Isolation of Polyvalent Bacteriophages by Sequential Multiple-Host Approaches

Abstract

Many studies on phage biology are based on isolation methods that may inadvertently select for narrow-host-range phages. Consequently, broad-host-range phages, whose ecological significance is largely unexplored, are consistently overlooked. To enhance research on such polyvalent phages, we developed two sequential multihost isolation methods and tested both culturedependent and culture-independent phage libraries for broad infectivity. Lytic phages isolated from activated sludge were capable of interspecies or even interorder infectivity without a significant reduction in the efficiency of plating (0.45 to 1.15). Two polyvalent phages (PX1 of the Podoviridae family and PEf1 of the Siphoviridae family) were characterized in terms of adsorption rate (3.54 ؋ 10 ؊10 to 8.53 ؋ 10 ؊10 ml/min), latent time (40 to 55 min), and burst size (45 to 99 PFU/cell), using different hosts. These phages were enriched with a nonpathogenic host (Pseudomonas putida F1 or Escherichia coli K-12) and subsequently used to infect model problematic bacteria. By using a multiplicity of infection of 10 in bacterial challenge tests, >60% lethality was observed for Pseudomonas aeruginosa relative to uninfected controls. The corresponding lethality for Pseudomonas syringae was ϳ50%. Overall, this work suggests that polyvalent phages may be readily isolated from the environment by using different sequential hosts, and this approach should facilitate the study of their ecological significance as well as enable novel applications.T he total bacteriophage (phage) population on Earth is estimated at 10 31 or more, making phages by far the most abundant biological entities on the planet (1, 2). As such, phages exert a significant influence over global biogeochemical cycles (3, 4) and are important drivers of bacterial diversity (5). Considering their ecological importance and value as a potential genetic resource, increasing our fundamental understanding of phage biology may facilitate the development of novel applications. However, research on phage diversity and ecology may be inadvertently limited by the use of biased isolation techniques that preferentially select for narrow-host-range phages (6, 7), while-broad hostrange phages are consistently overlooked.The classic approach to isolate and study phages is typically performed with high-density, nutrient-rich batch monocultures grown under planktonic conditions and most often results in the isolation of narrow-host-range phages (8)(9)(10).Previous studies have demonstrated the existence of polyvalent phages, among which the temperate phages P1 and Mu are the most studied (11, 12). However, their broad-host-range properties were discovered accidently, and few studies have explored methods for isolating and enriching polyvalent phages. Jensen et al. developed a multiple-host enrichment method to identify phages capable of interclass infectivity (13), and Bielke et al. used a sequential isolation method to isolate phages with intergenus infectivity (14). However, these phage isolation methods have...

Characterization of two polyvalent phages infecting Enterobacteriaceae

“…Other studies have shown that sequential or simultaneous addition of multiple hosts should be used to isolate phages with broader host range. However with these methods the host range of these phages seems limited to the bacterial species used during their isolation1646. The current study indicates that it is possible to isolate such phages using a single host.…”

Engineering Phage Host-Range and Suppressing Bacterial Resistance Through Phage Tail Fiber Mutagenesis

“…However, the latter strategy requires prior knowledge of related genetic elements with the desired host-range and the capacity to engineer those elements into a functional phage, which often results in laborious ad hoc trial and error periods (Ando et al, 2015; Chen et al, 2017; Gebhart et al, 2017; Hawkins et al, 2008; Heilpern and Waldor, 2003; Lin et al, 2012; Nguyen et al, 2012; Scholl et al, 2009; Yoichi et al, 2005). Harnessing natural evolution, on the other hand, requires elaborate co-culture and selection strategies that balance the need to have the phage population grow and mutate while also providing an alternative host for phage mutants to reproduce on (Qimron et al, 2006; Ross et al, 2016; Yu et al, 2015). A recent paper by Yosef and colleagues combined the approaches of rational host-range engineering with evolution.…”

MyoviridaePhagePDXKills EnteroaggregativeEscherichia coliwithout Human Microbiome Dysbiosis

“…Because of the disparate location of these EAEC isolates, and that PDX formed plaques on caseassociated EPEC strains from children in the Seattle area, the results suggested that the sequential method (21) for isolating phage that recognized more core bacterial antigens was successful.…”

“…Phage were sequentially isolated from E.coli strain MC4100, EPEC strain LRT9, and EAEC clinical isolate EN1E-0227 using a sequential isolation technique to enrich for core antigen recognition (21). Several clearly defined plaques from the final overlay were picked using a sterile inoculating needle and suspended in microcentrifuge tubes containing SM buffer.…”