Bacteriophage therapeutic development will clearly benefit from understanding the fundamental dynamics of in vivo phage-bacteria interactions. Such information can inform animal and human trials, and much can be ascertained from human cell-line work. We have developed a human cell-based system using Clostridium difficile, a pernicious hospital pathogen with limited treatment options, and the phage phiCDHS1 that effectively kills this bacterium in liquid culture. The human colon tumorigenic cell line HT-29 was used because it simulates the colon environment where C. difficile infection occurs. Studies on the dynamics of phage-bacteria interactions revealed novel facets of phage biology, showing that phage can reduce C. difficile numbers more effectively in the presence of HT-29 cells than in vitro. Both planktonic and adhered Clostridial cell numbers were successfully reduced. We hypothesise and demonstrate that this observation is due to strong phage adsorption to the HT-29 cells, which likely promotes phage-bacteria interactions. The data also showed that the phage phiCDHS1 was not toxic to HT-29 cells, and phage-mediated bacterial lysis did not cause toxin release and cytotoxic effects. The use of human cell lines to understand phage-bacterial dynamics offers valuable insights into phage biology in vivo, and can provide informative data for human trials.

Antimicrobial resistance (AMR) is a major problem globally. The main bacterial organisms associated with urinary tract infection (UTI) associated sepsis are E. coli and Klebsiella along with Enterobacter species. These all have AMR strains known as ESBL (Extended Spectrum Beta-Lactamase), which are featured on the WHO priority pathogens list as “critical” for research. Bacteriophages (phages), as viruses that can infect and kill bacteria, could provide an effective tool to tackle these AMR strains. There is currently no “gold standard” for developing a phage cocktail. Here we describe a novel approach to develop an effective phage cocktail against a set of ESBL-producing E. coli and Klebsiella largely isolated from patients in United Kingdom hospitals. By comparing different measures of phage efficacy, we show which are the most robust, and suggest an efficient screening cascade that could be used to develop phage cocktails to target other AMR bacterial species. A target panel of 38 ESBL-producing clinical strains isolated from urine samples was collated and used to test phage efficacy. After an initial screening of 68 phages, six were identified and tested against these 38 strains to determine their clinical coverage and killing efficiency. To achieve this, we assessed four different methods to assess phage virulence across these bacterial isolates. These were the Direct Spot Test (DST), the Efficiency of Plating (EOP) assay, the planktonic killing assay (PKA) and the biofilm assay. The final ESBL cocktail of six phages could effectively kill 23/38 strains (61%), for Klebsiella 13/19 (68%) and for E. coli 10/19 (53%) based on the PKA data. The ESBL E. coli collection had six isolates from the prevalent UTI-associated ST131 sequence type, five of which were targeted effectively by the final cocktail. Of the four methods used to assess phage virulence, the data suggests that PKAs are as effective as the much more time-consuming EOPs and data for the two assays correlates well. This suggests that planktonic killing is a good proxy to determine which phages should be used in a cocktail. This assay when combined with the virulence index also allows “phage synergy” to inform cocktail design.

A common cause of human food poisoning is through ingestion of pork products contaminated with Salmonella spp. Worryingly multi-drug resistant (MDR) Salmonella strains have been isolated from pigs, which motivates the need for alternative antimicrobials. In this study isolation and characterization of 21 lytic Salmonella phages is described. All 21 phages, labeled as SPFM phages were shown to efficiently infect MDR Salmonella strains isolated from United Kingdom pigs and phages SPFM1, SPFM3, SPFM10, SPFM14, SPFM15, SPFM17, and SPFM19 could lyse 100% of strains tested. The phage genome sizes range from 233 to 242 Kb, which qualifies them as jumbo phages. All SPFM phage genomes are approximately 95% similar to each other by average nucleotide identity, they encode between 258–307 coding sequences and share 188 core genes. Phylogenetic analysis shows these phages are most similar to phages of the genus Seoulvirus and to further characterize phages within the genus, genes under positive selection were identified. Several of the genes under evolutionary selection pressure were predicted to encode for proteins that interact with bacteria. We describe the phenotypic and genetic characterization of this novel Salmonella phage set. As the phages efficiently kill MDR Salmonella strains, they may offer a promising alternative to antibiotics.

