Sputum specimens were decontaminated by heat treatment (56°C for 30 minutes), and BAL samples were concentrated by centrifugation. The prepared specimens were cultured onto an unselective medium – buffered charcoal–yeast extract (BCYE) agar (Oxoid, Basingstoke, UK) – and a selective medium, modified Wadowsky–Yee agar (Oxoid) supplemented with l -cysteine. The plates were incubated in candle jars (3%–5% CO2) at 35°C in a humidified atmosphere and inspected for 4–14 days for the presence of Legionella spp. colonies. Colonies showing characteristics of Legionella spp., such as grayish-white, shiny colonies were selected. Gram staining was done to show the thin, faintly stained filamentous Gram-negative morphology. Suspected colonies were subcultured on BCYE agar with and without l -cysteine and unselective media, such as sheep-blood agar and MacConkey agar, for verification. Isolates that grew on BCYE agar with l -cysteine, but not on the other media, were considered Legionella. Strains unable to grow on media without l -cysteine were further identified by PCR and LAMP for the mip gene.14 (link)
3
Cultivation and Standardization of Bacterial Strains
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An Escherichia coli (Ec 039) strain and a Legionella pneumophila serogroup 1 (Lp1 008-GFP) GFP-modified strain were used [41 (link),42 (link)]. They were stored at -80°C in Cryobank tubes (Mast Diagnostic, France). After thawing, Lp1 008-GFP and Ec 039 were plated, respectively, onto BCYE agar (Buffered Charcoal Yeast Extract, SR0110 C, Oxoid, France) and Luria Broth (LB) agar supplemented with chloramphenicol (Sigma Aldrich, France) at 8 mg/mL (for GFP plasmid selection) for 24 h (Ec 039) or 72 h (Lp1 008-GFP) at 37°C. They were then re-plated onto the same medium and incubated at 37°C for another 24 h (Ec 039) or for 3 days (Lp1 008-GFP). These cultures were then used to achieve a 10-mL calibrated suspension (CS) in sterile normal saline (0.9% NaCl) water. The final concentrations tested were 2x106 and 2x105 CFU/mL for Legionella and 2x106 CFU/mL for E. coli.
4
Photoinactivation of Legionella pneumophila
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The photoinactivation studies were conducted with Legionella pneumophila serogroup 1, strain ATCC BAA-74 or 130b (clinical isolate). The bacteria were routinely cultured on buffered charcoal yeast extract (BCYE) agar (Oxoid, Altrincham, UK) for 3–5 days at 35 ± 2 °C. They were re-suspended in sterile tap water (STW) and the concentration was adjusted by measuring optical density (OD600). A stock solution that contained approximately 109 CFU of Legionella per mL was prepared. The bacterial concentrations of approximately 5 × 105 CFU/mL or 1 × 108 CFU/mL were used in the experiments. The number of cultivable bacteria in the experiments was confirmed by enumeration of the bacteria on BCYE-agar.
5
Choline Effect on Legionella gormanii Growth
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L. gormanii was cultured on BCYE agar (Oxoid, Basingstoke, UK) with and without 100 μg/mL choline chloride (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C and 5% CO2 in the humid atmosphere for 3 days. The bacteria were suspended in sterile MilliQ water (OD600 = 0.1), and the subsequent 10-fold dilutions were performed to 10−3. Then, 5 µL of the last dilution of the bacterial suspension was transferred into a sterile Eppendorf tube, mixed with 5 µL of sterile MilliQ water (control) or the LL-37 peptide solution in water (10 µM and 20 µM). After 1 h of incubation at 37 °C, the bacterial suspensions were plated on the BCYE medium and incubated for 3 days at the same temperature. Subsequently, the number of CFUs was enumerated from the BCYE plates. The studies were carried out in three independent experiments, with each experiment including three replicates.
