Development of a phage-based diagnostic test for the identification of Clostridium difficile

Clostridium difficile is the most common bacterial cause of infectious diarrhoea in healthcare environments and in 2014 was responsible for 13,785 infections in the UK. C. difficile infection (CDI) is spread via the faecal-oral route and by contact with contaminated surfaces. However, despite the healthcare concerns no tests are available to validate if sufficient cleaning has been conducted. In addition, Polymerase Chain Reaction (PCR) and Enzyme Immunoassays (EIAs)-based tests used to diagnose CDI lack sensitivity and specificity and hence false negative results are commonly obtained. To overcome these concerns the aim of the PhD research has been to develop the first diagnostic test that exploits the specific interactions of C. difficile bacteriophages (phages), viruses that specifically infect and kill C. difficile. In order to develop a C. difficile phage-based test, first suitable phages that can be used for the test were identified and this was conducted by screening 35 different C. difficile phages against 160 clinically relevant C. difficile isolates. Five phages were found to infect the most number of isolates and were investigated further to identify whether a phage-based diagnostic could be developed based on phages binding (adsorption) to different C. difficile subgroups. However, for all five phages, adsorption rates were not consistently high for C. difficile subgroups in comparison to other common bacteria found in similar locations to C. difficile. Therefore, to increase specificity of the phage-based diagnostic test a new approach was taken by tagging two phages with luminescence luxAB genes (reporter phages), which would be expressed once C. difficile cells were infected with the phages. To design the C. difficile reporter phages, non-essential phage genes were replaced with the luxAB genes, but this study revealed mutagenesis of C. difficile was troublesome and extensive optimisation was required. In addition, once the reporter phages had successfully been constructed the luxAB genes were unstable within the phage genome and were lost during phage replication. Despite extensive optimisation and due to time constrains the luxAB genes were not stabilised within the phages but future work will focus on stabilising the genes

Potential Roles for Bacteriophages in Reducing <em>Salmonella</em> from Poultry and Swine

This chapter discusses application of natural parasites of bacteria, bacteriophages (phages), as a promising biological control for Salmonella in poultry and swine. Many studies have shown phages can be applied at different points from farm-to-fork, from pre to post slaughter, to control the spread of Salmonella in the food chain. Pre-slaughter applications include administering phages via oral gavage, in drinking water and in feed. Post slaughter applications include adding phages to carcasses and during packaging of meat products. The research discussed in this chapter demonstrate a set of promising data that relate to the ability of phages to reduce Salmonella colonisation and abundance. Collectively the studies support the viability of phage as antimicrobial prophylactics and therapeutics to prevent and control Salmonella in the food chain

Prophylactic Delivery of a Bacteriophage Cocktail in Feed Significantly Reduces Salmonella Colonization in Pigs

Nontyphoidal Salmonella spp. are a leading cause of human food poisoning and can be transmitted to humans via consuming contaminated pork. To reduce Salmonella spread to the human food chain, bacteriophage (phage) therapy could be used to reduce bacteria from animals' preslaughter. We aimed to determine if adding a two-phage cocktail to feed reduces Salmonella colonization in piglets. This first required spray drying phages to allow them to be added as a powder to feed, and phages were spray dried in different excipients to establish maximum recovery. Although laboratory phage yields were not maintained during scale up in a commercial spray dryer (titers fell from 3 × 108 to 2.4 × 106 PFU/g respectively), the phage titers were high enough to progress. Spray dried phages survived mixing and pelleting in a commercial feed mill, and sustained no further loss in titer when stored at 4°C or barn conditions over 6 months. Salmonella-challenged piglets that were prophylactically fed the phage-feed diet had significantly reduced Salmonella colonization in different gut compartments (P < 0.01). 16S rRNA gene sequencing of fecal and gut samples showed phages did not negatively impact microbial communities as they were similar between healthy control piglets and those treated with phage. Our study shows delivering dried phages via feed effectively reduces Salmonella colonization in pigs. Infections caused by Salmonella spp. cause 93.8 million cases of human food poisoning worldwide, each year of which 11.7% are due to consumption of contaminated pork products. An increasing number of swine infections are caused by multidrug-resistant (MDR) Salmonella strains, many of which have entered, and continue to enter the human food chain. Antibiotics are losing their efficacy against these MDR strains, and thus antimicrobial alternatives are needed. Phages could be developed as an alternative approach, but research is required to determine the optimal method to deliver phages to pigs and to determine if phage treatment is effective at reducing Salmonella colonization in pigs. The results presented in this study address these two aspects of phage development and show that phages delivered via feed prophylactically to pigs reduces Salmonella colonization in challenged pigs

