Background: With advances in sequencing technology and decreasing costs, the number of phage genomes that have been sequenced has increased markedly in the past decade. Materials and Methods: We developed an automated retrieval and analysis system for phage genomes (https:// github.com/RyanCook94/inphared) to produce the INfrastructure for a PHAge REference Database (INPHARED) of phage genomes and associated metadata. Results: As of January 2021, 14,244 complete phage genomes have been sequenced. The INPHARED data set is dominated by phages that infect a small number of bacterial genera, with 75% of phages isolated on only 30 bacterial genera. There is further bias, with significantly more lytic phage genomes (*70%) than temperate (*30%) within our database. Collectively, this results in *54% of temperate phage genomes originating from just three host genera. With much debate on the carriage of antibiotic resistance genes and their potential safety in phage therapy, we searched for putative antibiotic resistance genes. Frequency of antibiotic resistance gene carriage was found to be higher in temperate phages than in lytic phages and again varied with host. Conclusions: Given the bias of currently sequenced phage genomes, we suggest to fully understand phage diversity, efforts should be made to isolate and sequence a larger number of phages, in particular temperate phages, from a greater diversity of hosts.

Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in health care settings across the world. Despite its pathogenic capacity, it can be carried asymptomatically and has been found in terrestrial and marine ecosystems outside hospital environments. Little is known about these environmental strains, and few studies have been conducted on estuarine systems. Although prophage abundance and diversity are known to occur within clinical strains, prophage carriage within environmental strains of C. difficile has not previously been explored. In this study, we isolated C. difficile from sites sampled in two consecutive years in an English estuarine system. Isolates were characterized by PCR ribotype, antibiotic resistance, and motility. The prevalence and diversity of prophages were detected by transmission electron microscopy (TEM) and a phage-specific PCR assay. We show that a dynamic and diverse population of C. difficile exists within these sediments and that it includes isolates of ribotypes which are associated with severe clinical infections and those which are more frequently isolated from outside the hospital environment. Prophage carriage was found to be high (75%), demonstrating that phages play a role in the biology of these strains.

Rapid identification and enumeration of the numerically important bacteriophages has been till recently a major limitation for studies of virus ecology. The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has changed this. The flow cytometric method allows the detection and discrimination of a wide variety of viruses of different morphology, genome type, and size. The present paper describes an optimized protocol for the enumeration of bacteriophages using a standard benchtop flow cytometer.

and a.d.millard@warwick.ac.uk Cyanophage abundance has been shown to £uctuate over long timescales and with depth, but little is known about how it varies over short timescales. Previous short-term studies have relied on counting total virus numbers and therefore the phages which infect cyanobacteria cannot be distinguished from the total count.In this study, an isolation-based approach was used to determine cyanophage abundance from water samples collected over a depth pro¢le for a 24 h period from the Indian Ocean. Samples were used to infect Synechococcus sp. WH7803 and the number of plaque forming units (pfu) at each time point and depth were counted. At 10 m phage numbers were similar for most time-points, but there was a distinct peak in abundance at 0100 hours. Phage numbers were lower at 25 m and 50 m and did not show such strong temporal variation. No phages were found below this depth. Therefore, we conclude that only the abundance of phages in surface waters showed a clear temporal pattern over a short timescale. Fifty phages from a range of depths and time points were isolated and puri¢ed. The molecular diversity of these phages was estimated using a section of the phage-encoded psbD gene and the results from a phylogenetic analysis do not suggest that phages from the deeper waters form a distinct subgroup.

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.

Acaryochloris marina strains have been isolated from several varied locations and habitats worldwide demonstrating a diverse and dynamic ecology. In this study, the whole cell photophysiologies of strain MBIC11017, originally isolated from a colonial ascidian, and the free-living epilithic strain CCMEE5410 are analyzed by absorbance and fluorescence spectroscopy, laser scanning confocal microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent protein analysis. We demonstrate pigment adaptation in MBIC11017 and CCMEE5410 under different light regimes. We show that the higher the incident growth light intensity for both strains, the greater the decrease in their chlorophyll d content. However, the strain MBIC11017 loses its phycobiliproteins relative to its chlorophyll d content when grown at light intensities of 40 microE m(-2) s(-1) without shaking and 100 microE m(-2) s(-1) with shaking. We also conclude that phycobiliproteins are absent in the free-living strain CCMEE5410.

Graphical abstract

Background: With advances in sequencing technology and decreasing costs, the number of bacteriophage genomes that have been sequenced has increased markedly in the last decade. Materials and Methods: We developed an automated retrieval and analysis system for bacteriophage genomes, INPHARED (https://github.com/RyanCook94/inphared), that provides data in a consistent format. Results: As of January 2021, 14,244 complete phage genomes have been sequenced. The data set is dominated by phages that infect a small number of bacterial genera, with 75% of phages isolated only on 30 genera. There is further bias with significantly more lytic phage genomes than temperate within the database, resulting in ~54% of temperate phage genomes originating from just three host genera. Within phage genomes putative antibiotics resistance genes were found in higher frequencies in temperate phage than lytic phages. Conclusion: We provide a mechanism to reproducibly extract complete phage genomes and highlight some of the biases within this data, that underpins our current understanding of phage genomes.