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Phage therapy has shown great promise for the treatment of multidrug-resistant bacterial infections. However, the lack of a thorough and organized understanding of phage-body interactions has limited its clinical application. Here, we administered different purified phages ( Salmonella phage SE_SZW1, Acinetobacter phage AB_SZ6, and Pseudomonas phage PA_LZ7) intravenously to healthy animals (rats and monkeys) to evaluate the phage-induced host responses and phage pharmacokinetics with different intravenous (IV) doses in healthy animals. The plasma and the organs were sampled after different IV doses to determine the phage biodistribution, phage-induced cytokines, and antibodies. The potential side effects of phages on animals were assessed. A non-compartment model revealed that the plasma phage titer gradually decreased over time following a single dose. Repeated doses resulted in a 2-3 Log 10 decline of the plasma phage titer at 5 min compared to the first dose, regardless of the type of phage administered in rats. Host innate immune responses were activated including splenic enlargement following repeated doses. Phage-specific neutralization antibodies in animals receiving phages were detected. Similar results were obtained from monkeys. In conclusion, the mammalian bodies were well-tolerant to the administered phages. The animal responses to the phages and the phage biodistribution profiles could have a significant impact on the efficacy of phage therapy.IMPORTANCEPhage therapy has demonstrated potential in addressing multidrug-resistant bacterial infections. However, an insufficient understanding of phage-host interactions has impeded its broader clinical application. In our study, specific phages were administered intravenously (IV) to both rats and monkeys to elucidate phage-host interactions and evaluate phage pharmacokinetics (PK). Results revealed that with successive IV administrations, there was a decrease in plasma phage concentrations. Concurrently, these administrations elicited both innate and adaptive immune responses in the subjects. Notably, the observed immune responses and PK profiles exhibited variation contingent upon the phage type and the mammalian host. Despite these variations, the tested mammals exhibited a favorable tolerance to the IV-administered phages. This underscores the significance of comprehending these interactions for the optimization of phage therapy outcomes., Competing Interests: The authors declare no conflict of interest.

Phages are highly abundant in the human gut, yet most of them remain uncultured. Here, we present a gut phage isolate collection (GPIC) containing 209 phages for 42 commensal human gut bacterial species. Genome analysis of the phages identified 34 undescribed genera. We discovered 22 phages from the Salasmaviridae family that have small genomes (∼10-20 kbp) and infect Gram-positive bacteria. Two phages from a candidate family, Paboviridae, with high prevalence in the human gut were also identified. Infection assays showed that Bacteroides and Parabacteroides phages are specific to a bacterial species, and strains of the same species also exhibit substantial variations in phage susceptibility. A cocktail of 8 phages with a broad host range for Bacteroides fragilis strains effectively reduced their abundance in complex host-derived communities in vitro. Our study expands the diversity of cultured human gut bacterial phages and provides a valuable resource for human microbiome engineering., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Efforts have been made to reduce the genomes of living cells, but phage genome reduction remains challenging. It is of great interest to investigate whether genome reduction can make phages obtain new infectious properties. We developed a CRISPR/Cas9-based iterative phage genome reduction (CiPGr) approach and applied this to four distinct phages, thereby obtaining heterogeneous genome-reduced mutants. We isolated and sequenced 200 mutants with loss of up to 8-23% (3.3-35 kbp) of the original sequences. This allowed the identification of non-essential genes for phage propagation, although loss of these genes is mostly detrimental to phage fitness to various degrees. Notwithstanding this, mutants with higher infectious efficiency than their parental strains were characterized, indicating a trade-off between genome reduction and infectious fitness for phages. In conclusion, this study provides a foundation for future work to leverage the information generated by CiPGr in phage synthetic biology research., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Bacteriophages (phages) are the most abundant and diverse biological entity on Earth. Due to the lack of universal gene markers and database representatives, there about 50-90% of genes of phages are unable to assign functions. This makes it a challenge to identify phage genomes and annotate functions of phage genes efficiently by homology search on a large scale, especially for newly phages. Portal (portal protein), TerL (large terminase subunit protein), and TerS (small terminase subunit protein) are three specific proteins of Caudovirales phage. Here, we developed a CNN (convolutional neural network)-based framework, DeephageTP, to identify the three specific proteins from metagenomic data. The framework takes one-hot encoding data of original protein sequences as the input and automatically extracts predictive features in the process of modeling. To overcome the false positive problem, a cutoff-loss-value strategy is introduced based on the distributions of the loss values of protein sequences within the same category. The proposed model with a set of cutoff-loss-values demonstrates high performance in terms of Precision in identifying TerL and Portal sequences (94% and 90%, respectively) from the mimic metagenomic dataset. Finally, we tested the efficacy of the framework using three real metagenomic datasets, and the results shown that compared to the conventional alignment-based methods, our proposed framework had a particular advantage in identifying the novel phage-specific protein sequences of portal and TerL with remote homology to their counterparts in the training datasets. In summary, our study for the first time develops a CNN-based framework for identifying the phage-specific protein sequences with high complexity and low conservation, and this framework will help us find novel phages in metagenomic sequencing data. The DeephageTP is available at https://github.com/chuym726/DeephageTP., Competing Interests: The authors declare there are no competing interests., (©2022 Chu et al.)