Salmonella spp. is a leading cause of gastrointestinal enteritis in humans where it is largely contracted via contaminated poultry and pork. Phages can be used to control Salmonella infection in the animals, which could break the cycle of infection before the products are accessible for consumption. Here, the potential of 21 myoviruses and a siphovirus to eliminate Salmonella in vitro and in vivo was examined with the aim of developing a biocontrol strategy to curtail the infection in poultry and swine. Together, the phages targeted the twenty-three poultry and ten swine prevalent Salmonella serotype isolates tested. Although individual phages significantly reduced bacterial growth of representative isolates within 6 h post-infection, bacterial regrowth occurred 1 h later, indicating proliferation of resistant strains. To curtail bacteriophage resistance, a novel three-phage cocktail was developed in vitro, and further investigated in an optimized Galleria mellonella larva Salmonella infection model colonized with representative swine, chicken and laboratory strains. For all the strains examined, G. mellonella larvae given phages 2 h prior to bacterial exposure (prophylactic regimen) survived and Salmonella was undetectable 24 h post-phage treatment and throughout the experimental time (72 h). Administering phages with bacteria (co-infection), or 2 h post-bacterial exposure (remedial regimen) also improved survival (73–100% and 15–88%, respectively), but was less effective than prophylaxis application. These pre-livestock data support the future application of this cocktail for further development to effectively treat Salmonella infection in poultry and pigs. Future work will focus on cocktail formulation to ensure stability and incorporation into feeds and used to treat the infection in target animals.

Antimicrobial resistance (AMR) is a major problem globally. The main bacterial organisms associated with urinary tract infection (UTI) associated sepsis are E. coli and Klebsiella along with Enterobacter species. These all have AMR strains known as ESBL (Extended Spectrum Beta-Lactamase), which are featured on the WHO priority pathogens list as critical for research. Bacteriophages (phages) as viruses that can infect and kill bacteria, could provide an effective tool to tackle these AMR strains. There is currently no gold standard for developing a phage cocktail. Here we describe a novel approach to develop an effective phage cocktail against a set of ESBL-producing E. coli and Klebsiella largely isolated from patients in UK hospitals. By comparing different measures of phage efficacy, we show which are the most robust, and suggest an efficient screening cascade that could be used to develop phage cocktails to target other AMR bacterial species. A target panel of 38 ESBL-producing clinical strains isolated from urine samples was collated and used to test phage efficacy. After an initial screening of 68 phages, six were identified and tested against these 38 strains to determine their clinical coverage and killing efficiency. To achieve this, we assessed four different methods to assess phage virulence across these bacterial isolates. These were the Direct Spot Test (DST), the Efficiency of Plating (EOP) assay, the planktonic killing assay and the biofilm assay. The final ESBL cocktail of six phages could effectively kill 23/38 strains (61%) for Klebsiella 13/19 (68%) and for E. coli 10/19 (53%) based on the planktonic killing assay data. The ESBL E. coli collection had six isolates from the prevalent UTI-associated ST131 sequence type, five of which were targeted effectively by the final cocktail. Of the four methods used to assess phage virulence, the data suggests that planktonic killing assays are as effective as the much more time-consuming EOPs and data for the two assays correlates well. This suggests that planktonic killing is a good proxy to determine which phages should be used in a cocktail. This assay when combined with the virulence index also allows phage synergy to inform cocktail design.

