Phage-antibiotic synergy: cell filamentation is a key driver of successful phage predation

Phages are promising tools to fight antibiotic-resistant bacteria, and as for now, phage therapy is essentially performed in combination with antibiotics. Interestingly, combined treatments including phages and a wide range of antibiotics lead to an increased bacterial killing, a phenomenon called phage-antibiotic synergy (PAS), suggesting that antibiotic-induced changes in bacterial physiology alter the dynamics of phage propagation. Using single-phage and single-cell techniques, each step of the lytic cycle of phage HK620 was studied in E. coli cultures treated with either ciprofloxacin or cephalexin, two filamentation-inducing antibiotics. In the presence of sublethal doses of antibiotics, multiple stress tolerance and DNA repair pathways are triggered following activation of the SOS response. One of the most notable effects is the inhibition of bacterial division. As a result, a significant fraction of cells forms filaments that stop dividing but have higher rates of mutagenesis. Antibiotic-induced filaments become easy targets for phages due to their enlarged surface areas, as demonstrated by fluorescence microscopy and flow cytometry techniques. Adsorption, infection and lysis occur more often in filamentous cells compared to regular-sized bacteria. In addition, the reduction in bacterial numbers caused by impaired cell division may account for the faster elimination of bacteria during PAS. We developed a mathematical model to capture the interaction between sublethal doses of antibiotics and exposition to phages. This model shows that the induction of filamentation by sublethal doses of antibiotics can amplify the replication of phages and therefore yield PAS. We also use this model to study the consequences of PAS on the emergence of antibiotic resistance. A significant percentage of hyper-mutagenic filamentous bacteria are effectively killed by phages due to their increased susceptibility to infection. As a result, the addition of even a very low number of bacteriophages produced a strong reduction of the mutagenesis rate of the entire bacterial population. We confirm this prediction experimentally using reporters for bacterial DNA repair. Our work highlights the multiple benefits associated with the combination of sublethal doses of antibiotics with bacteriophages.

Methods
Phage-Antibiotic Synergy measurements. PAS measurements in liquid cultures were carried out by OD600measurements in a 96-well microplate. Briefly, a log-phase E. coli TD2158 PL4 culture was diluted to a final OD600 = 0.025. Subinhibitory antibiotic concentrations were added at this time. Replicates of 200 μL of each condition were dispensed in each well. After two hours of incubation at 37ºC under 180 rpm agitation, the same number of phages (phage titer PF/mL) was added to each replicate and lysis was observed by the reduction in OD600. Conversely, PAS measurements in semi-solid cultures were performed using the double agar overlay assay. For this, 10 mL of a log-phase culture of E. coli TD2158 PL4 at OD600 = 1 were infected with a final titer of 103 PFU/mL of phage HK620 and incubated at 37°C under 180 rpm agitation. Twenty-five minutes post-infection, 100 μL of this culture was mixed with 3 mL of soft-LBA (0.75% agar) and plated over a 20 mL bottom layer of 1.5 % LBA on a petri dish. Antibiotics were added at sublethal concentrations in the bottom layer. Plates containing 100 PFU each were imaged using a digital camera (Nikon D5300). Plaque diameter was measured using a homemade software designed by Leon Espinosa (available upon request) and average plaque diameter was plotted for each condition. 

Single-cell infection. An overnight culture of E. coli TD2158 PL4 was diluted to final OD600 = 0.025 in 10 mL of LB medium, and supplemented with the appropriate sublethal antibiotic concentrations when required. At OD600 ≈ 0.8, phages were added to reach a final MOI between 0.1 to 10. Thirty minutes post-infection aliquots were sampled and fixed by diluting 1:1 in PBS buffer PFA 4% solution to stop phage replication and prevent cell lysis. For adsorption analysis, unabsorbed phages were washed by centrifugation followed by resuspension of the pellet in PBS-PFA 2% solution. For all samples, the mix was put on a coverslip, gently squeezed under a 1 mm thick 1% agarose pad, and directly imaged on an inverted epifluorescence microscope (Nikon TiE) using an oil immersion 100X NA 1.45 objective. Images were acquired using a cooled camera (Hamamatsu Orca Fusion). Acquisition was carried out using Nikon’s NIS-Element software.

Image analysis. Cell image analysis was performed using MicrobeJ [1]. Cell shape parameters were directly measured from phase-contrast microscopy. For phage adsorption quantification, automated foci detection was carried out using the maxima foci function of MicrobeJ, after background subtraction and thresholding the GFP channel to isolate individual fluorescent phages. To quantify phage infection of HK620 hkcEF::PrrnB-gfp the integrated fluorescence of each cell was measured in the GFP. Statistical analysis was conducted in R [2] and figures were produced using the package ggplot2 [3]. To calculate average phage adsorption per unit of surface, E. coli shape was considered as a cylinder with two half spheres on each extremity. Bacterial length (L), measured from pole to pole, and average width (d) was determined for each bacterium using the image analysis software MicrobeJ. We then used these measurements to approximate bacterial cell surface through the following equation: π*d*(L-d) + 4*π*(d/2)2. The first term represents the surface of the cylinder of length (L-d), and the second, the surface of the two hemispheres of radius (d/2) at each pole.

Mutation rates measurements. The frequency of RifR CFU after 20 hours of growth was determined as follows. A log-phase, OD600 = 1 culture was diluted 1:106 (final concentration 200–500 CFU/mL) and 100 μL were dispensed in each well of a U-shaped bottom 96-well microplate (Nunclon Delta-Treated, U-Shaped-Bottom Microplate, Nunc). Antibiotics, if used, were added at ½ of the MIC at this point. The plate was covered with a sealing tape and incubated at 37 °C, 180 rpm, in a humidity cassette to minimise culture evaporation. After 20 h of incubation, the total volume (100 µL) of 84 wells was plated onto separate LBA plates supplemented with 75 μg/mL rifampicin. The remaining 12 wells were serially diluted and plated on non-selective plates to count the total number of CFU. Mutation rates were estimated by the MSS-MLE algorithm provided by the FALCOR calculator (https://lianglab.brocku.ca/FALCOR/). For the experiments in which we assessed phage effect in RifR mutant frequencies, HK620 phages were added in all wells at 11 h post-inoculation with a final titer of 4x103 PFU/well. Each condition was repeated at least three times.

References

1.   Ducret A, Quardokus EM, Brun YV. MicrobeJ, a tool for high throughput bacterial cell detection and quantitative analysis. Nat Microbiol. 2016;1: 16077. doi:10.1038/nmicrobiol.2016.77

2.   R Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2021. Available: https://www.R-project.org/

3.   Wickham H. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York. 2016.