LES prophages have been suggested to contribute to the competitiveness of their bacterial host in vivo. LESB58 mutants, with disrupted prophage genes, exhibited 10 to 1000-fold decreased competitiveness in a rat model of chronic lung infection compared to wild type LESB58 . The LES phages are induced by exposure to clinically relevant antibiotics, e.g. ciprofloxacin , and free LES phages and other tailed-phage virions have been detected in CF patient sputa [25, 26]. Temperate phages are key vectors of horizontal gene transfer (HGT) . Therefore,
it is important to assess the ability of the LES phages to infect other bacterial hosts Proteases inhibitor to which they may confer traits beneficial to Belnacasan life in the CF lung environment. Here we describe the infection characteristics of three of the five LES prophages LESφ2, LESφ3 and LESφ4, induced from the sequenced CF lung isolate LESB58. Results LES phage morphology Three different Siphoviridae phages were induced from LESB58 cultures and visualised using electron microscopy. The phages possessed icosahedral heads (50–60 nm diameter) and long flexible tails (approximately 200 nm). Plaque assay of each phage on PAO1 resulted in the formation of small
turbid plaques with different phage-specific morphologies. LESφ3 plaques were the largest (2–3 mm), with well-defined lysogen mTOR inhibitor islands, whereas LESφ2 plaques were considerably smaller (0.5-1.5 mm). LESφ4 produced plaques with small, clear centres surrounded by a turbid halo. The identity of each LES
phage responsible for the different plaque morphologies was confirmed using a multiplex PCR assay. Differential induction of LES phages from LESB58 The sensitivity of the LES phages to induction into the lytic cycle was determined and compared. Real-time quantitative (Q)-PCR was used to measure relative increases in phage DNA copy number following induction by exposure of LESB58 to norfloxacin. After exposure to norfloxacin for 60 min and recovery for 2 h, LESφ2 was the most abundant free phage detected (6.2 x 107 copies μl-1), compared to LESφ3 (6.9 x 106 copies μl-1) and LESφ4 (1 x 107 copies μl-1) (Figure 1). Furthermore, the increase in LESφ2 production between 30 and 60 min exposure times Selleckchem Verteporfin was higher (3.67 fold increase) than that for LESφ3 (1.74 fold increase) and LESφ4 (2.06 fold increase). Thus while norfloxacin induction caused a significant increase in the replication of all three phages (LESφ2 – F1, 8 56.97, P 0.001; LESφ3 – F1, 8 14.02, P 0.006; LESφ4 – F1, 8 16.88, P 0.003), only LESφ2 showed significantly greater phage production after 60 min compared to 30 min norfloxacin exposure (induction*time interaction, F1, 8 20.90, P 0.002); by contrast, the duration of exposure had no effect on phage production in LESφ3 and LESφ4 (induction*time interaction, LESφ3 – F1, 8 1.05, P 0.