Andrew R. Timms

Quantitative models of in vitro bacteriophage-host dynamics and their application to phage therapy.

Phage therapy is the use of bacteriophages as antimicrobial agents for the control of pathogenic and other problem bacteria. It has previously been argued that successful application of phage therapy requires a good understanding of the non-linear kinetics of phage–bacteria interactions. Here we combine experimental and modelling approaches to make a detailed examination of such kinetics for the important food-borne pathogen Campylobacter jejuni and a suitable virulent phage in an in vitro system. Phage-insensitive populations of C. jejuni arise readily, and as far as we are aware this is the first phage therapy study to test, against in vitro data, models for phage–bacteria interactions incorporating phage-insensitive or resistant bacteria. We find that even an apparently simplistic model fits the data surprisingly well, and we confirm that the so-called inundation and proliferation thresholds are likely to be of considerable practical importance to phage therapy. We fit the model to time series data in order to estimate thresholds and rate constants directly. A comparison of the fit for each culture reveals density-dependent features of phage infectivity that are worthy of further investigation. Our results illustrate how insight from empirical studies can be greatly enhanced by the use of kinetic models: such combined studies of in vitro systems are likely to be an essential precursor to building a meaningful picture of the kinetic properties of in vivo phage therapy.

Application of a group II Campylobacter bacteriophage to reduce strains of Campylobacter jejuni and Campylobacter coli colonizing broiler chickens.

Members of the genus Campylobacter are frequently responsible for human enteric disease worldwide. Persistent Campylobacter contamination of poultry meat is a common problem that represents a significant food safety risk through the consumption of undercooked poultry meat or through cross-contamination of other foods during the preparation of poultry. Bacteriophage therapy is one possible means by which this colonization of poultry could be controlled, thus limiting the entry of Campylobacter into the human food chain. Previously group III phages with genome sizes of approximately 140 kb had been administered to Campylobacter jejuni-colonized poultry. The application of a group II Campylobacter phage, CP220, with a genome size of 197 kb is described here. Phage CP220 was administered to both C. jejuni- and C. coli-colonized birds. A 2-log CFU/g decline in cecal Campylobacter counts was observed after 48 h in birds colonized with C. jejuni HPC5 and administered with a single 7-log PFU dose of CP220. The incidence of phage resistance developing in Campylobacter-colonized chickens upon exposure to virulent phages was determined to be 2%, and the resistant types remained a minor component of the population. To achieve a similar reduction in Campylobacter numbers in C. coli OR12-colonized birds, a 9-log PFU dose of CP220 was required. Using phage to reduce Campylobacter colonization in poultry offers the prospect of a sustainable intervention measure that may limit the entry of these pathogens into the human food chain.

Campylobacter bacteriophages and bacteriophage therapy

Members of the genus Campylobacter are frequently responsible for human enteric disease with occasionally very serious outcomes. Much of this disease burden is thought to arise from consumption of contaminated poultry products. More than 80% of poultry in the UK harbour Campylobacter as a part of their intestinal flora. To address this unacceptably high prevalence, various interventions have been suggested and evaluated. Among these is the novel approach of using Campylobacter‐specific bacteriophages, which are natural predators of the pathogen. To optimize their use as therapeutic agents, it is important to have a comprehensive understanding of the bacteriophages that infect Campylobacter, and how they can affect their host bacteria. This review will focus on many aspects of Campylobacter‐specific bacteriophages including: their first isolation in the 1960s, their use in bacteriophage typing schemes, their isolation from the different biological sources and genomic characterization. As well as their use as therapeutic agents to reduce Campylobacter in poultry their future potential, including their use in bio‐sanitization of food, will be explored. The evolutionary consequences of naturally occurring bacteriophage infection that have come to light through investigations of bacteriophages in the poultry ecosystem will also be discussed.

