DOI: 10.5061/dryad.3ffbg79q8
title: Phage-antibiotic synergy: cell filamentation is a key driver of successful phage predation

description: 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.
description: <strong>Phage-Antibiotic Synergy
 measurements. </strong>PAS measurements in liquid cultures were carried
 out by OD<sub>600</sub>measurements in a 96-well microplate.
 Briefly, a log-phase <em>E. coli</em> TD2158 PL4 culture was
 diluted to a final OD<sub>600 </sub>= 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 OD<sub>600</sub>. 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 <em>E.
 coli</em> TD2158 PL4 at OD<sub>600</sub> = 1 were
 infected with a final titer of 10<sup>3</sup> 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.  <strong>Single-cell
 infection. </strong>An overnight culture of <em>E.
 coli</em> 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 OD<sub>600</sub> ≈ 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. <strong>Image
 analysis. </strong>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 <em>hkcEF::P<sub>rrnB</sub>-gfp</em> 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, <em>E. coli</em> shape was considered as a
 cylinder with two half spheres on each extremity. Bacterial length
 (<em>L</em>), measured from pole to pole, and average width
 (<em>d</em>) 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: <em>π*d*(L-d)
 + 4*π*(d/2)<sup>2</sup>. </em>The first term represents
 the surface of the cylinder of length (<em>L-d</em>), and the
 second, the surface of the two hemispheres of radius
 (<em>d</em>/2) at each pole. <strong>Mutation rates
 measurements. </strong>The frequency of Rif<sup>R</sup> CFU
 after 20 hours of growth was determined as follows. A log-phase,
 OD<sub>600</sub> = 1 culture was diluted
 1:10<sup>6</sup> (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 Rif<sup>R</sup> mutant frequencies, HK620 phages were added in all wells at 11 h post-inoculation with a final titer of 4x10<sup>3</sup> PFU/well. Each condition was repeated at least three times. <strong>References</strong> 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. 
description: # Phage-antibiotic synergy: cell filamentation is a key driver of
 successful phage predation ## PATHOGENS-D-22-02110R2
 "Phage-antibiotic synergy: cell filamentation is a key driver of
 successful phage predation" 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. ## Description of the Data and file structure
 Data corresponding to the figures in the manuscript are arranged in
 individual tabs included in the same file Datafile\_figures\_2.xlsx * Fig.
 1b: Radii measurements of phage HK620 lysis plaques in the presence of
 subinhibitory antibiotics concentrations (indicated in the “Treatment”
 column). * Fig. 1c: *E. coli* TD2158 PL4 growth/lysis dynamics in the
 presence/absence of antibiotics and phage HK620. Each row represents one
 of the four replicates carried out for each antibiotic/phage treatment.
 Individual values represent the OD600 measurement at the given timepoint
 (Time row). * Fig. 2a: *E. coli* MG1655 growth/lysis dynamics in the
 presence/absence of antibiotics and phage T4. Each row represents one of
 the four replicates carried out for each antibiotic/phage treatment.
 Individual values represent the OD600 measurement at the given timepoint
 (Time row). * Fig. 2b: *E. coli* MG1655 growth/lysis dynamics in the
 presence/absence of antibiotics and phage T5. Each row represents one of
 the four replicates carried out for each antibiotic/phage treatment.
 Individual values represent the OD600 measurement at the given timepoint
 (Time row). * Fig. 2a: *E. coli* MG1655 growth/lysis dynamics in the
 presence/absence of antibiotics and phage T7. Each row represents one of
 the four replicates carried out for each antibiotic/phage treatment.
 Individual values represent the OD600 measurement at the given timepoint
 (Time row). * Fig. 3b, 3c: Measurements of HK620 adsorption on individual
 *E. coli* TD2158 PL4 bacterium. Cells are numbered in the “Cell” column,
 antibiotic treatment is indicated in the “Treatment” column, measured
 bacterial length is indicated in the “Cell length (µm)” column, and the
 number of adsorbed HK620 is marked in “Number of adsorbed phages” column.
 * Fig. 3d: Mean and s.e.m. values of HK620 adsorption obtained from the
 dataset indicated in “Fig. 3b, 3c” for each antibiotic condition. * Fig.
