There is an accepted general protocol for the characterization of a newly isolated phage, viz.: selection of the phage from the external source / DNA isolation / genome sequencing / genome annotation / and a recommendation that the phage is suitable for use in therapy. The biological characterization of phages that are deemed suitable for use in therapy is usually limited to an estimation of their host range, and their designation as temperate or virulent. Some further features can be identified from an annotation of the genome. However, only a direct comparative study of a new phage in relation to other phages including visual observation of phages interactions on the surface of infected bacterial biofilm, can provide a reliable indication regarding its safety for therapeutic application. This is now illustrated below, using some new findings.
Until June 2014, the NCBI database contained information on 66 P. aeruginosa phages in the Caudata group. Most of these phages (47 out of 66) are considered as virulent and can be classified into a small number of species. It is evident that new species will be found, but it is also clear that the number of such new species will not be excessive. The presently known various species of P. aeruginosa virulent phages are presented in Table 1. All in all, there are 11 species of supposedly virulent phages for which the genomes have been annotated (genome of Lin68 has not been sequenced yet).
Table 1. Summary of species of virulent bacteriophages active against P. aeruginosa
The species that are evident candidates for use in therapy are the first five in Table 1, which are also the most frequently occurring: the PB1-, phiKMV-, PaP1-, KPP10-and PaP3/LUZ24-like phages. The frequency of their isolation may reflect their wide lytic spectra. We detected these species of phages in three different commercially available phage mixtures (produced by Microgen in Perm, Ufa and Nizhnii Novgorod – all in the Russian Federation). We have noted that some of the phages from these commercial mixtures exhibit a specific phenotype (opales cence) that may be related to that of phage phiKZ and other giant phages. As it transpires, phages of species phiKZ are permanent components of commercial mixtures, although EL-like and Lin68-like phages have not been yet found.
It is noteworthy, that in general, the sizes of phage DNAs of every species have their specific confined range. The largest volume may reflect the maximum capacity of the capsid, and the smallest may correspond to the size of the DNA molecule that is required in order to contain all of the important phage genes for that species, although there may be specific exceptions (Sokolova et al., 2014). The s ize difference between the DNAs of phages within the same species perhaps suggests the presence of excessive nucleotide sequences that are not an essential component of the phage genome. This might imply that phages which carrying minimum number of ORFs open preferable for use in therapy.
phiKMV-like phages are present in all commercial preparations. DNA restriction analysis for several phages reveals an appreciable degree of similarity to phiKMV (Burkal'tseva et al., 2006). It would appear that their presence in commercial phage mixtures reflects their high growth rate and their wide range of lytic activity. According to data (Ceyssens et al., 2011), the duration of their latent period is 21-28 min, and their spectrum of lytic activity ranges from 5% to 58% when assessed against more than 100 randomly selected clinical isolates of P. aeruginosa. In both size and structure, the genomes of phages of this species are very conservative and DNA homology is at the level of 83–97%. It is believed that the genomes of phiKMV-like phages have emerged to a substantial extent as a consequence of vertical evolution. In some cases, the differences between genomes may be so minimal as to be detectable only by a detailed comparison of the nucleotide sequences (Kulakov et al., 2009).
We have observed that some phiKMV-like phages, e.g. phiNFS (Figure 1) present in commercial preparations, when plated on a lawn of P. aeruginosa strain PAO1 (which is considered to be a Standard host for P. aerugunosa phages), exhibit instability, with segregation of secondary mutants occurring as a result of adaptation to the new host. The secondary mutants are stable and show plaque morphology similar to that of phiKMV.
Figure 1. Phage phiNFS isolated from a commercial mixture and plated on a lawn of P. aeruginosa strain PAO1, showing the presence of plaques of unusual morphology. Repeated subcultures confirmed phage instability under these conditions. The scale bar is 1 cm.
Some newly revealed properties of phiKMV-like phages, described below, permit a deeper understanding of the interrelations of phiKMV-like phages with P. aeruginosa and alter the perception of these phages as promising candidates for use in phage therapy. Following a single day of incubation on lawns in Petri dishes, phiKMV-like phages produce large clear plaques with narrow halos. With continued incubation, however, an increasing number of bacterial colonies can be seen. In a plaque of phage phiKMV after three to four days of incubation, range of bacterial colonies can be seen, varying in appearance (Figure 2A). Two types of colonies isolated from such plaques produced phage after several repeated cycles of re-plating (Figure 2B). We consider this phenomenon to reflect a transition of the bacterial cells into a pseudolysogenic state. It is may be due to an infection of some survivors within the plaques that are in a specific physiological state.
