Characterization of a Novel Group of Listeria Phages That Target Serotype 4b Listeria monocytogenes

Abstract

Listeria monocytogenes serotype 4b strains are the most prevalent clinical isolates and are widely found in food processing environments. Bacteriophages are natural viral predators of bacteria and are a promising biocontrol agent for L. monocytogenes. The aims of this study were to characterize phages that specifically infect serotype 4b strains and to assess their ability to inhibit the growth of serotype 4b strains. Out of 120 wild Listeria phages, nine phages were selected based on their strong lytic activity against the model serotype 4b strain F2365. These nine phages can be divided into two groups based on their morphological characteristics and host range. Comparison to previously characterized phage genomes revealed one of these groups qualifies to be defined as a novel species. Phages LP-020, LP-027, and LP-094 were selected as representatives of these two groups of phages for further characterization through one-step growth curve and inhibition of serotype 4b L. monocytogenes experiments. Listeria phages that target serotype 4b showed an inhibitory effect on the growth of F2365 and other serotype 4 strains and may be useful for biocontrol of L.monocytogenes in food processing environments.

Keywords: Listeria monocytogenes; bacteriophage; phage; serotype 4b.

Figure 1

Transmission electron microscopy images of Listeria phages representing two morphologies: (A) LP-020 and (B) LP-094, characterized by an icosahedral capsid and a flexible, non-contractile tail; (C) LP-027, characterized by an icosahedral capsid and a long, flexible, non-contractile tail. Phages were stained with 1% phosphotungstic acid (pH &) and imaged at a final magnification of ×69,700–83,600. Images were analyzed using FIJI 3 (v2.0.0-rc-69/1.52p).

Figure 2

Linear BLASTn comparisons of representative P35-like Listeria phages. Genes are represented by arrows and are colored based on putative function (see key at bottom). The shaded region between genomes represents nucleotide similarity, with the darker gray representing higher similarity and lighter gray indicating lower similarity (see scale at bottom right).

Figure 3

Host range analysis of Listeria phages against a panel of Listeria monocytogenes strains that represent different serotypes. Panel (A) represents efficiency of plaquing (EOP) results where values represent the log transformed efficiencies of plaquing of each phage against each bacterial strain compared to the phage propagation host strain. Panel (B) represents efficiency of activity (EOA) results where values represent the greatest dilution factor where phage activity was observed against each strain relative to the phage propagation host strain. Values are the mean of data from three biological replicates.

Figure 4

One-step growth curve of Listeria monocytogenes F2365 treated with (A) LP-20, (B) LP-0-27, or (C) LP-094 at a MOI = 0.1 at 25 °C. Filled triangles represent the phage titer in chloroform treated samples and unfilled triangles represent the phage titer in untreated samples. Data are mean values of three biological replicates and error bars represent standard error.

Figure 4

One-step growth curve of Listeria monocytogenes F2365 treated with (A) LP-20, (B) LP-0-27, or (C) LP-094 at a MOI = 0.1 at 25 °C. Filled triangles represent the phage titer in chloroform treated samples and unfilled triangles represent the phage titer in untreated samples. Data are mean values of three biological replicates and error bars represent standard error.

Figure 5

Inhibition growth curve of Listeria monocytogenes F2365 treated with (A) LP-020, (B) LP-027 or (C) LP-094 at different MOIs. Unfilled circles represent SM buffer control, unfilled triangles represent samples treated at an MOI of 0.1, unfilled squares represent samples treated at an MOI of 1, and diamonds represent samples treated at a MOI of 10. Data are mean values of three biological replicates and error bars represent the standard error.

Figure 6

Inhibition growth curve of a cocktail of Listeria monocytogenes serotype 4 strains (F2365 (4b), FSL J1-208 (4a), FSL F2-695 (4a), FSL F2-501 (4b), FSL J2-071 (4c), FSL W1-110 (4b), and FSL J1-148 (4b)) treated with (A) LP-020 and (B) LP-094 at different MOIs. Unfilled circles represent SM buffer control, unfilled triangles represent samples treated at a MOI of 0.1, unfilled squares represent samples treated at a MOI of 1, and diamonds represent samples treated at a MOI of 10. Data are mean values of three biological replicates and error bars represent the standard error.