Fig 1.
Electron micrograph showing the morphology of three different V. cholerae specific phages.
Note the hexagonal head and tails of various lengths.
Table 1.
Phenotypic and genetic characteristics of JSF3, JSF4 and JSF7 vibriophages.
Fig 2.
Degradation of biofilms by JSF7 phage and concomitant increase in corresponding V. cholerae count.
Biofilms of the indicated strains were established on the sides of glass tubes by static incubation in LB medium. After washing away planktonic cells, the biofilm was measured by staining with crystal violet followed by dye-extraction and measurement of OD at 550nm. Viable count of V. cholerae cells in the aqueous phase was determined at the indicated times after addition of JSF7 phage (1.5 x 107 pfu/ml). Note also that strains V51 and MO1220 are resistant to JSF7 phage, whereas strain C6706 is susceptible. Thus JSF7 phage degrades the biofilm matrix of both phage resistant and susceptible strains and releases planktonic cells. Each data-point represents the mean value and standard deviation of three independent observations.
Fig 3.
Resistance of biofilm-associated Vibrio cholerae to lytic phages JSF3 and JSF4.
Phage susceptibility of biofilm-associated cells were compared to that of respective freshly grown planktonic cells or cells physically dispersed from biofilms by shaking with glass beads and suspended in LB. Different cell suspensions and intact biofilms were exposed to 1 x 103 pfu/ml of JSF3 or JSF4 phage in LB supplemented with 5 mM CaCl2 and 5 mM MgCl2 and incubated at 37°C with shaking. At different time intervals, the residual bacterial cells were estimated. The survival rate of planktonic and biofilm-associated cells of V. cholerae O1 El Tor strain C6706 and O139 strain MO1220 after exposure to specific phages JSF4 and JSF3 respectively for different time periods are shown. Data points represent mean values and standard deviations of three different observations. The p values for the difference between survival of biofilm associated cells and planktonic cells when exposed to JSF3 (*) and JSF4 (**), and those for V. cholerae cells dissociated from biofilms by physical agitation vs fresh cultures of the bacteria exposed to JSF3 (†) and JSF4 (††), are shown.
Table 2.
Activity of diverse phages on biofilms of V. cholerae O1 or O139 in microcosms.
Fig 4.
Schematic representation of the structure, size and direction of transcription of the open reading frames (ORFs) of phage JSF3, JSF4, and JSF7.
The two ORFs designated ORF29 and ORF30 which are predicted to encode GDSL-like Lipase and Polysaccharide (Cycloalternan/pectin)-degrading enzymes respectively in JSF7 phage are shown with grey arrows.