Diversity, Dynamics and Therapeutic Application of Clostridioides difficile Bacteriophages

Clostridioides difficile causes antibiotic-induced diarrhoea and pseudomembranous colitis in humans and animals. Current conventional treatment relies solely on antibiotics, but C. difficile infection (CDI) cases remain persistently high with concomitant increased recurrence often due to the emergence of antibiotic-resistant strains. Antibiotics used in treatment also induce gut microbial imbalance; therefore, novel therapeutics with improved target specificity are being investigated. Bacteriophages (phages) kill bacteria with precision, hence are alternative therapeutics for the targeted eradication of the pathogen. Here, we review current progress in C. difficile phage research. We discuss tested strategies of isolating C. difficile phages directly, and via enrichment methods from various sample types and through antibiotic induction to mediate prophage release. We also summarise phenotypic phage data that reveal their morphological, genetic diversity, and various ways they impact their host physiology and pathogenicity during infection and lysogeny. Furthermore, we describe the therapeutic development of phages through efficacy testing in different in vitro, ex vivo and in vivo infection models. We also discuss genetic modification of phages to prevent horizontal gene transfer and improve lysis efficacy and formulation to enhance stability and delivery of the phages. The goal of this review is to provide a more in-depth understanding of C. difficile phages and theoretical and practical knowledge on pre-clinical, therapeutic evaluation of the safety and effectiveness of phage therapy for CDI

Development of a Phage Cocktail to Target Salmonella Strains Associated with Swine

Infections caused by multidrug resistant Salmonella strains are problematic in swine and are entering human food chains. Bacteriophages (phages) could be used to complement or replace antibiotics to reduce infection within swine. Here, we extensively characterised six broad host range lytic Salmonella phages, with the aim of developing a phage cocktail to prevent or treat infection. Intriguingly, the phages tested differed by one to five single nucleotide polymorphisms. However, there were clear phenotypic differences between them, especially in their heat and pH sensitivity. In vitro killing assays were conducted to determine the efficacy of phages alone and when combined, and three cocktails reduced bacterial numbers by ~2 &times; 103 CFU/mL within two hours. These cocktails were tested in larvae challenge studies, and prophylactic treatment with phage cocktail SPFM10-SPFM14 was the most efficient. Phage treatment improved larvae survival to 90% after 72 h versus 3% in the infected untreated group. In 65% of the phage-treated larvae, Salmonella counts were below the detection limit, whereas it was isolated from 100% of the infected, untreated larvae group. This study demonstrates that phages effectively reduce Salmonella colonisation in larvae, which supports their ability to similarly protect swine

Development of a Phage Cocktail to Target <i>Salmonella</i> Strains Associated with Swine

Infections caused by multidrug resistant Salmonella strains are problematic in swine and are entering human food chains. Bacteriophages (phages) could be used to complement or replace antibiotics to reduce infection within swine. Here, we extensively characterised six broad host range lytic Salmonella phages, with the aim of developing a phage cocktail to prevent or treat infection. Intriguingly, the phages tested differed by one to five single nucleotide polymorphisms. However, there were clear phenotypic differences between them, especially in their heat and pH sensitivity. In vitro killing assays were conducted to determine the efficacy of phages alone and when combined, and three cocktails reduced bacterial numbers by ~2 × 10(3) CFU/mL within two hours. These cocktails were tested in larvae challenge studies, and prophylactic treatment with phage cocktail SPFM10-SPFM14 was the most efficient. Phage treatment improved larvae survival to 90% after 72 h versus 3% in the infected untreated group. In 65% of the phage-treated larvae, Salmonella counts were below the detection limit, whereas it was isolated from 100% of the infected, untreated larvae group. This study demonstrates that phages effectively reduce Salmonella colonisation in larvae, which supports their ability to similarly protect swine

