Background

The field of phage therapy, which is the clinical use of viruses to kill bacteria, has grown exponentially over recent decades (Gordillo Altamirano and Barr, 2019). Prior to their selection for therapeutic use, phages are typically subjected to a thorough characterization pipeline (Terwilliger et al., 2020). Lysogenic or temperate phages are those with the capacity to integrate themselves into their host’s genome, and are routinely excluded from therapeutic use due to their subpar bactericidal ability, the emergence of homoimmunity, and the potentially harmful consequences of lysogenic conversion (Fortier and Sekulovic, 2013; Hyman, 2019).

Temperate behavior is first suspected in phages that produce turbid plaques on bacterial lawns. However, plaque morphology can vary greatly depending on a variety of factors, including the growth medium used and the physiological state of the bacterial host (Ramesh et al., 2019). Delayed or substandard bactericidal activity in bacterial growth assays can also serve as an indicator, but their interpretation is not always clear-cut. Alternatively, genotypic screening for genes that are essential for lysogeny, such as integrase and excisionase enzymes, are further indicators of temperate behavior (Hyman, 2019). However, extraction and sequencing of phage genomes can be challenging, and the accuracy of homology-based analyses is highly dependent on the quality of the sequencing data, assemblies, and available databases (Russell, 2018). Importantly, the presence of a predicted integrase in the genome of a phage may not be definite confirmation of the ability to lysogenize a host in vitro or in vivo (Howard-Varona et al., 2017).

Here, we outline a simple protocol, modified from Pope et al. (2013), that evaluates the ability of a given phage to lysogenize its host in vitro. The assay can be reliably used in cases where no sequencing data are available. Furthermore, this protocol has the advantage of providing confirmation of lysogenic behavior for a given phage, instead of prediction, given by genome sequencing-based tests. It does not require specialized equipment and can complement analysis of plaque morphology and growth. Although we describe the protocol using an Acinetobacter baumannii-specific phage (Gordillo Altamirano et al., 2021), we also provide data from an Enterococcus faecium-specific phage (Figure 1), demonstrating that the methodology is broadly applicable to any phage with a bacterial host culturable on solid media. As such, incubation and media conditions may need be adapted to suit the host. Finally, in addition to excluding lysogenic phages from therapeutic use, the protocol can be used to produce lysogens (bacteria harboring a prophage) for downstream applications in phage biology and biotechnology research.