Figure 2. Pseudolysogeny of P. aeruginosa PAO1 cells after phiKMV infection. The growth of phiKMV (four days of incubation) on a PAO1 lawn is shown. The different types of resistant and sensitive colonies in the zone of lysis (A). Some of the isolated clones are phiKMV-resistant and do not produce phage (B-1); they do not affect the growth of most other phage species, including phage PB1. This means that the resistance of such mutants is not associated with loss of the surface lipopolysaccharide (LPS) (Jarrell and Kropinski, 1977). Other clones with different colony phenotypes produce phage after several re-plates (B-2 and B-3). The scale bar is 1 cm
Within a few days, the sizes of phiKMV plaques increase. Thus, unlike other phage species, phiKMV is capable of ov ercoming for a period of time the conditions occurring in an a ging biofilm, which prevent the growth of phages of other species (Figure 3). Growth of phages of several other phage species produces halos of different sizes and appearances. The halo of phiKMV phage is narrow in comparison with the halos of other phages. Halos that are especially large and mucous are formed around phiKZ-like phages (phiKZ, Lin68 and phi10/2). The formation of halos by P. aeruginosa phages is caused by the activity of depolymerases that act on polysaccharides. These enzymes, produced by a range of phage species, are structural components of the tail (Castillo and Bartell, 1974; 1976) and participate in the adsorption of phage particles to lipopolysaccharides (LPS) in the bacterial cell wall.
Figure 3. The growth of phage KMV and phages of other species from our collection after four days of incubation on a P. aeruginosa PAO1 lawn. The different interactions of the halo-producing enzymes of the various phages can be seen. They confirm the differences in their specificities, which is important in selecting the composition of phage mixtures to achieve a maximal biofilm-disrupting effect. The scale bar is 1 cm.
It is useful to compare the differences in growth patterns of phages belonging to different species in areas of confluent growth, so as to estimate the potential compatibility of the phages concerned, with a view to designing phage mixtures that can exert an optimal lytic effect in biofilm without the drawback of mutual growth inhibition. Figure 4 demonstrates the incompatibility of some phages.
Figure 4. Mutual incompatibilities of phages, on examples shown between phiKMV (in center) and PB1 (upper left), phiKZ (upper right), phi11 (lower left), PMG1 (lower right). The scale bar is 1 cm.
Currently, it is thought that phage enzymes that contribute to halo formation during the growth of phages on lawns of non-mucoid P. aeruginosa strains can be assumed to be enzymes that are specific for the disruption of LPS in the external part of the bacterial cell wall. On the other hand, Hanlon and co-authors (Hanlon et al., 2001) have shown that although purified temperate bacteriophage F116 is capable of migrating through P. aeruginosa biofilms and that this may be facilitated by a reduction in alginate viscosity, the source of the enzyme may in fact be the bacterial host itself.
The authors of one study (Glonti et al., 2010) have suggested that a member of the phiKMV-like phages, PT6, produces an enzyme that depolymerizes the alginic acid capsule. The genome of PT6 is as yet unse quenced and unannotated, but in the annotated genomes of other phiKMV-like phages there are no genes that encode an alginate lyase. It will of interest to continue this line of enquiry, however. The possibility cannot be excluded that phage PT6 infection stimulates the production of a new bacterial alginase that is capable of destroying acetylated alginate. Further studies are clearly necessary to clarify this issue, given that the formation of acetylated alginate is the main precondition for the production of a stable biofilm of P. aeruginosa. In order to prove the hypothesis that a phage produces an alginate lyase, it is necessary to use bacterial strains that generate an excess of alginate. In experiments with strain P. aeruginosa Pse163, which produces such an excess of alginate, we could not detect evidence of anti-alginate activity in any of several phages that were tested. Thus, following infection of the stable alginate-producing strain Pse163 with phiKMV, there were no visible halos around the spot of phage after three days of incubation (Figure 5) (see also in Krylov and Shaburova, 2012).
Figure 5. Comparison of the growth of several phages after overnight incubation and after three days of incubation on lawns of P. aeruginosa strains PAO1 (A, B) and PSE163 (C, D). The disposition of the phages on the bacterial lawns is given in the first panel: 1-SL2; 2-Lin68; 3-EL; 4-φC17; 5-D3112; 6-B3; 7-phiKMV; 8-E79; 9-phiMK. The spot of phiKMV is greatly increased on lawn PAO1 (A, B) with increased incubation period. The sizes of the spots for the other phages remain unchanged. On the PSE163 lawn (C, D), none of the spots for the phages, including KMV, have increased in size. Thus, phage phiKMV, an common with the other phages, is incapable of digesting the alginate of the permanently alginate-producing strain, PSE163. The scale bar is 1 cm.
