Investigation of Salmonella Phage–Bacteria Infection Profiles: Network Structure Reveals a Gradient of Target-Range from Generalist to Specialist Phage Clones in Nested Subsets


Bacteriophages that lyse Salmonella enterica are potential tools to target and control Salmonella infections. Investigating the host range of Salmonella phages is a key to understand their impact on bacterial ecology, coevolution and inform their use in intervention strategies. Virus–host infection networks have been used to characterize the “predator–prey” interactions between phages and bacteria and provide insights into host range and specificity. Here, we characterize the target-range and infection profiles of 13 Salmonella phage clones against a diverse set of 141 Salmonella strains. The environmental source and taxonomy contributed to the observed infection profiles, and genetically proximal phages shared similar infection profiles. Using in vitro infection data, we analyzed the structure of the Salmonella phage–bacteria infection network. The network has a non-random nested organization and weak modularity suggesting a gradient of target-range from generalist to specialist species with nested subsets, which are also observed within and across the different phage infection profile groups. Our results have implications for our understanding of the coevolutionary mechanisms shaping the ecological interactions between Salmonella phages and their bacterial hosts and can inform strategies for targeting Salmonella enterica with specific phage preparations.

1. Introduction

Salmonella enterica serovars Enteritidis and Typhimurium are major foodborne pathogens of worldwide concern, which often cause severe diarrheal diseases sometimes with fatal outcomes. According to the WHO report, 550 million people are infected annually, including 220 million children under the age of five [1]. The majority of these cases of domestically acquired salmonellosis are caused by various Salmonella serovars transmitted through the food chain (The US Centers for Disease Control and Prevention) [2]. Increasing antibiotic-resistance of this bacterium is aggravating the epidemic situation [3]. Due to the increasing problem of antibiotic resistance, the development of new strategies to sustainably control food-borne pathogens is urgently needed. Bacteriophages (phages) are potential alternative tools to target Salmonella infection. In post-Soviet Union countries, phage preparations have a long history of application for treatment and prophylaxis against dysenterial diseases, such as shigellosis, escherichiosis, and salmonellosis. These preparations have been successfully used for prophylaxis of salmonellosis among civilian population as well as in Red Army units [4,5,6]. In Western countries phage applications are used for biocontrol in foods [3]. Better understanding of phage–bacteria interactions will facilitate the development of a more rational approach to select appropriate phages for therapy, prophylaxis or biocontrol of Salmonella infections. Therefore, investigating the host range of Salmonella phages is a key to understand their impact on bacterial ecology, coevolution and inform intervention strategies.
One way to study a phage’s host ranges and ecological interactions is through the investigation of phage–bacteria infection networks [7,8,9] (PBINs). Essentially, these networks (or graphs) represent predator–prey interactions between phages and bacteria, respectively. These ecological relationships are graphed as a bipartite network where edges connect nodes from two different subsets, i.e., phages and bacteria. In such networks, an interaction between a single bacterium and a phage could have an impact on other members of the network too. Studying the structure of PBINs essentially addresses the question of “who-kills-whom” and provides important information regarding the ecological, biological and coevolutionary mechanisms underlying the network’s structure [10,11,12,13,14]. For example, large variation in host range species suggests that some phages have evolved to infect many hosts (generalists with wide host ranges), while others have evolved to infect a few select hosts (specialists with narrow host ranges). Equally, large variation in susceptibility host species suggests that the network consists of hosts with generalist and specialist resistance repertoires. At an evolutionary scale, large variation in host and susceptibility range sizes could indicate that particular modes of coevolution are occurring [10,15].
In this study, we investigate the in vitro infection profiles of different Salmonella phages against a wide range of Salmonella bacterium strains. We further use these data to construct a Salmonella phage–bacteria infection network (PBIN) and study its structure. Based on the above, we define a set of different Salmonella phage infection groups (or clusters) and explore their association to known Salmonella phage characteristics. Our aim is to better understand the underlying ecological and evolutionary dynamics that shape the Salmonella phages’ host-range.

2. Materials and Methods

2.1. Phages and Bacterial Strains

Thirteen Salmonella-specific individual phage clones previously isolated from environmental sources (sewage, the river and sea water samples, milk) [16,17] were used for testing the Salmonella strains for susceptibility, these are GEC_vB_B1, GEC_vB_B3, GEC_vB_NS7, GEC_vB_BS, GEC_vB_MG, GEC_vB_7A, GEC_vB_N5, GEC_vB_N8, GEC_vB_N3, GEC_vB_M4, GEC_vB_M5, GEC_vB_HIL, and GEC_vB_TR. The genomes of eight out of the above phages were previously sequenced and annotated by short-read high throughput sequencing [16] (MiniSeq Illumina NGS platform, Illumina, San Diego, CA, USA) (Supplementary Table S1). Six genomes have been deposited in NCBI and are available via GenBank accession numbers in Supplementary Table S1. Five of these phages belong to the Myoviridae family with two different genera: Felixounavirus (GEC_vB_B1, GEC_vB_B3, GEC_vB_NS7, and GEC_vB_BS), and Seunavirus (GEC_vB_MG). Three phages belong to the Demerecviridae family of the genus Tequintavirus (GEC_vB_N3, GEC_vB_N5, GEC_vB_N8). The second group of phages, sequenced using long-read sequencing [18] (MinION, Oxford Nanopore Technology, ONT, Oxford, UK), is composed of five members belonging to Siphoviridae family, genus Jerseyvirus (GEC_vB_M4, GEC_vB_M5, GE_vB_HIL), Myoviridae family genus Felixounavirus (GEC_vB_7A), and Podoviridae family genus Lederbergvirus (GEC_vB_TR) (Supplementary Table S1).
Altogether, 141 Salmonella strains originating from Ireland [19] were included in this study. These strains are related to various serovars: S. Typhimurium (n = 40), S. Dublin (n = 22), S. Enteritidis (n = 21), S. Anatum (n = 10), S. Infantis (n = 8), S. Newport (n = 7), S. Bredeney (n = 5), S. Derbey (n = 4), S. Braenderup (n = 2), S. Germinara (n = 2), S. Uganda (n = 2), S. Senftenberg (2), S. Kentucky (n = 1), S. Java (1), S. Branderburg (n = 1), S. Bareilly (n = 1), S. Virchow (n = 1), S. Goldcost (n = 1), S. Poona (n = 1), and unknown serotypes (n = 9). Overall, 19 serotypes of Salmonella were included into the work. The majority of these strains (n = 88) were of veterinary origin (bovine n = 36, porcine n = 34, poultry n = 17, duck n = 1), 27 were isolated from humans, 6 strains were obtained from food products (cheese, vegetables, fish) and 1 from terpene.

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