In order to examine to which extent phage encoded virulence factors could be captured and spread within an existing E. coli biofilm, we decided to make acquisition of the naturally stx1-encoding temperate H-19B phage [32] readily detectable through selective plating by equipping it with an antibiotic resistance marker. Since only a small fragment of the H-19B genome sequence is currently known [32], a random transposon mutagenesis procedure was followed to tag this (pro)phage. More specifically, E. coli MG1655 was first lysogenized with H-19B, after which a random Tn10-transposon library of ca. 10,000 clones was constructed in this lysogen using the λNK1324 hop protocol described by Kleckner et al. [33]. Assuming some of the Tn10 insertions to be located within the H-19B prophage, mitomycin C was subsequently used to induce the prophage in the obtained pool of transposon mutants. To isolate H-19B::Tn10 mutants within the corresponding phage lysate, it was first plaqued on E. coli MG1655. Since the Tn10 transposon codes for the cat gene and confers chloramphenicol resistance, lysogens arising in the middle of turbid plaques were scored for the presence or absence of chloramphenicol resistance. As such, nine chloramphenicol resistant lysogens were obtained, which were further confirmed to simultaneously have acquired the stx1 operon of H-19B. The main advantage of this procedure is that it automatically disregards phage mutants compromised in lytic or lysogenic development. Moreover, in contrast to phage recombineering protocols [34], this protocol can be applied without prior knowledge of the phage’s genome sequence and resembles the method used previously by Acheson et al. [35] to look for phage encoded exported proteins.

From one of the obtained H-19B::Tn10 lysogens, the Tn10 insertion site was determined and found to map within a gene bearing homology to the nleG virulence genes. NleG proteins are effectors of the type 3 secretion system that are thought to mimic eukaryotic E3 ubiquitin ligases [36], and are generally found in the late region of phage genomes [37]. This insertion underscores the presence of additional virulence genes to be present in H-19B, supports the observation that neither lytic nor lysogenic behaviour is affected in H-19B::Tn10 and due to the uptake of an additional piece of DNA (i.e,. the transposon) adds evidence to the plasticity of Stx genomes [38]. Subsequently, a mature three-day old E. coli biofilm was only once exposed to a small number (i.e., 150 viral particles spread over one hour) of the corresponding H-19B::Tn10 derivative in order to mimic an accidental exposure. Subsequently, the spread of this virulence conferring phage genome throughout the biofilm population was tracked. More specifically, the effluent of the biofilm was examined for the presence of (i) cells lysogenized with H-19B::Tn10, and (ii) free H-19B::Tn10 phage (Figure 1). Interestingly, from this analysis it became clear that lysogenic conversion of the biofilm proceeded very rapidly, with the emergence of ca. 10⁶ CFU/mL of H-19B::Tn10 lysogens on a total effluent cell count of circa 10⁹ CFU/mL (i.e., 0.06% conversion) after 24 h, and an above 50% conversion reached after five days. Moreover, at the end of the experiment the ratio between total and lysogenized cells within the actual biofilm itself corresponded to that observed in the effluent, demonstrating that, at least at the end of the experiment, lysogenic conversion had occurred in the entire biofilm Furthermore, during the first two days after phage exposure, the concentration of free H-19B::Tn10 phage in the effluent quickly rose from the applied 50 PFU/mL to 10⁵–10⁶ PFU/mL, after which it remained stable at this level. It can be anticipated that part of the biofilm is being lysed, leading to a local enrichment in H-19B::Tn10 concentration. Nevertheless, it remains unclear exactly to what extent the emergence of H-19B::Tn10 lysogens is the result of de novo lysogenic conversion of pre-existing wild-type cells or of clonal enrichment of the first converted cells.

In a second approach, biofilm dynamics were examined upon single exposure of a 3-day old mature E. coli biofilm to ca. 10⁸ particles per milliliter of the obligatory lytic T7 phage. The high phage titer selected in this approach was considered to best reflect the clinical application of phage in combatting biofilm-related infections. After three additional days of incubation, cells within the biofilm were harvested and enumerated. A comparison between the number of viable cells within the T7-exposed or -unexposed biofilm revealed an almost 100-fold reduction (p-value in two tailed homoscedastic T-test <0.05) in the phage exposed biofilm (Figure 2). However, in between the normal colonies observed during plating, colonies with a mucoid appearance were observed in biofilms treated with T7 phages (Figure 3A), but not in untreated biofilms. This phenotype was earlier shown to be correlated with T7 resistance due to a mutation resulting in the production of excess capsular polysaccharide preventing T7 adhesion [39] due to physical blocking of the phage binding site [40], and actual T7 resistance of these colonies could be confirmed as in Figure 3B. After examining ca. 400 colonies of both types, phage resistant clones with a non-mucoid phenotype or phage sensitive clones with a mucoid phenotype were not observed. In turn, this enabled us to accurately determine the number of resistant cells in the biofilm. However, a large variation in the number of resistant cells was found between biological replicates (Table 1), ranging from circa 0.05% to over 28%. As in Luria-Delbruck fluctuation experiments, this variation likely stemmed from clonal enrichment of stochastically pre-existing T7-resistant mutants that spontaneously arise within a population. Since 3 days after initial phage exposure T7 particles still remained present in the effluent and the biofilm, on-going selection for T7-resistance development seems warranted upon longer incubation of the biofilm.