 3e: Mean and s.e.m. values of HK620 adsorption obtained from the dataset
 indicated in “Fig. 3b, 3c” separating each antibiotic-treated culture in
 filaments (cell length &gt; 5.2 µm) and regular-sized cells (cell
 length &lt; 5.2 µm). The morphological category is indicated in the
 “Morphology” column. * Fig. 3f: Mean and s.e.m. values of HK620 adsorption
 per unit of surface separating each antibiotic-treated culture in
 filaments (cell length &gt; 5.2 µm) and regular-sized cells (cell
 length &lt; 5.2 µm). The morphological category is indicated in the
 “Morphology” column. * Fig. 4b, 4c: Measurements of T5 adsorption on
 individual *E. coli* MG1655 bacterium. Cells are numbered in the “Cell”
 column, antibiotic treatment is indicated in the “Treatment” column,
 measured bacterial length is indicated in the “Cell length (µm)” column,
 and the number of adsorbed T5 is marked in “Number of adsorbed phages”
 column. * Fig. 3d: Mean and s.e.m. values of T5 adsorption obtained from
 the dataset indicated in “Fig. 4b, 4c” for each antibiotic condition. *
 Fig. 3e: Mean and s.e.m. values of T5 adsorption obtained from the dataset
 indicated in “Fig. 4b, 4c” separating each antibiotic-treated culture in
 filaments (cell length &gt; 5.2 µm) and regular-sized cells (cell
 length &lt; 5.2 µm). The morphological category is indicated in the
 “Morphology” column. * Fig. 3f: Mean and s.e.m. values of T5 adsorption
 per unit of surface separating each antibiotic-treated culture in
 filaments (cell length &gt; 5.2 µm) and regular-sized cells (cell
 length &lt; 5.2 µm). The morphological category is indicated in the
 “Morphology” column. * Fig. 5b: Percentages of infected cells using HK620
 hkcEF::PrrnB-gfp in the presence of ciprofloxacin at different
 multiplicities of infection (MOIs). The morphological category is
 indicated in the “Subpopulation” column, and the multiplicity of infection
 in the “MOI” column. * Fig. 5b: Percentages of infected cells using HK620
 hkcEF::PrrnB-gfp in the presence of cephalexin at different MOIs. The
 morphological category is indicated in the “Subpopulation” column, and the
 multiplicity of infection in the “MOI” column. * Fig. 6b: Number of T7
 foci detected in individual ciprofloxacin-treated *E. coli* MG1655 at two
 timepoints (10 and 30 minutes). Cell length and foci number are indicated
 in their respective column. * Fig. 7: One-step growth curve data of phage
 HK620 in the presence of subinhibitory concentrations of antibiotics.
 Antibiotics are indicated in the “Treatment" column. Plaque-forming
 units observed for each timepoint are indicated in three columns under the
 "PFU/cell" header, each column belongs to one of the three
 replicates performed for each antibiotic treatment. Resulting mean and
 s.e.m are also indicated. * Fig. 10a: Fluctuation test data of *E. coli*
 TD2158PL4 in the presence/absence of ciprofloxacin and HK620. The
 presence/absence of ciprofloxacin is indicated in the “Antibiotic” column,
 the presence/absence of phage HK620 is indicated in the “Phage” column.
 The mutagenesis rate for each replicate is indicated in its own column and
 the mean and s.e.m. values are also indicated. * Fig. 10b: Fraction of
 SOS-triggered cells in the presence of ciprofloxacin at increasing
 concentrations (“[Ciprofloxacin]” column). The replicate number for each
 condition is indicated in the "Replicate" column. The
 morphological categories are indicated in the “population” column. The
 mean percentages of SOS-triggered cells and their respective s.e.m. are
 also indicated. * Fig. 10c: Simultaneous measurements of phage HK620
 hkcEF::PrrnB-gfp infection and SOS-induction in individual E. coli TD2158
 PL4 bacteria in the presence of subinhibitory ciprofloxacin. Cells are
 numbered in the “Cell” column. GFP intensity due to phage infection is
 indicated on the “GFP intensity” column. Fold-change of mCherry intensity
 (reporter of SOS activation) is indicated on “SOS fold-change” column.
 Measured bacterial length is indicated in the “Cell length (µm)” column.
 The status of infected/non-infected for each bacteria is determined from
 the GFP intensity levels and is indicated in the last column. ##
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