New Bacteriophages with Podoviridal Morphotypes Active against Yersinia pestis: Characterization and Application Potential

Abstract

Phages of highly pathogenic bacteria represent an area of growing interest for bacterial detection and identification and subspecies typing, as well as for phage therapy and environmental decontamination. Eight new phages-YpEc56, YpEc56D, YpEc57, YpEe58, YpEc1, YpEc2, YpEc11, and YpYeO9-expressing lytic activity towards Yersinia pestis revealed a virion morphology consistent with the Podoviridae morphotype. These phages lyse all 68 strains from 2 different sets of Y. pestis isolates, thus limiting their potential application for subtyping of Y. pestis strains but making them rather promising in terms of infection control. Two phages-YpYeO9 and YpEc11-were selected for detailed studies based on their source of isolation and lytic cross activity towards other Enterobacteriaceae. The full genome sequencing demonstrated the virulent nature of new phages. Phage YpYeO9 was identified as a member of the Teseptimavirus genus and YpEc11 was identified as a member of the Helsettvirus genus, thereby representing new species. A bacterial challenge assay in liquid microcosm with a YpYeO9/YpEc11 phage mixture showed elimination of Y. pestis EV76 during 4 h at a P/B ratio of 1000:1. These results, in combination with high lysis stability results of phages in liquid culture, the low frequency of formation of phage resistant mutants, and their viability under different physical-chemical factors indicate their potential for their practical use as an antibacterial mean.

Keywords: bacteriophage; phage genome; phage therapy; phage typing; plague.

Figure 1

Virion morphology of phage active against Y. pestis: (a) YpEc1; (b) YpYeO9; (c) YpEc56D; (d) YpEc58; (e) YpEc57; (f) YpEc2; (g) YpEc11; and (h) YpEc56. TEM JEM 100SX (JEOL), instrumental magnification × 40,000.

Figure 2

Genome map of phage YpYeO9. Gene functions are color-coded (orange: DNA and RNA metabolism; green: tail; blue: DNA packaging and head; light blue: head to tail; fuchsia: lysis; red: auxiliary metabolic and host takeover genes; gray: hypothetical protein).

Figure 3

Intergenomic similarities heat map constructed by VIRIDIC showing the average nucleotide identity between the following phages: YpYeO9, YpEc11 phage Berlin (NC_008694.1), Yepe2 (NC_011038.1), phiA1122 (AY247822.1), T7 (NC_001604.1), T3 (NC_047864.1), fPS-59 (NC_047935.1), and YpP-R (JQ965701.1). Indeed, they are completely different to L-413C (NC_004745.1).

Figure 4

Genome map of phage YpEc11. Gene functions are color-coded (orange: DNA and RNA metabolism; green: tail; blue: DNA packaging and head; light blue: head to tail; fuchsia: lysis; red: auxiliary metabolic and host takeover genes; gray: hypothetical protein).

Figure 5

Annotation and comparison of the phages’ genomes. Gene functions are color-coded (orange: DNA and RNA metabolism; green: tail; blue: DNA packaging and head; light blue: head to tail; fuchsia: lysis; red: auxiliary metabolic and host takeover genes; gray: hypothetical protein).

Figure 6

The viral proteomic tree constructed with the VIP tree.

Figure 7

The viability of phage active against Y. pestis in BHI broth, SM buffer, and PSB solution: (a) phage YpYeO9 and (b) phage YpEc11. The results are the averages of three parallel experiments with geometric SD shown as the vertical lines.

Figure 8

Phage–host interaction: (a) adsorption curve of YpEc11, YpYeO9, YpEc11*E, and YpYeO9*Ye phage and (b) single-step growth curve of YpEc11 and YpYeO9 phage. The results are the mean values of three independent tests. Standard deviations (SD) are indicated.

Figure 9

Survival of phage active against Y. pestis in acidic and alkaline environments during different exposure times: (a) 30 min exposure and (b) 60 min exposure. The results are the averages of three parallel experiments with geometric SD shown as the vertical lines.

Figure 10

The survival of phage active against Y. pestis at different temperatures at (a) 10 min exposure and (b) 30 min exposure. The results are the averages of three parallel experiments with geometric SD shown as the vertical lines.

Figure 11

The survival of phage active against Y. pestis in 1% sodium hypochlorite solution. The results are the averages of three parallel experiments with geometric SD shown as the vertical lines.

Figure 12

Antibacterial efficacy of phage active against Y. pestis in liquid microcosm: (a) phage/bacteria 100:1 and (b) phage/bacteria 1000:1. The results are the averages of three parallel experiments with geometric SD shown as the vertical lines.