No abstract

In this study we isolated novel bacteriophages, infecting the zoonotic bacterium Campylobacter jejuni. These phages may be used in phage therapy of C. jejuni colonized poultry to prevent spreading of the bacteria to meat products causing disease in humans. Many C. jejuni phages have been isolated using NCTC12662 as the indicator strain, which may have biased the selection of phages. A large group of C. jejuni phages rely on the highly diverse capsular polysaccharide (CPS) for infection and recent work identified the O-methyl phosphoramidate modification (MeOPN) of CPS as a phage receptor. We therefore chose seven C. jejuni strains each expressing different CPS structures as indicator strains in a large screening for phages in samples collected from free-range poultry farms. Forty-three phages were isolated using C. jejuni NCTC12658, NCTC12662 and RM1221 as host strains and 20 distinct phages were identified based on host range analysis and genome restriction profiles. Most phages were isolated using C. jejuni strains NCTC12662 and RM1221 and interestingly phage genome size (140 kb vs. 190 kb), host range and morphological appearance correlated with the isolation strain. Thus, according to C. jejuni phage grouping, NCTC12662 and NCTC12658 selected for CP81-type phages, while RM1221 selected for CP220-type phages. Furthermore, using acapsular ∆kpsM mutants we demonstrated that phages isolated on NCTC12658 and NCTC12662 were dependent on the capsule for infection. In contrast, CP220-type phages isolated on RM1221 were unable to infect non-motile ∆motA mutants, hence requiring motility for successful infection. Hence, the primary phage isolation strain determines both phage type (CP81 or CP220) as well as receptors (CPS or flagella) recognised by the isolated phages.

Summary The host range of phages is a key to understand their impact on bacterial ecology and evolution. Because of the complexity of phage–host interactions, the variables that determine the breadth of a phage host range remain poorly understood. Here, we propose a novel holistic approach to identify the host range determinants of a new collection of phages infecting Salmonella, isolated from animal, environmental and wastewater samples that were able to infect 58 of the 71 Salmonella strains in our collection. By using a set of statistic approaches (non‐metric dimensional scaling, Bray–Curtis distance, PERMANOVA), we analysed phenotypic (host range on wild‐type and receptor mutants) and genetic data (taxonomic assignment and receptor binding proteins) to evaluate the impact of isolation strain and niche, phage receptor and genus on the host range. Statistical analysis revealed that two phage characteristics influence the host range by explaining the most variance: the receptor by 45% and the genus by 51%. Interestingly, phage genus and receptor in combination explained 79% of the variance, establishing these characteristics as the major determinants of the host range. This study demonstrates the power and the novelty of applying statistical approaches to phenotypic and genetic data to investigate the ecology of phage–host interactions.

Campylobacter jejuni NCTC12662 is sensitive to infection by many Campylobacter bacteriophages. Here we used this strain to investigate the molecular mechanism behind phage resistance development when exposed to a single phage and demonstrate how phase variable expression of one surface component influences phage sensitivity against many diverse C. jejuni phages. When C. jejuni NCTC12662 was exposed to phage F207 overnight, 25% of the bacterial cells were able to grow on a lawn of phage F207, suggesting that resistance develops at a high frequency. One resistant variant, 12662R, was further characterized and shown to be an adsorption mutant. Plaque assays using our large phage collection showed that seven out of 36 diverse capsular polysaccharide (CPS)-dependent phages could not infect 12662R, whereas the remaining phages formed plaques on 12662R with reduced efficiencies. Analysis of the CPS composition of 12662R by high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) showed a diminished signal for O-methyl phosphoramidate (MeOPN), a phase variable modification of the CPS. This suggested that the majority of the 12662R population did not express this phase variable modification in the CPS, indicating that MeOPN serves as a phage receptor in NCTC12662. Whole genome analysis of 12662R showed a switch in the length of the phase variable homopolymeric G tract of gene 06810, encoding a putative MeOPN-transferase located in the CPS locus, resulting in a non-functional protein. To confirm the role of 06810 in phage resistance development of NCTC12662, a 06810 knockout mutant in NCTC12662 was constructed and analyzed by HR-MAS NMR demonstrating the absence of MeOPN in the CPS of the mutant. Plaque assays using NCTC12662Δ06810 demonstrated that seven of our CPS-dependent Campylobacter phages are dependent on the presence of MeOPN for successful infection of C. jejuni, whereas the remaining 29 phages infect independently of MeOPN, although with reduced efficiencies. Our data indicate that CPS-dependent phages uses diverse mechanisms for their initial interaction with their C. jejuni host.

Here, we describe the methods for isolation, purification, and propagation of Campylobacter jejuni bacteriophages from samples expected to contain high number of phages such as chicken feces. The overall steps are (1) liberation of phages from the sample material; (2) observation of plaque-forming units on C. jejuni lawns using a spot assay; (3) isolation of single plaques; (4) consecutive purification procedures; and (5) propagation of purified phages from a plate lysate to prepare master stocks.