We have two contradictory views on the value of phiKMV for phage therapy. Although the ability of phage phiKMV to lyse bacteria in aging biofilms can be considered as a highly useful feature, it cannot be excluded that there is an intrinsic relationship with pseudolysogeny (as it is seen on Figure 2) and, as a result, with the survival of infected bacteria in the microbiota of the infection site. Perhaps this special feature of phiKMV-like phages has been the cause of the failure to use them for the eradication of P. aeruginosa in a mouse model of cystic fibrosis (Henry et al., 2013).
Seemingly, should it prove impossible to select mutants of phiKMV-like phages without pseudolysogenic effects, it will be advisable to abandon the use of phiKMV-like phages as therapeutic agents.
PB1-like phages are a usual component of phage therapeutic commercial mixtures. This species is considered to be one of the most promising for application in phage therapy. One of its major advantages is the ability to generate mutants with extended lytic activity (Pleteneva et al., 2008 and 2009; Ceyssens et al., 2009), an absence of pseudolysogenic effects (complete lysis of infected bacteria), and low frequencies of phage-resistant bacterial mutants. Phages of this species are adsorbed onto LPS and produce a halo around the plaque as a result of the activities of LPS-destroying enzymes that are synthesized by bacteria in the course of phage infection.
Phages of species PaP3/LUZ24-like phages are permanent components of commercial mixtures; but in some of them, we found unusual properties. Their possible influence on the behavior of the phage during the process of phage therapy is not clear. Further comparative studies are required before phages of this species can be introduced into phage therapy as components of mixtures that are well-studied and safe for extended use. The first phage of this species to be described, PaP3, was isolated as a temperate phage (Tan et al., 2007). However, no repressor gene has been found in its genome. The other phage of this species, LUZ24, shows a high degree of relatedness to PaP3 and also does not encoding repressor protein. A remarkable property of LUZ24 is the presence of an intron in its genome (Ceyssens et al., 2008). The isolation of phages of related species, infecting other pseudomonads, P. putida and P. fluorescens (Glukhov et al., 2012; Eller et al., 2014), suggests a possibility for the migration of these phages between different soil pseudomonades.
Two new phages, TL and CHU, were isolated in our laboratory from natural sources. According to the results of genome sequencing, TL is closely related to other phages of the species. But, unlike other related phages, it encodes a transposase. Phage CHU shows a positive PCR response with TL primers, and produces identical fragments after DNA digestion by endonucleases. A feature common to TL and CHU is weak growth on lawns of the Standard P. aeruginosa strain PAO1. However, they grow well and selectively on lawns of some clinical isolates of P. aeruginosa (P. aeruginosa strain 8-20) or of mutants of PAO1 (P. aeruginosa strain PAO-ELR2), showing a high level of instability (Figure 6); however, the role of transposase in phage TL instability is not evident. Introduction of the plasmid pMG53 (IncP2) into P. aeruginosa strain PAO1 restores the growth of both phages, with simultaneous loss of instability. From the point of view of their use in phage therapy, this is a desirable feature because IncP2 group plasmids frequently inhibit the growth of various phage species. In addition, in the regions of the halos formed during the growth of various other phages on lawns of P. aeruginosa strain PAO1, the lytic activity of phage TL increases significantly, thereby allowing the identification of the halo-forming phages (Pleteneva et al., 2011). CHU, on the hand, does not exhibit this behavior (Figure 7).
Figure 6. Genetic instability of plaque morphology in TL (A) and CHU (B) phages, as revealed on P. aeruginosa strain PAO-ELR2 (for TL) and clinical strain P. aeruginosa 8-20 (for CHU). The scale bar is 1 cm.
Figure 7. The growth of TL (A) and CHU (B) in the halos of some halo-forming phages on a lawn of P. aeruginosa strain PAO1. The phage TL acts as a "developer" of halos of phages phi297, E79 and F8 when grow in close proximity to their plaques. This is related to better growth in cells in the halo (Pleteneva et al., 2011). The nature of halos, produced by phages phiMK, SL2 and phiKZ is different and they do not interact with TL. In contrast, the closely related phage CHU has no such activity. The scale bar is 1 cm.
It is unclear why phage PaP3, having no repressor in its genome, has been isolated from the bacterial strain as a temperate phage. It is possible that in this case, the bacteria were in a pseudolysogenic state. Thus, only after further studies of the cause and the consequences of the growth instability of these phages will it be possible to assess their potential utility.