A bacteriophage cocktail delivered in feed significantly reduced Salmonella colonization in challenged broiler chickens

ABSTRACTNontyphoidal Salmonella spp. are a leading cause of human gastrointestinal infections and are commonly transmitted via the consumption of contaminated meat. To limit the spread of Salmonella and other food-borne pathogens in the food chain, bacteriophage (phage) therapy could be used during rearing or pre-harvest stages of animal production. This study was conducted to determine if a phage cocktail delivered in feed is capable of reducing Salmonella colonization in experimentally challenged chickens and to determine the optimal phage dose. A total of 672 broilers were divided into six treatment groups T1 (no phage diet and unchallenged); T2 (phage diet 106 PFU/day); T3 (challenged group); T4 (phage diet 105 PFU/day and challenged); T5 (phage diet 106 PFU/day and challenged); and T6 (phage diet 107 PFU/day and challenged). The liquid phage cocktail was added to mash diet with ad libitum access available throughout the study. By day 42 (the concluding day of the study), no Salmonella was detected in faecal samples collected from group T4. Salmonella was isolated from a small number of pens in groups T5 (3/16) and T6 (2/16) at ∼4 × 102 CFU/g. In comparison, Salmonella was isolated from 7/16 pens in T3 at ∼3 × 104 CFU/g. Phage treatment at all three doses had a positive impact on growth performance in challenged birds with increased weight gains in comparison to challenged birds with no phage diet. We showed delivering phages via feed was effective at reducing Salmonella colonization in chickens and our study highlights phages offer a promising tool to target bacterial infections in poultry

Use of single molecule sequencing for comparative genomics of an environmental and a clinical isolate of Clostridium difficile ribotype 078.

BACKGROUND: How the pathogen Clostridium difficile might survive, evolve and be transferred between reservoirs within the natural environment is poorly understood. Some ribotypes are found both in clinical and environmental settings. Whether these strains are distinct from each another and evolve in the specific environments is not established. The possession of a highly mobile genome has contributed to the genetic diversity and ongoing evolution of C. difficile. Interpretations of genetic diversity have been limited by fragmented assemblies resulting from short-read length sequencing approaches and by a limited understanding of epigenetic regulation of diversity. To address this, single molecule real time (SMRT) sequencing was used in this study as it produces high quality genome sequences, with resolution of repeat regions (including those found in mobile elements) and can generate data to determine methylation modifications across the sequence (the methylome). RESULTS: Chromosomal rearrangements and ribosomal operon duplications were observed in both genomes. The rearrangements occurred at insertion sites within two mobile genetic elements (MGEs), Tn6164 and Tn6293, present only in the M120 and CD105HS27 genomes, respectively. The gene content of these two transposons differ considerably which could impact upon horizontal gene transfer; differences include CDSs encoding methylases and a conjugative prophage only in Tn6164. To investigate mechanisms which could affect MGE transfer, the methylome, restriction modification (RM)  and the CRISPR/Cas systems were characterised for each strain. Notably, the environmental isolate, CD105HS27, does not share a consensus motif for (m4)C methylation, but has one additional spacer  when compared to the clinical isolate M120. CONCLUSIONS: These findings show key differences between the two strains in terms of their genetic capacity for MGE transfer. The carriage of horizontally transferred genes appear to have genome wide effects based on two different methylation patterns. The CRISPR/Cas system appears active although perhaps slow to evolve. Data suggests that both mechanisms are functional and impact upon horizontal gene transfer and genome evolution within C. difficile