Vancomycin hcl

As a first screening assay, 60 Paramecium strains were infected with L. pneumophila Ofk308 in 1.5 mL tubes at an multiplicities of infection (MOI) of 10,000 according to a previously reported method (Watanabe et al., 2016 (link)) with slight modifications. Tubes were incubated at 25°C for 24 h. After incubation, samples were treated for 30 min at 50°C to purge the K. pneumoniae fed to the Paramecium. Ten microliters of each sample was spotted onto GVPC agar (BCYE agar supplemented with glycine; Wako, 3 mg/mL), containing vancomycin HCl (Wako, 1 μg/mL), polymyxin B (Wako, 80 IU/mL), and sulfate cycloheximide (Wako, 80 μg/mL). As a second screening assay, the Paramecium strains selected during the first screening assay were infected with L. pneumophila Ofk308 in the same manner as a first screening assay. Incubated samples were cultured on GVPC agar after serial dilution to determine the colony forming units (CFU). The reduction of bacterial loads were evaluated by classification into three classes: -; no reduction of bacterial load (50–100%), ±; low reduction of bacterial load (less than 50%), +; high reduction of bacterial load (less than 10%) compared to control (cultured Legionella in the medium without Paramecium).

One hundred milliliters of sample was collected from each site in sterile bottles and centrifuged at 3,000 × g for 30 min. The deposits were resuspended in 1 mL phosphate-buffered saline (PBS) as concentrates. Concentrated samples were heated at 50°C for 30 min and spread onto the surface of GVPC. GVPC is BCYE supplemented with glycine (Wako, Osaka, Japan, 3 mg/mL), vancomycin HCl (Wako, 1 μg/mL), polymyxin B (Wako, 80 IU/mL), and sulfate cycloheximide (Wako, 80 μg/mL). Plates were incubated at 37°C and inspected daily. Isolated bacteria were grown on BCYE or BCYE without cysteine at 37°C. Cysteine auxotrophic bacteria were used for PCR analysis.