Isolation and Characterization of phiLLS, a Novel Phage with Potential Biocontrol Agent against Multidrug-Resistant Escherichia coli

Abstract

Foodborne diseases are a serious and growing problem, and the incidence and prevalence of antimicrobial resistance among foodborne pathogens is reported to have increased. The emergence of antibiotic-resistant bacterial strains demands novel strategies to counteract this epidemic. In this regard, lytic bacteriophages have reemerged as an alternative for the control of pathogenic bacteria. However, the effective use of phages relies on appropriate biological and genomic characterization. In this study, we present the isolation and characterization of a novel bacteriophage named phiLLS, which has shown strong lytic activity against generic and multidrug-resistant Escherichia coli strains. Transmission electron microscopy of phiLLS morphology revealed that it belongs to the Siphoviridae family. Furthermore, this phage exhibited a relatively large burst size of 176 plaque-forming units per infected cell. Phage phiLLS significantly reduced the growth of E. coli under laboratory conditions. Analyses of restriction profiles showed the presence of submolar fragments, confirming that phiLLS is a pac-type phage. Phylogenetic analysis based on the amino acid sequence of large terminase subunits confirmed that this phage uses a headful packaging strategy to package their genome. Genomic sequencing and bioinformatic analysis showed that phiLLS is a novel bacteriophage that is most closely related to T5-like phages. In silico analysis indicated that the phiLLS genome consists of 107,263 bp (39.0 % GC content) encoding 160 putative ORFs, 16 tRNAs, several potential promoters and transcriptional terminators. Genome analysis suggests that the phage phiLLS is strictly lytic without carrying genes associated with virulence factors and/or potential immunoreactive allergen proteins. The bacteriophage isolated in this study has shown promising results in the biocontrol of bacterial growth under in vitro conditions, suggesting that it may prove useful as an alternative agent for the control of foodborne pathogens. However, further oral toxicity testing is needed to ensure the safety of phage use.

Keywords: bacteriophage phiLLS; biotechnological applications; genome sequence; in silico.

Figure 1

Transmission electron microscopy images of phiLLS negatively stained using 2% uranyl acetate. Negatively stained electron micrographs of phiLLS virions showing the typical morphology of phages within the family Siphoviridae. (A) Broad view of the phage particles. (B) High magnification of a single phage particle.

Figure 2

One-step growth curve of phage phiLLS. Shown are the pfu per infected cell in the cultures at different time points. Each data point represent mean from three independent experiments, and the error bars indicate standard deviations. (A) The latent period is 15 min and (B) burst size was estimated to be 176 PFU per one infected cell.

Figure 3

Bacterial challenge test of phage phiLLS with E. coli O157:H7 CECT 4076. E. coli log-phase culture was infected with phage phiLLS at 100 (Line black), 1.0 (Line green), and 0.1 (Line blue), when the OD at 600 nm was 1.0. The growth curve of bacterial was used as a control (Line red). The graphs show viable-cell counts of samples collected every 30 min. The error bars indicate standard deviations from the results of triplicate experiments.

Figure 4

Neighbor-joining phylogenetic tree of terminase large subunit of phiLLS and their comparison to other coliphages with known packaging mechanisms. Bootstrap analysis was performed with 1,000 repetitions. The terminase large subunits were compared using the ClustalW in Geneious program version R9. Colored boxes indicate the phages grouped into similar cluster that share same packaging strategy.

Figure 5

Enzymatic analysis of phiLLS genomic DNA. A restriction map of the genomic DNA of phage phiLLS was constructed using the restriction endonucleases BamHI, HindIII, and the products were separated by agarose gel electrophoresis. Phage DNA digested with BamHI without heat treatment (Lane 1), phage DNA digested with BamHI with heat treatment (Lane 2). Genome of bacteriophage Lambda digested with HindIII was used as control, the size of the bands is indicated on the right and red arrows indicate the fragment containing the cos sequence (Lane 3). Phage DNA digested with HindIII without heat treatment (Lane 4), phage DNA digested with HindIII with heat treatment (Lane 5).

Figure 6

Map of the genome organization of bacteriophage phiLLS (A) and Comparative genomic maps of phages phiLLS, vB_EcoS_FFH1 and bV_EcoS_AKFV33 using the Mauve progressive alignments to determine conserved sequence regions (B). (A) The predicted ORFs are indicated as arrows, the orientation of which shows the direction of transcription. Different colors identify predicted molecular function for ORF. DNA regulation module (Green arrows), packaging module (yellow arrows), phage structural proteins (blue arrows), host lysis proteins (red arrows), hypothetical proteins (black arrows), and accessory genes (orange arrows). Other genetic elements are shown, including putative promoters (pink), and tRNAs (dark blue). (B) Boxes with identical colors represent local colinear blocks (LCB), indicating homologous genomic regions shared by phage chromosomes without sequence rearrangements.

Figure 7

Cumulative GC skew analysis of the phage genome sequence. The global minimum and maximum are displayed in the cumulative graph were calculated by using a window size of 1,000 bp and a step size of 100 bp. The GC-skew and the cumulative GC-skew are represented by blue and red lines, respectively. The minimum and maximum of a GC-skew can be used to predict the origin of replication (27179 nt) and the terminus location (103791 nt).

Figure 8

Comparison of codon usage and tRNAs between phiLLS and host. (A) Rose plot show the possible association between tRNAs and codon usage in phage and their host. The frequency scale is represented at the center of the rose plot. (B) Ten tRNAs present in phage genome tend to correspond to codons that are highly used by the phage genes, while rare in the host genome.