The group of phiKZ-like phages active on now includes three species, phiKZ, EL and Lin68, and they attract the attention of investigators because of the unusual structure of their phage particles and some specific features in their infection of bacterial cells (Krylov and Zhazykov, 1978 and 1984; Hertveldt et al., 2005; Thomas et al., 2008; Pleteneva et al., 2010; Krylov et al., 2011; Cornelissen et al., 2012; Sokolova et al., 2014). The common features for all phages in this group are: 1) identical morphology and size of the phage particle; 2) the specific packaging of DNA ("inner body" -helical coil with supercoiled DNA wound around it); 3) the absence of any enzyme recognized by genome annotation as a phage DNA polymerase; 4) the ability following infection with a high multiplicity to convert all of the bacterial cells into a pseudolysogenic condition (carrier state). Supposedly, the generality of these characteristics reflects the phylogenetic relationship of the various species within this group.
Pseudolysogeny in the case of the phiKZ-like phages displays very specific features that differentiate it from the cases of pseudolysogeny previously mentioned. Thus, in a one-step growth cycle experiment following the infection of sensitive cells with a multiplicity of infection (m.o.i.) in the range of one to five particles, these phages exhibit features of virulent phages – all infected cells were killed, with the liberation of a modest number of phage particles (Krylov and Zhazykov, 1978). At higher m.o.i. values, however, infection with phiKZ-like phages leads eventually to a special state in which bacterial cells continue to divide, leading to the formation of colonies that can grow for several days in Petri dishes. In consequence, these colonies produce huge amounts of phage particles (Krylov and Zhazykov, 1978 and 1984; 2004; Burkal'tseva et al., 2002; Pleteneva et al., 2009). This may be of particular significance, in view of the fact that phages of species phiKZ have been found in various commercial therapeutic blends. Phages of species EL and Lin68 are infrequent (we have isolated two phages, RU and CHE, which are closely related to EL, and a single phage LBG22, which is related to Lin68) (Burkal'tseva et al., 2002).
To date, all the phiKZ-like phages that have been sequenced do not encode "Standard" DNA-polymerases (Mesyanzhinov et al., 2002; Hertveldt et al., 2005). Nevertheless, a very unusual DNA polymerase activity of a new type has been found to be encoded in the genomes of phiKZ-like phages (Cornelissen et al., 2012). It is possible that under the conditions that arise following the high-multiplicity infection of cells, phage development is turned off for a period of time.
The precise mechanism for such an effect of m.o.i. is not yet clear, but it is evident that such phages, containing the phage genome in its wild-type state, can facilitate HGT (because pseudolysogenic cells can support the development of other phages, including temperate phages). Thus, pseudolysogeny is one of the reasons why phiKZlike phages in their wild-type state are not desirable components of phage mixtures.
Such unusual behavior could not have been predicted from the sequencing and annotation of the phage genomes. Furthermore, a study of phiKZ transcription (Ceyssens et al., 2014) has found one other unique feature of this phage: phiKZ does not require a functionally active bacterial transcriptional system. The mechanism of temporary lysis inhibition is not yet elucidated. It is of interest that in both the phiKZ and EL genomes there exist genes that encode proteins similar to the repressors of phages that are specific for unrelated bacterial species (Mesyanzhinov et al., 2002; Hertveldt et al., 2005). These may function as a repressor-type activity, blocking the lytic cycle and leading the infected cells into a pseudolysogenic state. Bacterial cells in such a state, being infected with phiKZ, are capable of movement, division and the production of cells sensitive to phage infection (Krylov et al., 2013). In the case of cells infected with phiKZ-like phages at high m.o.i., the pseudolysogenic state offers a biological advantage (to the phage) because, in the absence of a phage-coded DNA polymerase and given that these phages are independent of bacterial transcriptional activity, pseudolysogeny leads to a great increase in phage production. Some of the cells become temporarily resistant to phage infection and are able transport phage particles (phage as a "rider") (Krylov et al., 2013).
Unconditional evidence of true but unstable lysogeny is provided by the selection of mutants with properties similar to those of virulent mutants of temperate phages (Krylov et al., 2011). Such virulent variants kill cells that are in a pseudolysogenic condition. The use of such mutants in therapeutic mixtures instead of wild-type phiKZlike phages will prevent the possibility of HGT (Krylov et al., 2010; Pleteneva et al., 2010; Krylov et al., 2011). The dominance of mutant phage phiKZ in mixed infections with EL shows that even in choosing between related phages for phage-therapy application, it is necessary to take into attention the possibilities of mutual inhibition (Krylov et al., 2013).
Excellent evidence that phiKZ-like phages cannot be used in phage therapy in their wild-type state has been reported in a recent study (Henry et al., 2013) that showed that it was impossible to eradicate P. aeruginosa in a mouse-lung infection model.