comK prophage junction fragments as markers for Listeria monocytogenes genotypes unique to individual meat and poultry processing plants and a model for rapid niche-specific adaptation, biofilm formation, and persistence

Abstract

Different strains of Listeria monocytogenes are well known to persist in individual food processing plants and to contaminate foods for many years; however, the specific genotypic and phenotypic mechanisms responsible for persistence of these unique strains remain largely unknown. Based on sequences in comK prophage junction fragments, different strains of epidemic clones (ECs), which included ECII, ECIII, and ECV, were identified and shown to be specific to individual meat and poultry processing plants. The comK prophage-containing strains showed significantly higher cell densities after incubation at 30°C for 48 h on meat and poultry food-conditioning films than did strains lacking the comK prophage (P < 0.05). Overall, the type of strain, the type of conditioning film, and the interaction between the two were all highly significant (P < 0.001). Recombination analysis indicated that the comK prophage junction fragments in these strains had evolved due to extensive recombination. Based on the results of the present study, we propose a novel model in which the concept of defective comK prophage was replaced with the rapid adaptation island (RAI). Genes within the RAI were recharacterized as "adaptons," as these genes may allow L. monocytogenes to rapidly adapt to different food processing facilities and foods. If confirmed, the model presented would help explain Listeria's rapid niche adaptation, biofilm formation, persistence, and subsequent transmission to foods. Also, comK prophage junction fragment sequences may permit accurate tracking of persistent strains back to and within individual food processing operations and thus allow the design of more effective intervention strategies to reduce contamination and enhance food safety.

Fig. 1.

Schematic diagram of phage A118 integration into and excision out of the L. monocytogenes chromosome at comK. (A) Phage A118 with attP attachment site. Horizontal arrows indicate PCR priming sites for amplifying the fragment containing attP. (Modified from reference with permission of John Wiley & Sons.) (B) L. monocytogenes comK gene containing attB attachment site. Horizontal arrows indicate priming sites for amplifying comK with attB attachment site. (C) Lysogenized L. monocytogenes showing the locations of the forward and reverse primers (horizontal arrows) for PCR amplification of upstream and downstream comK prophage junction fragments. The location of gene locus LMOh7858_2426 in the lysogenized strain (20) is shown with an upward-pointing arrow. (D) Lysogenized L. monocytogenes showing the PCR targets for the upstream and downstream comK prophage junction fragment PCR (RBS, ribosomal binding protein; NC, noncoding region; att, attachment site). The partial gene targets are underlined.

Fig. 2.

Cluster diagrams based on upstream and downstream junction fragment sequences in L. monocytogenes isolates described in Table S1 in the supplemental material; the plant information and prophage type are labeled in the figure. (A) Upstream junction fragment cluster diagram. (B) Downstream junction fragment cluster diagram. Nodes are labeled with bootstrap values. *, plant information is not available.

Fig. 3.

(A) Epifluorescence photomicrographs showing different cell densities of L. monocytogenes on food-conditioning films. The number in the upper right corner of each picture indicates the cell density score, with 0 indicating absence of cells (turkey, no cells added) and 1 indicating very small (glass only, no food-conditioning film), 2 indicating small (hot dog with strain J1703), 3 indicating moderate (hot dog with strain OB020790), 4 indicating large (chicken with strain J1703), and 5 indicating very large (turkey with strain 08-5923) amounts of cells observed on the slides. Bars, 20 μm. (B) Epifluorescence photomicrographs showing the degradation of food-conditioning films and biofilm formation by the ECV strain (08-5923). Red arrows indicate undegraded food-conditioning films, and yellow arrows indicate biofilm formation. Bars, 40 μm.

Fig. 4.

Sequences of putative adaptons within comK prophages in epidemic clone strains of L. monocytogenes show 100% sequence identity within a processing plant over a 12-year period (plant D) but vary between processing plants (plants A and E). The putative adaptons ORFs HP1, HP2, gp15, gp13, and partial int (locus designations LMOh7858_2410, -2411, -2421, -2426, and -2475, respectively) are present in sequenced genomes of 1/2a and 4b serotypes of L. monocytogenes that contain the comK prophage and show 100% sequence identity within 1988 and 2000 ECIII isolates. The putative adaptons ORFs gp27 (LMOf6854_2338) and HP3 (LMOf6854_2375) (arrows shaded gray) indicate those comK prophage genes that are unique to serotype 1/2a strains but are not present in the ECII serotype 4b strain. comK′, N-terminal comK fragment; HP1, hypothetical protein 1; HP2, hypothetical protein 2; gp27, phage gp27 protein; gp15, phage gp15 protein; gp13, major tail protein; HP3, hypothetical protein 3; int, integrase; ′comK, C-terminal comK fragment. Arrows point to the corresponding positions of these loci in the comK prophage in ECII, ECIII, and ECV strains. Blocks (adaptons) with different shading indicate nonidentical sequences, and black blocks indicate 100% sequence identity within ECIII isolates.

Fig. 5.

Comparison of comK prophage genes in 1/2a and 4b serotypes of Listeria monocytogenes with sequenced bacteriophage genomes. Regions that correspond to different bacteriophages are shaded.

Fig. 6.

Proposed model for rapid niche-specific adaptation and persistence of L. monocytogenes. The cycle starts at the top with spontaneous induction of a rapid adaptation island (RAI) in the donor cell, followed by RAI phage formation and transduction of donor RAI into a recipient cell, which also contains a defective RAI or infective lysogenized phage integrated into its chromosome. Recombination between donor RAI and recipient RAI/comK prophage generates numerous RAI recombinants. Natural selection then acts on RAI recombinants to yield unique persistent prophage types that are adapted to individual processing plants or multiple plants manufacturing the same type of food product and thus produce the same type of food-conditioning film.