The infection of a bacterial cell by multiple, different bacteriophages (phages) constitutes an important part of phage ecology that influences phage evolution in many ways.1-5
To study interactions among phages, methods are required that can discriminate and quantify different phage types in the same sample. The double agar overlay assay,6
although considered to be the gold standard for phage quantification, falls short of what is required here. Its capability to differentiate phages is limited as it depends on differences in phenotypic markers such as plaque morphology or host range of the phages to be distinguished.5
Real-time quantitative PCR (qPCR) is an interesting alternative and has been successfully used to enumerate phages as well as other microparasites.7-10
Concerning phages, current applications of qPCR fall broadly in two groups, neither of which requires a highly specific assay. Either the specificity of the method is not an issue as samples are expected to contain only one type of phage, or the method has been deliberately adapted to be unspecific such that a broader group of phages is detected.11-13
Here, a method is presented where qPCR is used to achieve the opposite, namely the discrimination of closely related phages in mixed lysates.
To develop and validate this method, a set of ten lambdoid phages of Escherichia coli
str. K-12 is used. These phages have been used previously to study the competitive interactions that occur when two prophages are induced in the same lysogenic cell.5
Pairs of these phages can be distinguished by differential plating on bacteria that exclude one phage by its receptor specificity or immunity group. However, this approach fails to discriminate most combinations of three or more phages. This can be overcome by a qPCR approach where individual phages types are specifically targeted.
The assay is based on an earlier protocol,9
a modified version of which has been used previously to quantify variants of phage λ that did not produce plaques or whose plaques were difficult to count.14
It consists of two steps, an initial treatment with DNase to remove phage DNA that is not enclosed in capsids (which would cause an overestimation of the phage titer), and the qPCR itself. While the method performs very robustly, its extension to achieve the discriminatory power to accurately quantify individual phages in mixed samples has never been explored and required further experiments for validation.
Together, these experiments show that the assay detects all viable phages in a sample and clearly discriminates individual phage types. The good correspondence to the plaque assay suggests that the method can be used by digital PCR15
for absolute quantification without an external standard and for the quantification of phages which are difficult or impossible to quantify otherwise (e.g., because they form no or barely visible plaques, or no cells suitable for plating are available).14,16
The method is used to quantify individual phages in lysates that were obtained from the induction of lysogens containing either one, two, or three different lambdoid prophages. Specific pair-wise combinations of these phages have previously been shown to strongly interfere during prophage induction, leading to substantial declines in productivity for both phages involved.5
It is therefore of interest to investigate whether this mutual interference also affects other prophages that partake in the induction, and whether they can mediate the outcome.
The method presented here is hoped to be a useful tool to conduct experiments in the area of phage ecology and evolution. Data on viral diversity in various environments accumulate at a staggering pace,17-20
and it is important to test whether concepts from ecological and evolutionary theory can be applied to understand the processes that give rise to this diversity.21-24
Another field where the application of qPCR is promising is phage therapy, the treatment of bacterial infections with phages. The ability to discriminate different phages will allow better understanding of phage population kinetics when phage cocktails instead of single phages are used.25-27