Luria agar la

MRSA isolates relating to staphylococcal bacteremia cases were identified and collected from the University Hospitals of Leicester (UHL) archive and stored in tryptic soy broth (TSB; BD Diagnostics Systems) with 20% (vol/vol) glycerol at −80°C. Unless otherwise stated, isolates were cultured on Luria agar (LA; Oxoid) at 37°C in air followed by Luria broth (LB; Oxoid) and were incubated with shaking at 37°C. For nutrient-restrictive conditions, 6% horse blood agar (HBA; Oxoid) was used and strains were cultured at 37°C in 5% CO₂ followed by CRPMI medium (CRPMI medium is RPMI 1640 medium [Sigma-Aldrich] depleted of metal ions via treatment with 6% [wt/vol] Chelex 100 [Sigma-Aldrich] but with 10% [vol/vol] untreated RPMI 1640 medium reapplied to provide the minimum elements required for growth) (23 (link)) cultured statically at 37°C in 5% CO₂.

We used six phyllosphere bacteria isolates (JB 3B, JB 16B, JB 20B, JB 26B, JB 12 F, and EJB 5 F) were obtained from previous study by Juliana [9 ], which were isolated from leaf surface of Psidium guajava in Karanganyar, Jakarta. Chromobacterium violaceum wild type and Chromobacterium violaceum 026 were used as indicator bacteria. In addition, this study also used several fish pathogenic bacteria, namely S. agalactiae ATCC279956, while A. hydrophila, strain OF 83 (GenBank accession number MW624435.1) and V. harveyi isolated from infected shrimp were obtained from Health Aquatic Organism Laboratory, Department of Aquaculture, Faculty of Fisheries and Marine Sciences, Bogor Agricultural University.
The phyllosphere isolates streaked onto King’s B 10% (20 g Protease Peptone; 1.5 g K₂HPO₄; 1.5 g Mg₂So₄. 7H₂O; 10 mL Glycerol; 10 g Agar Bacto; 1 L distilled water) then incubated at 28 °C for 48 h. C. violaceum wild type and C. violaceum 026 were streaked onto Luria Agar (LA) (Oxoid) then incubated 28 °C for 48 h. A. hydrophila was inoculated onto LA and incubated at 28 °C for 24 h. S. agalactiae was inoculated onto LA and incubated at 37 °C for 24 h. Meanwhile, V. harveyi was inoculated onto LA supplemented with 2% of NaCl (w/v) and incubated at 28 °C for 24 h.

S. enterica serovar Typhimurium ATTC strain 14028 s for which the complete genome sequence is available [39] (link) was used as wild-type and isogenic strains were constructed from it. Transductions were made using phage P22 HT [40] (link). Deletions were made by λ-red recombineering [10] (link), [11] (link) replacing the relevant genes with an FRT-tetRA-FRT cassette as described previously [41] (link). The genotypes of the bacterial strains used are listed in Table 1. Oligonucleotides used for recombineering and/or DNA sequencing across deletion junctions are listed (Table S5). Drug resistance markers were removed from recombineered strains by site-specific recombination after transduction with a P22 phage lysate grown on a strain carrying the plasmid pCP20 (Amp^R) expressing Flp recombinase [42] (link), leaving one FRT sequence at the site of deletion. Bacteria were grown at 37°C in Luria-Bertani broth (LB) or on Luria Agar, LA (LB supplemented with 1.5% agar; Oxoid, Basingstoke, England; 0.2% glucose; 3 mM CaCl₂). Antibiotics were used at the following final concentrations; tetracycline 15 µg/mL, ampicillin 100 µg/mL, rifampicin 100 µg/mL. For colony-aging experiments agar plates were incubated at 37°C in sealed plastic bags to minimize dehydration.