Fig 1.
Possible interactions between two treatments on the rate of mortality of a target pathogen.
Antagonism—in combination, the two kill the pathogen at a lower rate than the best of the treatments alone. Facilitation—together, the treatments kill at a rate greater than the best of the treatments alone but less than that if the treatments were acting independently, where they would kill at a rate equal to the product of the rates at which they act alone. Synergy—the pathogen is killed at a greater rate than that when the treatments are acting independently.
Fig 2.
Schematic representation of the in-vitro procedure used to quantitatively explore the efficacy of antibiotics and phage for treating biofilm populations of bacteria.
(1) The biofilms were established by inoculating 1 X106 cells/mL in 2 mL LB in 24 well polystyrene plates. (2) These cultures were incubated for 48 hours without shaking to establish the biofilm. (3) The planktonic cells were removed with aspirator and wells were washed twice with saline. (4) These biofilm cultures were treated in one of two ways, (4a) Fresh LB with the phage (106 PFU/mL and antibiotics were simultaneously added to the wells. (4b) Fresh LB with the phage mixture (106 PFU/mL) was added first and then antibiotics were added with a delay of 4 or 24 hours. These cultures were incubated for total 48 hours starting from the addition of phages. (5) Following treatment, the cells were rendered planktonic by scraping the biofilm from walls of the well with the wooden applicator and later forced through a syringe needle to homogenize the cultures. The densities of viable bacteria and of phage were estimated by serial dilution and plating.
Fig 3.
Treatment of intact biofilms grown on plastic.
Viable cell densities (mean ± standard error, three replicates) in 48 hours, intact biofilm populations of P. aeruginosa PA14 on plastic then treated for 48 hours in various combinations of two phages (NP1, NP3) and/or five antibiotics (ceftazidime, ciprofloxacin, colistin, gentamicin, and tobramycin). Antibiotics were used at 1X and 8X MIC concentrations. Abbreviations are given by the first 3 letters of the drug name, and the number following the abbreviation indicates 1X or 8X MIC. A prefix N- indicates inclusion of both phages. “Con” is the untreated control with no antibiotic or phage added as treatment. A ‘P’ above the bar indicates that the phage titer at 48 hours exceeded the inoculum density by at least a factor of 10. ‘S’ indicates statistical support for synergy, ‘F’ for facilitation. Raw data of these experiments can be found in (S1 Excel File).
Fig 4.
Effect of treatment order in killing P. aeruginosa PA14 on plastic.
Bacteria alone were grown 48 hours, then treated with the two phages (N) for 0, 4 or 24 hours. They were then treated with the drug (8X MIC, abbreviations as in Fig 3) for the duration of treatment. Starting from the time phages were added, the culture was grown 48 hours, so the duration of treatment following antibiotic addition was shorter with the longer phage pretreatments. (A) Densities of viable P. aeruginosa PA14 at the end of treatment. The horizontal dashed line is the limit of detection (102/mL), and yellow boxes indicate that estimates were below the limit of detection. (B) Densities of phage at the end of treatment. The bold black line in (B) is the initial density of phage introduced. * Indicates that the 24 hours delay of gentamycin and tobramycin each have statistically significant effects on cell density compared to simultaneous treatment (P< 0.04, when correcting for multiple comparisons; tests of significance were equivalent for a Mann-Whitney U test and a median test using a Fisher’s exact test calculation—parametric tests were not possible because of some censored data). Means and standard errors from data obtained from two independent experiments, with a combined total of 5 replicate cultures. Raw data of these experiments can be found in (S2 Excel File).
Fig 5.
Combination of phage and antibiotic treatment on the ascent of antibiotic resistance.
Treatment of P. aeruginosa PA14 biofilm populations grown for 48 hours either fully resistant to the treating drug (CipR, GenR) or containing an initial mix of susceptible and drug-resistant bacteria (designated as—Mix). Abbreviations are given by the first 3 letters of the treating drug name followed by the status of the initial population (mixed or fully drug resistant). A prefix N- indicates inclusion of both phages with the drug. A- Viable cell density after 48 hours of treatment with ciprofloxacin (Cip) and ciprofloxacin with phage mixture (NCip) B- Viable cell density after 48 hours of treatment with gentamicin (Gen) and gentamicin with phage mixture (NGen) C- Titers of phage in (A). D- Titers of phage in (B). The horizontal line in (C) and (D) is the initial density of phage. For ciprofloxacin, the differences in total cell density were not statistically significant between Cip-Mix and Cip-CipR nor between NCip-Mix and NCip-CipR. The difference in the drug resistant fraction between Con-Mix and NCip-Mix is highly significant (P << 10−4). For gentamicin, the differences in total cell density were not statistically significant between NGen-Mix and NGen-GenR but were statistically significant between Gen-Mix and Gen-GenR (P < 10−4); however the magnitude of difference in this latter case is not large. The difference in the drug resistant fraction between Con-Mix and NGen-Mix is highly significant (P < 10−4). Raw data of these experiments can be found in (S3 Excel File).
Fig 6.
Treatment of biofilm grown on epithelial cells.
Treatment of 8 hours old biofilm population of P. aeruginosa PA14 on human epithelial cells treated with a mixture of NP1 and NP3 phage (N) and 1X MIC concentrations of ceftazidime, ciprofloxacin, colistin, gentamicin and tobramycin. A- Viable cell densities of Pseudomonas estimated in two independent experiments (red, blue) at 12 hours of exposure to the treatments. ‘S’ indicates statistical support for synergy and ‘F’ for facilitation. B- Densities of phage in two independent experiments sampled 12 hours after treatment. The bold black line in B is the density of the mixture of NP1 and NP3 introduced to the biofilm. Mean ± standard error for 3 replicates. Raw data of these experiments can be found in (S4 Excel File).