Combination of genetically diverse Pseudomonas phages enhances the cocktail efficiency against bacteria

Abstract

Phage treatment has been used as an alternative to antibiotics since the early 1900s. However, bacteria may acquire phage resistance quickly, limiting the use of phage treatment. The combination of genetically diverse phages displaying distinct replication machinery in phage cocktails has therefore become a novel strategy to improve therapeutic outcomes. Here, we isolated and studied lytic phages (SPA01 and SPA05) that infect a wide range of clinical Pseudomonas aeruginosa isolates. These relatively small myophages have around 93 kbp genomes with no undesirable genes, have a 30-min latent period, and reproduce a relatively high number of progenies, ranging from 218 to 240 PFU per infected cell. Even though both phages lyse their hosts within 4 h, phage-resistant bacteria emerge during the treatment. Considering SPA01-resistant bacteria cross-resist phage SPA05 and vice versa, combining SPA01 and SPA05 for a cocktail would be ineffective. According to the decreased adsorption rate of the phages in the resistant isolates, one of the anti-phage mechanisms may occur through modification of phage receptors on the target cells. All resistant isolates, however, are susceptible to nucleus-forming jumbophages (PhiKZ and PhiPA3), which are genetically distinct from phages SPA01 and SPA05, suggesting that the jumbophages recognize a different receptor during phage entry. The combination of these phages with the jumbophage PhiKZ outperforms other tested combinations in terms of bactericidal activity and effectively suppresses the emergence of phage resistance. This finding reveals the effectiveness of the diverse phage-composed cocktail for reducing bacterial growth and prolonging the evolution of phage resistance.

Nucleus-forming jumbophage PhiKZ therapeutically outcompetes non-nucleus-forming jumbophage Callisto

“…To observe the progress of the replication cycle of the phage Callisto in comparison to the well-studied chimalliviruses PhiKZ, we performed a single-cell infection assay and observed DAPI-staining DNA to follow the phage reproduction process in the host cells. 41 , 52 The results showed that, during the phage infections, the infected Pseudomonas cells had their morphology altered throughout the time, which might be a result of the phage infections compared to the uninfected cell control ( Figures 3 A, 3C, and 3D). During infection with phage PhiKZ ( Figure 3 A; PhiKZ), the phage assembled the phage nucleus localized close to the midcell of the host ( Figure 3 A; PhiKZ, green arrows).…”

“… 64 For example, utilizing genetically diverse phages that exhibit distinct mechanisms of pre-killing (MOK) is shown to be a superior approach compared to employing closely related phages or single phage alone. 41 In particular, our jumbophages Callisto and PhiKZ, which are proven here to display different MOKs, would possibly be potential candidates for an effective phage combination for further investigation of their use in clinical settings. It is worth noting that some jumbophages might have drawbacks for application due to their nature: a long latent period and small burst size, 27 , 28 and these factors should definitely be taken into account when selecting jumbophage candidates for therapy.…”

“…To further explore the potential of phage Callisto for medical and biotechnological applications, we tested the host spectrum, investigated the efficiency of plating (EOP) of the phage against lab strains and clinical strains of Pseudomonas spp. 41 , 42 and examined the presence of unwanted genes in the phage genome associated with bacterial virulence, antibiotic resistance, and the lysogenic life cycle that are disadvantageous for phage therapy. The results revealed that phage Callisto had a relatively broad host range compared to phage PhiKZ, 41 as indicated by its potential to form a clear zone in approximately half of the Pseudomonas spp.…”

“… 41 , 42 and examined the presence of unwanted genes in the phage genome associated with bacterial virulence, antibiotic resistance, and the lysogenic life cycle that are disadvantageous for phage therapy. The results revealed that phage Callisto had a relatively broad host range compared to phage PhiKZ, 41 as indicated by its potential to form a clear zone in approximately half of the Pseudomonas spp. tested (11/23) ( Table 1 ).…”

Bacteriophages as a potential substitute for antibiotics: A comprehensive review

“…66 SPA01 and SPA05 phages were used to treat infection in vitro caused by P. aeruginosa, which showed phage resistance due to the prevention of phage adsorption and phage-host interaction, possibly via receptor modification by the host immune system or genetic modification. 67 In myophage, T4 spike protein gp5 is a lysozyme responsible for hydrolyzing glycosidic bonds in peptidoglycan, forming stable complexes along with the help of gp27 protein. 68 Phage encounters a restriction-modification (R-E) system which allows restriction endonuclease to cleave foreign genome.…”