Bacteriophage (phage) therapy is a promising alternative to antibiotics for the treatment of bacterial pathogens, including Clostridium difficile. However, as for many species, in C. difficile the physical interactions between phages and bacterial cells have not been studied in detail. The initial interaction, known as phage adsorption, is initiated by the reversible attachment of phage tail fibers to bacterial cell surface receptors followed by an irreversible binding step. Therefore binding can dictate which strains are infected by the phage. In this study, we investigated the adsorption rates and irreversible binding of three C. difficile myoviruses: CDHM1, CDHM3 and CDHM6 to ten strains that represent ten prevalent C. difficile ribotypes, regardless of their ability to infect. CDHM1 and CDHM3 phage particles adsorbed by ~75% to some strains that they infected. The infection dynamics for CDHM6 are less clear and ~30% of the phage particles bound to all strains, irrespective of whether a successful infection was established. The data highlighted adsorption is phage-host specific. However, it was consistently observed that irreversible binding had to be above 80% for successful infection, which was also noted for another two C. difficile myoviruses. Furthermore, to understand if there is a relationship between infection, adsorption and phage tail fibers, the putative tail fiber protein sequences of CDHM1, CDHM3 and CDHM6 were compared. The putative tail fiber protein sequence of CDHM1 shares 45% homology at the amino acid level to CDHM3 and CDHM6, which are identical to each other. However, CDHM3 and CDHM6 display differences in adsorption, which highlights that there is no obvious relationship between putative tail fiber sequence and adsorption. The importance of adsorption and binding to successful infection is often overlooked, and this study provides useful insights into host-pathogen interactions within this phage-pathogen system.

The bacterium Clostridium difficile is a significant cause of nosocomial infections worldwide. The pathogenic success of this organism can be attributed to its flexible genome which is characterized by the exchange of mobile genetic elements, and by ongoing genome evolution. Despite its pathogenic status, C. difficile can also be carried asymptomatically, and has been isolated from natural environments such as water and sediments where multiple strain types (ribotypes) are found in close proximity. These include ribotypes which are associated with disease, as well as those that are less commonly isolated from patients. Little is known about the genomic content of strains in such reservoirs in the natural environment. In this study, draft genomes have been generated for 13 C. difficile isolates from estuarine sediments including clinically relevant and environmental associated types. To identify the genetic diversity within this strain collection, whole-genome comparisons were performed using the assemblies. The strains are highly genetically diverse with regards to the C. difficile “mobilome,” which includes transposons and prophage elements. We identified a novel transposon-like element in two R078 isolates. Multiple, related and unrelated, prophages were detected in isolates across ribotype groups, including two novel prophage elements and those related to the transducing phage φC2. The susceptibility of these isolates to lytic phage infection was tested using a panel of characterized phages found from the same locality. In conclusion, estuarine sediments are a source of genetically diverse C. difficile strains with a complex network of prophages, which could contribute to the emergence of new strains in clinics.

Phage-bacterial recognition is species-specific, determined by interactions between phage receptor-binding proteins (RBPs) and corresponding bacterial receptors. RBPs are diverse, and we present data demonstrating the identification and characterisation of a novel C. difficile phage RBP. Putative RBP was identified for CDHS-1, and overexpressed, purified, and polyclonal antibodies were raised and used in phage neutralization assays. Anti-gp22 neutralised CDHS-1, indicating it is the RBP. Immunogold-labelling and transmission electron microscopy confirmed this, enabling visualization of the protein locations. A detailed structural understanding was obtained from determining the three-dimensional structure of gp22 by X-ray crystallography. gp22 is a new RBP class consisting of an N-terminal L-shaped α-helical superhelix domain and a C-terminal Mg2+-binding domain. The protein is a stable homodimer in solution mediated via reciprocal contacts between an α-helical hairpin located within the superhelix domain and additional asymmetrical contacts between the ends of the short arm of each L-shaped protomer. The dimer resembles U-shape with a crossbar formed from the hairpin of each partner. C. difficile binding is Mg2+-dependent. CDHS-1 could not infect a C. difficile S-layer mutant suggesting the bacterial receptors are within the S-layer. These findings provide novel insights into phage biology and extend our knowledge of RBPs.