E. coli MG1655 [41], MG1655 lacZ::Tc [42], and MG1655 H-19B (i.e., lysogenized with stx1 encoding phage H-19B; [43]) were used in this study, and were cultured at 37 °C under shaking conditions in Lysogeny Broth (LB). In order to determine the number of bacteria (as colony forming units or CFU) in a sample, a dilution series was prepared and plated on LB agar (1.5% agar), after which the concentration was expressed as CFU/mL.

To obtain a H-19B phage lysate from lysogens, cells from 1 mL bacterial culture were pelleted by centrifugation (6000 × g, 15 min), after which 50 µL of CHCl₃ was added to sterilize the supernatant and to release progeny phage from infected but non-lysed cells. In case high phage titers were required, the cultures were grown in the presence of 2 µg/mL mitomycin C which induces the release of temperate phages. To obtain a T7 phage [44] lysate, T7 was propagated on MG1655, after which 1 mL of cells were pelleted by centrifugation (6000 × g, 15 min) and the resulting supernatant was sterilized by addition of 50 µL of CHCl₃. In order to determine the number of phages (as plaque forming units or PFU) in a sample of lysate or effluent, a dilution series was prepared and plated on E. coli MG1655 grown in LB soft agar (0.7% agar), after which the concentration was expressed as PFU/mL.

Where necessary, growth media were supplemented with tetracycline (20 µg/mL) and/or chloramphenicol (30 µg/mL) (Applichem, Darmstadt, Germany).

After its construction as described in the text, the Tn10 insertions site in H-19B::Tn10 was mapped by subcloning of the Tn10 encoded chloramphenicol resistance marker in pUC18. Subsequently, genomic DNA flanking the Tn10 transposon was sequenced using a primer (5’-AAGCACCGCCGGACATC-3’) reading outwards of the transposon. In addition, the isolated H-19B::Tn10 phage was also confirmed by PCR (directly on plaques or crude lysate) to still carry its stx1 locus, by using primers flanking this region (5’-CAGTGGATCCTGGCACGGAAACATGGGT-3’ and 5’-TCAGTCTAGATTACGTCTTTGCAGT CGAGAAGTC-3’).

Biofilms were grown at 30 ºC in three-channel flow chambers (Biocentrum DTU: Technical University of Denmark, Soltofts Plads 221 DK-2800 Kgs, Lyngby) with individual channel dimensions of 1 × 4 × 40 mm³ [45] that were covered with a microscope glass coverslip (st1; Knittel Gläser, Braunschweig, Germany). The setup makes use of a 16-channel peristaltic pump (Watson Marlow 205S, Zellik, Belgium) that feeds each channel with a flow of 3 mL/h (flow rate of 0.2 mm/s) of AB-trace medium [2 g/L (NH₄)₂SO₄; 6 g/L Na₂HPO₄.2H₂0; 3 g/L KH₂PO₄; 3 g/L NaCl; 9.5 mg/L MgCl₂; 11.1mg/L CaCl₂ and 0.1 mL/L of the following trace metal mixture (200 mg/L CaSO₄.2H₂O; 200 mg/L FeSO₄.7H₂O; 20 mg/L MnSO₄.H₂O; 20 mg/L CuSO₄.5H₂O; 20 mg/L ZnSO₄.7H₂O; 10 mg/L CoSO₄.7H₂O; 10 mg/L NaMoO₄.H₂O; 5 mg/L H₃BO₃)] supplemented with 0.3 mM glucose and 1 µg/mL thiamine dichloride. Bubble traps were placed in each channel before the flow cell to remove air bubbles. Before use, the flow system was sterilized by flushing with a solution of 0.5% sodium hypochlorite for 4 h, and rinsed with approximately 0.2 L of sterile water before the medium was pumped through.

Bacterial cultures for inoculation were prepared by diluting an overnight LB broth culture 1/100 in fresh LB medium and regrowing it for 4 h at 30 ºC under shaking conditions. To inoculate the flow cells the medium flow was stopped, flow chambers were turned with the glass coverslip down and 250 µL of the diluted cell suspension was carefully injected through the silicon tubes into each flow channel with a small syringe. After 1 h, to allow adsorption of the cells to the coverslip surface, the flow channels were turned upright and the flow was resumed. For experiments with H-19B::Tn10 and T7, E. coli MG1655 lac::Tc and E. coli MG1655 were used, respectively. When macroscopically visible mature E. coli biofilms were formed after 3 days, they became exposed for 1 h to either sterile water, 50 PFU/mL of H-19B::Tn10 or ca. 10⁸ PFU/mL of T7 via a separate input channel.

Biofilms were analyzed by determining the number of viable cells (CFU) or phage particles (PFU) in either the effluent or in the biofilm itself. Effluent was collected using a small connector inserted in the tubing behind the flowcell, while the actual biofilm was harvested by carefully disconnecting the flowcells and vigorously pipetting up and down the channels with 250 µL potassium phosphate buffer (10 mM, pH 7.0).

After vortexing, dilution series of collected samples were plated on LB agar (in case E. coli MG1655 was used) or LB agar supplemented with tetracycline (in case E. coli MG1655 lac::Tc was used) to enumerate total cell counts, and on LB agar supplemented with chloramphenicol to enumerate H-19B::Tn10 lysogens. Phages were enumerated by first sterilizing the collected samples with CHCl₃, and subsequently plating them on E. coli MG1655.