From:
"Bacteria, Phages and Septicemia
Aušra Gaidelyté 1, Martti Vaara 2, Dennis H.Bamford 1
1 Department of Biological and Environmental Sciences, Institute of Biotechnology, University of Helsinki, Helsinki, Finland, 2 Department of Clinical Microbiology, Helsinki University Hospital, Helsinki, Finland
We observed that the majority of the bacterial isolates from septicemia patients spontaneously secreted phages active against other isolates of the same bacterial strain, but not to the strain causing the disease.
Such phages were also detected in the initial blood cultures, indicating that phages are circulating in the blood at the onset of sepsis. The fact that most of the septicemic bacterial isolates carry functional prophages suggests an active role of phages in bacterial infections. Apparently, prophages present in sepsis causing bacterial clones play a role in clonal selection during bacterial invasion.”
1-For UV induction, bacteria were grown to 200 Klett units and collected by centrifugation for 10 min at 6,000 rpm using a Sorvall SS-34 rotor at 4°C. Bacteria were suspended in the same volume of M9 broth and transferred to a glass Petri dish.
The bacterial suspension was irradiated for 42 sec at A254 followed by dark storage on ice for 1 h. Cells were collected by centrifugation for
5 min at 13,000 rpm using a Heraeus Biofuge at 22°C. Bacteria were suspended in 3 volumes of LB and the number of plaques was determined after additional two h incubation at 37°C.
2a-For MitC (mitomycin C) induction experiments, cells were grown to 200 Klett units and induced with MitC at a final concentration of 5 mg/ml. Cells were incubated for 15 min at 37°C and the growth medium was then replaced with fresh LB. Plaques were determined after additional two h incubation at 37°C.
or
2b-To confirm the absence of temperate bacteriophages,originating from the bacterial hosts a standard technique for bacteriophage induction using the DNA-damaging antimicrobial agent mitomycin C was carried out, as described by Miller.
Bacterial cultures were aliquoted in 1 ml volumes in sterile eppendorf tubes, covered with aluminium foil thus protecting the bacteria from photoreactivation of drug-induced DNA damage. Mitomycin C (Sigma-Aldrich) was added to final concentrations of 1 or 5 mg/ml . A control tube without mitomycin C was added to evaluate the presence of ‘nondrug induced’ bacteriophages. The tubes were incubated for 3 h at 37°C. Subsequently, twenty ml of chloroform was added to the control tubes to lyse the bacteria. The lysates were centrifuged in order to separate the intact bacterial cells from the supernatant. The final titre of bacteriophages in the supernatant was determined using the double agar overlay method.
3-To test if antibiotics could induce phage production, the same procedure was used as in the previous paragraph with the following
modifications. Three different antibiotic concentrations were tested depending on the antibiotic used and bacterial strain employed.
For E. coli strains, 1, 10, and 30 mg/ml final concentrations of tobramycin (tomycin, Orion Pharma) and 0.03, 0.3, and 3 mg/ml of ciprofloxacin (Bayer) were used. For S. aureus strains, 1, 10, 20 mg/ml final concentrations of tobramycin and 1, 10, 30 mg/ml of ciprofloxacin were used.
Viable counts of cell suspensions were determined to evaluate the antibacterial activity of UV, MitC or antibiotic treatment.
Methods for Phage induction
Bacterial Virulence properties altered by Phages
From:Bacteriophage Control of Bacterial VirulencePatrick L. Wagner and Matthew K. Waldor