6
Legionella pneumophila Green Fluorescent Strain
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A Legionella pneumophila serogroup 1 GFP (green fluorescent protein)-modified strain was used39 (link)40 (link). This LP1 008-GFP strain was stored at −80 °C in Cryobank tubes (Mast Diagnostic, Amiens, France). After thawing, the strain was plated onto BCYE agar (Buffered Charcoal Yeast Extract, Oxoid, France) supplemented with chloramphenicol (Sigma Aldrich, France) at 8 mg/mL for 72 h at 37 °C (for plasmid selection) and then re-plated onto the same medium and incubated at 37 °C for another 2–3 days to obtain Lp1 008 EPF cells or for 5–6 days to obtain Lp1 008 SPF cells. These cultures were then used to achieve 5 mL suspensions in sterile normal saline (0.9% NaCl) water at a final concentration of 2.106 or 2.107 CFU/mL. An optical density of 0.2 at 600 nm (Biomate TM3; Avantec, Illkirch, France) was taken as a reference for a suspension containing 108 CFU/mL. The 5 mL suspensions were immediately placed in the nebulizer for cascade impactor experiments to determine the size distribution of airborne particles.
7
Intracellular Growth of L. pneumophila
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Human isolate L. pneumophila Philadelphia I (JR32), equipped with a complete dot/icm gene set encoding a type IV secretion system, which is required for intracellular amoebal growth [48] (link), was kindly provided by Dr. Masaki Miyake of the University of Shizuoka, Japan. L. pneumophila was cultured on BCYE agar (OXOID, Hampshire, UK) at 37°C for 2 days.
8
Cultivation and Preservation of L. pneumophila
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Model organism used in experiments was L. pneumophila serogroup 1, strain 130b or ATCC BAA-74 (clinical isolate). This bacterial strain was obtained from the collection of the Department of Microbiology and Parasitology, University of Rijeka. The bacterium was kept in 10% glycerol broth, stored in a freezer at -80 °C. After cultivation for 3-5 days on buffered charcoal yeast extract (BCYE) agar (Oxoid, Altrincham, UK) at 35 ± 2 °C in an aerobic atmosphere, the bacterium was used in experiments.
9
Genetic Manipulation of Legionella
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L. pneumophila with distinct genetic backbones, but originally derived from the Philadelphia-1 progenitor strain, [JR32 (T4ASS + /T4BSS + /Tra -), JR32
(T4ASS + /T4BSS -/ Tra -), Lp01 (T4ASS -/T4BSS + /Tra + ), Lp02 (T4ASS -/T4BSS -/Tra + )]
were used [15, 16] . All the bacteria were genetically modified to carry a GFP-expressing plasmid for ease of visualization. The bacteria were cultured on B-CYE agar (OXOID) at 37 °C for 2 days. Parachlamydia Bn 9 (ATCC VR-1476) was purchased from ATCC. The bacteria were propagated and stored at -80°C until used according to methods described previously [17] . Also, Mimivirus Kasaii, isolated from the mouth of the Arakawa River, which is close to Kasai Rinkai Park in Tokyo [18] , was used for this study. The virus was propagated in C3 amoebae, and the supernatant of ameobal lytic culture was used as the solution of virus. GFP-expressing Escherichia coli was constructed by transformation of E. coli (DH5α) with pBBR122 (Funakoshi) carrying gfp gene.
(T4ASS + /T4BSS -/ Tra -), Lp01 (T4ASS -/T4BSS + /Tra + ), Lp02 (T4ASS -/T4BSS -/Tra + )]
were used [15, 16] . All the bacteria were genetically modified to carry a GFP-expressing plasmid for ease of visualization. The bacteria were cultured on B-CYE agar (OXOID) at 37 °C for 2 days. Parachlamydia Bn 9 (ATCC VR-1476) was purchased from ATCC. The bacteria were propagated and stored at -80°C until used according to methods described previously [17] . Also, Mimivirus Kasaii, isolated from the mouth of the Arakawa River, which is close to Kasai Rinkai Park in Tokyo [18] , was used for this study. The virus was propagated in C3 amoebae, and the supernatant of ameobal lytic culture was used as the solution of virus. GFP-expressing Escherichia coli was constructed by transformation of E. coli (DH5α) with pBBR122 (Funakoshi) carrying gfp gene.
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