Table 1.
Transduction and lysogenization in cells when infected with temperate phages (upper part) or lytic phages (lower part).
Fig 1.
Time course experiment of S. aureus infected with ϕ11-ERM transducing lysate.
At time 0, S. aureus cells (RN4220) were infected at MOI = 1 with ϕ11-ERM propagated on JH930 harboring the pRMC2 plasmid encoding chloramphenicol resistance. At regular intervals (1, 3 and 9h), the total number of cells was determined as was the number of transductants (being resistant to chloramphenicol) and the number of transductants being lysogens and thus resistant to erythromycin.
Fig 2.
Simulations of three different experimental setups involving infection by one single type of phage show an advantage of transducing temperate phages in changing environments.
A) Phages infect bacteria carrying an antibiotic resistance gene (bacteria in red), and a sample of these phage particles infects bacteria initially susceptible to antibiotics (in green). Antibiotics are applied after the initial 7 iterations. 4 different scenarios are explored, using different types of phages: temperate and transducing phages in orange; temperate, and non-transducing phages in purple; virulent and transducing phages in blue; and virulent and non-transducing phages in pink. B) The number of bacteria (antibiotic resistant bacteria in red, and–initially–antibiotic sensitive bacteria in green) and the different types of phage particles are followed over time for the 4 different scenarios (each scenario corresponds to a column). The two different phases of the experiment (infection of antibiotic resistant bacteria and subsequently infection of antibiotic sensitive bacteria) are indicated in the x-axis. Lines correspond to the median of 100 different simulations with similar parameters, and the shaded areas correspond to a confidence interval of 95%. The model used in the simulations, as well as the values assumed for the parameters, are detailed in S1 Text.
Fig 3.
Identification of relevant parameters for phage survival.
A) Random Forest Analysis (RFA) is based on 5000 randomized combinations of parameter values and 20 repeated simulations for each combination. Parameters with a higher % in increased minimum square error have greater impact on the measured outcome: the fraction of simulations (for the 20 replicate simulations with a similar parameter combination) where phage are found alive. The parameters varied in the RFA are shown in S1 Text, section 3. B) Detailed effects of 3 variables in the survivability of initially antibiotic sensitive bacteria. The outcomes of simulations are shown in function of the probability of generalized transduction (y-axis) and genome size of the antibiotic resistant bacteria on which the phage was propagated (first panel), phage burst size (second panel) and number of phage particles sampled from the first part of the experiment (third panel) used for the second part of the experiment (see Fig 2). Each bin corresponds to the median of 20 replicate simulations for each parameter combination.
Fig 4.
Generalized transduction (GT) allows phage to persist in the presence of antibiotics.
RN4220 was infected with GT pac phage, ϕ11 (A) or non-GT, cos-phage ϕ12 (B) propagated on strain JH930 harboring pRMC2 encoding resistance to chloramphenicol. After 8 generations of growth the total number of bacteria and the number of chloramphenicol-resistant transductants were determined. Then, each culture was diluted 1000 fold in media containing a lethal concentration (30μg/ml) chloramphenicol and grown for 8 generations. The dilution step was repeated and the cultures were allowed to grow for additional 8 generations after which CFU of all cultures was determined on plates with or without chloramphenicol. Hundred transductants (or just survivors in case of ϕ12) were tested for lysogeny. Percentage over graphs indicate percentage of lysogens of total number of colonies tested.
Fig 5.
Efficient transduction transfers unlinked plasmid and chromosomal markers.
(A) S. aureus RN4220 after overnight infection with a ϕ11-ERM lysate propagated on a donor strain (JH1064) containing pRMC2 plasmid (CmR30) and a chromosomal marker (TetR5) inserted in the agr operon. (B) S. enterica Typhimurium LT2 after overnight infection with a phage P22 lysate propagated on a donor strain (JP14365) containing plasmid pET28a (KmR30) and a chromosomal marker (TetR20) inserted into the prpR gene. In both cases bar diagrams represent CFU ml-1 after incubation on selective plates. Grey bars represent phage infected cultures and white bars represent non-infected control cultures. From 66 to 150 transductants in each category were tested for lysogeny. Numbers above graphs indicate percentage of lysogens of total number of colonies tested.
Fig 6.
Gene-shuffling between genetically different strains lysogenized by the same phage.
(A) The experiment includes two lysogens carrying the same generalized transducing temperate phage, but with different antibiotic resistance genes in their chromosome (lysogen A resistant to erythromycin, lysogen B resistant to choloramphenicol). (B) Population dynamics of bacteria and phage when bacterial strains are subjected to a cocktail of two antibiotics (ERM+CAM) at iteration 10. Bacteria resistant to one antibiotic only are shown in faded symbols (crosses or circles, first column), with full colored symbols indicating bacteria resistant to both antibiotics (third column). Phages particles are shown in orange (second column), with full lines for active particles and dashed lines for transducing particles. Lines correspond to the median of 100 different simulations with similar parameters, and the shaded areas correspond to a confidence interval of 95%. (C) Two strains of 8325–4 (AA001) and USA300 (AA002) background, respectively, harboring different plasmids with unique antibiotic resistance markers were mixed 1:1 to OD600 = 0.01. After incubation over-night, number of cells from the two different strain backgrounds carrying one or both plasmid-encoded antibiotic resistance markers were determined.