A suggested classification for two groups of Campylobacter myoviruses

Most Campylobacter bacteriophages isolated to date have long contractile tails and belong to the family Myoviridae. Based on their morphology, genome size and endonuclease restriction profile, Campylobacter phages were originally divided into three groups. The recent genome sequencing of seven virulent campylophages reveal further details of the relationships between these phages at the genome organization level. This article details the morphological and genomic features among the campylophages, investigates their taxonomic position, and proposes the creation of two new genera, the “Cp220likevirus” and “Cp8unalikevirus” within a proposed subfamily, the “Eucampyvirinae”

Salmonella Typhimurium-specific bacteriophage ΦSH19 and the origins of species specificity in the Vi01-like phage family

BackgroundWhole genome sequencing of bacteriophages suitable for biocontrol of pathogens in food products is a pre-requisite to any phage-based intervention procedure. Trials involving the biosanitization of Salmonella Typhimurium in the pig production environment identified one such candidate, ΦSH19.ResultsThis phage was sequenced and analysis of its 157,785 bp circular dsDNA genome revealed a number of interesting features. ΦSH19 constitutes another member of the recently-proposed Myoviridae Vi01-like family of phages, containing S. Typhi-specific Vi01 and Shigella-specific SboM-AG3. At the nucleotide level ΦSH19 is highly similar to phage Vi01 (80-98% pairwise identity over the length of the genome), with the major differences lying in the region associated with host-range determination. Analyses of the proteins encoded within this region by ΦSH19 revealed a cluster of three putative tail spikes. Of the three tail spikes, two have protein domains associated with the pectate lyase family of proteins (Tsp2) and P22 tail spike family (Tsp3) with the prospect that these enable Salmonella O antigen degradation. Tail spike proteins of Vi01 and SboM-AG3 are predicted to contain conserved right-handed parallel β-helical structures but the internal protein domains are varied allowing different host specificities.ConclusionsThe addition or exchange of tail spike protein modules is a major contributor to host range determination in the Vi01-like phage family.

The complete plasmid sequences of Salmonella enterica serovar Typhimurium U288

Salmonella enterica Serovar Typhimurium U288 is an emerging pathogen of pigs. The strain contains three plasmids of diverse origin that encode traits that are of concern for food security and safety, these include antibiotic resistant determinants, an array of functions that can modify cell physiology and permit genetic mobility. At 148,711 bp, pSTU288-1 appears to be a hybrid plasmid containing a conglomerate of genes found in pSLT of S. Typhimurium LT2, coupled with a mosaic of horizontally-acquired elements. Class I integron containing gene cassettes conferring resistance against clinically important antibiotics and compounds are present in pSTU288-1. A curious feature of the plasmid involves the deletion of two genes encoded in the Salmonella plasmid virulence operon (spvR and spvA) following the insertion of a tnpA IS26-like element coupled to a blaTEM gene. The spv operon is considered to be a major plasmid-encoded Salmonella virulence factor that is essential for the intracellular lifecycle. The loss of the positive regulator SpvR may impact on the pathogenesis of S. Typhimurium U288. A second 11,067 bp plasmid designated pSTU288-2 contains further antibiotic resistance determinants, as well as replication and mobilization genes. Finally, a small 4675 bp plasmid pSTU288-3 was identified containing mobilization genes and a pleD-like G-G-D/E-E-F conserved domain protein that modulate intracellular levels of cyclic di-GMP, and are associated with motile to sessile transitions in growth.

Complete Genome Sequence of Salmonella enterica Serovar Typhimurium U288

ABSTRACT Salmonella enterica serovar Typhimurium U288 has firmly established itself within the United Kingdom pig production industry. The prevalence of this highly pathogenic multidrug-resistant serovar at such a critical point in the food chain is therefore of great concern. To enhance our understanding of this microorganism, whole-genome and plasmid sequencing was performed.

Using antimicrobial cultures, bacteriocins and bacteriophages to reduce carriage of food-borne bacterial pathogens in poultry

Abstract: This chapter will focus on the use of antimicrobial cultures, bacteriocins and bacteriophages to reduce carriage of food-borne bacterial pathogens in poultry. The characteristics, practical considerations together with potential shortcomings of each type of treatment will be described, along with specific examples of their application. Pathogenic bacteria from the genera Campylobacter and Salmonella constitute a common challenge to the poultry industry world wide in terms of reducing human food-borne disease. Because of the pre-eminence of these genera, this chapter will focus on research aimed at controlling these food-borne pathogens.