Bypassing Evolution of Bacterial Resistance to Phages: The Example of Hyper-Aggressive Phage 0524phi7-1

Abstract

The ideal bacteriophages (phages) for the treatment of bacterial disease (phage therapy) would bypass bacterial evolution to phage resistance. However, this feature (called a hyper-aggression feature) has never been observed to our knowledge. Here, we microbiologically characterize, fractionate, genomically classify, and perform electron microscopy of the newly isolated Bacillus thuringiensis phage 0524phi7-1, which we find to have this hyper-aggression feature. Even visible bacterial colonies are cleared. Phage 0524phi7-1 also has three other features classified under hyper-aggression (four-feature-hyper-aggressive phage). (1) Phage 0524phi7-1 forms plaques that, although sometimes beginning as semi-turbid, eventually clear. (2) Clear plaques continue to enlarge for days. No phage-resistant bacteria are detected in cleared zones. (3) Plaques sometimes have smaller satellite plaques, even in gels so concentrated that the implied satellite-generating phage motion is not bacterial host generated. In addition, electron microscopy reveals that phage 0524phi7-1 (1) is a myophage with an isometric, 91 nm-head (diameter) and 210 nm-long contractile tail, and (2) undergoes extensive aggregation, which inhibits typical studies of phage physiology. The genome is linear double-stranded DNA, which, by sequencing, is 157.103 Kb long: family, Herelleviridae; genus, tsarbombavirus. The data suggest the hypothesis that phage 0524phi7-1 undergoes both swimming and hibernation. Techniques are implied for isolating better phages for phage therapy.

Keywords: bacteriophage, aggregation of; bacteriophage, hibernation and swimming of; bacteriophage, plaque morphology of; bacteriophage, screening of; electron microscopy; ultracentrifugation.

Figure 1

Plating of phage 0524phi7-1 in a 0.2% agarose supporting gel. The plate was incubated for 18.0 h.

Figure 2

Plating of phage 0524phi7-1 for 18.0 h vs. plaque-supporting agarose gel concentration. The percentage of agarose is indicated at the upper left of the Petri plate. The M indicates a magnified region.

Figure 3

Plating of 0524phi7-1 vs. time in a 0.4% agarose plaque-supporting gel. The time in hour is indicated at the upper left of the Petri plate. The M indicates a magnified region.

Figure 4

Rate zonal centrifugation in a sucrose gradient of a preparation of phage T4 (centrifuge tube labeled T4) and phage 0524phi7-1 (centrifuge tube labeled 7-1). The image for T4 is reproduced from Ref. [35]. C, capsid; ϕ, phage.

Figure 5

Electrophoresis of the DNA packaged in particles in fractions of the sucrose gradient of Figure 4. DNase-resistant DNA was expelled from capsids and fractionated by agarose gel electrophoresis. The arrow indicates the direction of electrophoresis. Lanes are labeled by the average percentage of the sucrose gradient traversed. The DNA length standards are (1) the DNA of phages T3 (T3) and T4 (T4) and (2) a restriction endonuclease Hind III digest of phage lambda DNA (LH). Phage infectivity titers were the following (lane label, followed by titer ×10⁹ in parentheses): 3, (0.44); 13, (0.52); 18, (10.0); 24, (10.0); 32, (6.4); 47, (4.6); 61, (2.9); 70, (4.3); 79, (3.0); and 93, (4.1).

Figure 6

Electron microscopy of a single particle of negatively stained phage 0524phi7-1.

Figure 7

Electron microscopy of aggregated, DNA-emptied capsids of negatively stained phage 0524phi7-1.

Figure 8

Re-propagation on a mature lawn of in-gel propagating phages: (a) 18.0 and (b) 40.5 h incubation of six transfers of a plaque to a 1-day-old lawn and (c) 18.0 h incubation of three plaque transfers (right) and one 100 PFU liquid transfer (center of red circle).

Figure 9

Streaking for bacteria from the following zones: (a) clear, 18 h incubation, (b) semi-turbid, 18 h incubation, (c) turbid, 18 h incubation, and (d) turbid, 48 h incubation [of the Petri plate in (c)].