Bacteriophages isolated from activated sludge processes and their polyvalency

115

126

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...

Section: Discussionsupporting

confidence: 84%

Isolation of Polyvalent Bacteriophages by Sequential Multiple-Host Approaches

Mathieu

et al. 2016

115

126

“…Interestingly, phages isolated from activated sludge have been reported to display unusually broad host ranges, with some being reported to lyse both Gram-negative and Gram-positive bacteria (20,21,38). To date, none of these polyvalent phages have had their genomes completely sequenced.…”

Section: Resultsmentioning

confidence: 99%

Characterization of the Genome of the Polyvalent Lytic Bacteriophage GTE2, Which Has Potential for Biocontrol of Gordonia-, Rhodococcus-, and Nocardia-Stabilized Foams in Activated Sludge Plants

Petrovski

Seviour

Tillett

2011

Hydrophobic Actinobacteria are commonly associated with the stabilization of foams in activated sludge systems. One possible attractive approach to control these foam-stabilizing organisms is the use of specific bacteriophages. We describe the genome characterization of a novel polyvalent DNA phage, GTE2, isolated from activated sludge. This phage is lytic for Gordonia terrae, Rhodococcus globerulus, Rhodococcus erythropolis, Rhodococcus erythropolis, Nocardia otitidiscaviarum, and Nocardia brasiliensis. Phage GTE2 belongs to the family Siphoviridae, possessing a characteristic icosahedral head encapsulating a double-stranded DNA linear genome (45,530 bp) having 10-bp 3-protruding cohesive ends. The genome sequence is 98% unique at the DNA level and contains 57 putative genes. The genome can be divided into two components, where the first is modular and encodes phage structural proteins and lysis genes. The second is not modular, and the genes harbored there are involved in DNA replication, repair, and metabolism. Some have no known function. GTE2 shows promising results in controlling stable foam production by its host bacteria under laboratory conditions, suggesting that it may prove useful in the field as a biocontrol agent.

“…Therefore, to increase the phage infection range and inhibition efficiency, phage cocktails containing different phages have been developed and used for inhibition of pathogens such as Salmonella and E. coli O157:H7 (52,67,69). Although a few broad-host-range phages have been reported (6,31,34), the bacteriophage AR1 is the only phage reported to date that can inhibit both Salmonella and E. coli O157:H7 (24,42). AR1 is reported to infect many serotypes of E. coli and S. enterica serovar Enteritidis and serovar Choleraesuis, but not serovar Typhimurium.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, treatment of vegetables, such as cantaloupe and lettuce, with a phage cocktail to control E. coli O157:H7 showed a significant reduction in the bacteria (64). Although broad-host-range phages infecting several different genera of bacteria were previously reported (6,31,34) and many phages have been developed and evaluated for control of food-borne Salmonella and E. coli O157:H7, bacteriophages that simultaneously control these two pathogens have been rarely reported (24).…”

mentioning

confidence: 99%

Characterization and Comparative Genomic Analysis of a Novel Bacteriophage, SFP10, Simultaneously Inhibiting both Salmonella enterica and Escherichia coli O157:H7

Park

Lee

Shin

et al. 2012

166

130

Salmonella enterica and Escherichia coli O157:H7 are major food-borne pathogens causing serious illness. Phage SFP10, which revealed effective infection of both S. enterica and E. coli O157:H7, was isolated and characterized. SFP10 contains a 158-kb double-stranded DNA genome belonging to the Vi01 phage-like family Myoviridae. In vitro adsorption assays showed that the adsorption constant rates to both Salmonella enterica serovar Typhimurium and E. coli O157:H7 were 2.50 ؋ 10 ؊8 ml/min and 1.91 ؋ 10 ؊8 ml/min, respectively. One-step growth analysis revealed that SFP10 has a shorter latent period (25 min) and a larger burst size (>200 PFU) than ordinary Myoviridae phages, suggesting effective host infection and lytic activity. However, differential development of resistance to SFP10 in S. Typhimurium and E. coli O157:H7 was observed; bacteriophage-insensitive mutant (BIM) frequencies of 1.19 ؋ 10 ؊2 CFU/ml for S. Typhimurium and 4.58 ؋ 10 ؊5 CFU/ml for E. coli O157:H7 were found, indicating that SFP10 should be active and stable for control of E. coli O157:H7 with minimal emergence of SFP10-resistant pathogens but may not be for S. Typhimurium. Specific mutation of rfaL in S. Typhimurium and E. coli O157:H7 revealed the O antigen as an SFP10 receptor for both bacteria. Genome sequence analysis of SFP10 and its comparative analysis with homologous Salmonella Vi01 and Shigella phiSboM-AG3 phages revealed that their tail fiber and tail spike genes share low sequence identity, implying that the genes are major host specificity determinants. This is the first report identifying specific infection and inhibition of Salmonella Typhimurium and E. coli O157:H7 by a single bacteriophage.