3. Results

We decided to use LPS profiling and the sensitivity to the bacteriophages with known adsorption mechanisms for evaluation of the O antigen production status of the φ24B lysogens generated in environmental E. coli isolates. To do so, liquid cultures of the O antigen producing strains 4s, HS1/2, HS3-104, F5, F17, UP1 and UP11 and of the rough strains 4sR and C600 were challenged with phage φ24B:cat as described by James23. The lysogens were then selected by plating the mixture on LB plates supplemented with 34 µg/ml of chloramphenicol. The lysogens were obtained for strains 4s, HS1/2, HS3-104, F5 and F17. No lysogens were observed on strain UP11. The lysogenization frequency was about 10-4 lysogen cfu/ phage pfu for the rough strains and about 10-7 – 10-6 in O antigen-producing strains. The latter value is comparable to the level of spontaneous mutations in E. coliinactivating a medium-sized gene (e.g. phage-resistant mutants).
We selected 3 lysogen clones per strain and confirmed the φ24B prophage presence using PCR for gene 61 (the tailspike protein gene). ForE. coli 4s lysogens we also performed mitomycin C induction followed by transmission electron microscopy that confirmed that a phage morphologically identical to φ24B was produced.
LPS profiling of the lysogens obtained indicated that in all cases these strains did not produce O-antigen at all or the O-chain synthesis was greatly decreased compared to the parental strains (Fig. 1).
The bacteriophages that are potentially able to infect the strain but are restrained by its O-antigen can be successfully used as a probe for testing the efficacy of the O-antigen-mediated protection31. We developed the use of a T5-like bacteriophage DT571/2 mutant FimX lacking lateral tail fibers (LTFs) as such a probe30. We tested the ability of phage FimX to grow on the lawns of the lysogens obtained. This phage was not able to form plaques on the parental O-antigen – producing strains, except for F5 on which it formed plaques with an efficiency of plating (EOP) of 10-4 compared to the C600 strain used for FimX propagation 35. At the same time the EOP of FimX phage on all the lysogenic cultures tested was in the range of 0.1 – 1.0 compared to the E. coli C600 strain. The gain of sensitivity to the phage FimX observed after the lysogenization was not distinguishable from other methods of rough mutant generation previously used by us in 4s or F17 strains29, 31.
The other T5-like phages (DT57C, DT571/2, ABF and Gostya9) as well as the siphovirus 9g demonstrated the gain of the infectivity on the lysogens derivatives of some strains that were initially resistant to these phages. Phage G7C that is dependent on the specific O antigen recognition for infection of E. coli 4s cells30was not able to infect E. coli 4s (φ24B:cat) lysogenic strains in good agreement with O antigen production loss detected by the LPS profiling (Fig. 1).
Since the O antigen synthesis compromised strains are believed to be more vulnerable to immunity factors, we decided to measure the susceptibility of the lysogens obtained to the bactericidal activity of the horse serum (SBA). All the wild type strains were resistant to SBA in our conditions (Fig. 2). Their cultures grew in presence of the serum as well or even slightly more rapidly than in the control experiment. In the absence of the serum the lysogenic strains showed the growth rates close to their cognate wild type strains. At the same time the growth of the lysogens was almost completely abolished in the presence of the serum (Fig. 2). Only one of the lysogenic clones tested, the derivative of the strain HS3-104, was able to grow significantly in presence of the horse serum, though the rise of the optical density was delayed and the growth rate was significantly lower than in the parental strain (Fig.2). This result can be explained by the fact that in HS3-104 lysogens the O antigen synthesis was strongly decreased but not completely abolished (Fig.1). So, the actual synthesis of O-polysaccharide could be upregulated in this particular clone in the conditions of the experiment of SBA sensitivity measurement.