Advances in synthetic genomics have led to a great demand for genetic manipulation. Trimming any process to simplify and accelerate streamlining of genetic code into life holds great promise for synthesizing and studying organisms. Here, we develop a simple but powerful stepping-stone strategy to promote genome refactoring of viruses in one pot, validated by successful cross-genus and cross-order rebooting of 90 phages infecting 4 orders of popular pathogens. Genomic sequencing suggests that rebooting outcome is associated with gene number and DNA polymerase availability within phage genomes. We integrate recombineering, screening, and rebooting processes in one pot and demonstrate genome assembly and genome editing of phages by stepping-stone hosts in an efficient and economic manner. Under this framework, in vitro assembly, yeast-based assembly, or genetic manipulation of native hosts are not required. As additional stepping-stone hosts are being developed, this framework will open doors for synthetic phages targeting more pathogens and commensals., Competing Interests: The authors have filed a patent application., (© 2022 The Author(s).)

AsXd-1, a bacteriophage that infects Aeromonas salmonicida, was isolated from the wastewater of a seafood market in Shenzhen, China. The 39,014-bp genome of this phage contains 52 open reading frames (ORFs), 30 of which were found to be homologous to reference sequences that putatively encode functional phage proteins. Nine out of the remaining 22 ORFs with unknown functions were unique to AsXd-1. Gene annotation suggests that AsXd-1 has both lysogenic and lytic life cycles. Furthermore, both phylogenetic analysis based on the large subunit of terminase and genome sequence comparisons show that AsXd-1 is closely related to phages belonging to the genus Hk97virus. We thus propose AsXd-1 as a new member of the genus Hk97virus within the family Siphoviridae.

In this study, two bacteriophage isolates, AhSzq-1 and AhSzw-1 that specifically infect Aeromonas hydrophila strain KT998822, were isolated from seawater and characterized. One-step growth curves showed that the latent period of AhSzq-1 and AhSzw-1 are 50 min and 60 min, respectively. The sequence similarities between AhSzq-1 and AhSzw-1 were 88% at the DNA and 83% at the protein level, suggesting that these two phages are representatives of two different species. The virion morphology, DNA genome size and terminal repeats of these two phages are similar to those of viruses classified as T5virus phages. Both phylogenetic analyses and proteomic comparison show that AhSzq-1 and AhSzw-1 group with members of the T5virus genus. We thus propose these two phages as representative isolates of two new species within the T5virus genus.

Background: Substantial efforts have been made to link the gut bacterial community to many complex human diseases. Nevertheless, the gut phages are often neglected., Results: In this study, we used multiple bioinformatic methods to catalog gut phages from whole-community metagenomic sequencing data of fecal samples collected from both type II diabetes (T2D) patients (n = 71) and normal Chinese adults (n = 74). The definition of phage operational taxonomic units (pOTUs) and identification of large phage scaffolds (n = 2567, ≥ 10 k) revealed a comprehensive human gut phageome with a substantial number of novel sequences encoding genes that were unrelated to those in known phages. Interestingly, we observed a significant increase in the number of gut phages in the T2D group and, in particular, identified 7 pOTUs specific to T2D. This finding was further validated in an independent dataset of 116 T2D and 109 control samples. Co-occurrence/exclusion analysis of the bacterial genera and pOTUs identified a complex core interaction between bacteria and phages in the human gut ecosystem, suggesting that the significant alterations of the gut phageome cannot be explained simply by co-variation with the altered bacterial hosts., Conclusions: Alterations in the gut bacterial community have been linked to the chronic disease T2D, but the role of gut phages therein is not well understood. This is the first study to identify a T2D-specific gut phageome, indicating the existence of other mechanisms that might govern the gut phageome in T2D patients. These findings suggest the importance of the phageome in T2D risk, which warrants further investigation.

Cyanophages are abundant in the oceanic environment and directly impact cyanobacterial distributions, physiological processes and evolution. Two samples collected from coastal Maine in July and September 2009 were enriched for Synechococcus cells using flow cytometry and examined through metagenomic sequencing. Homology-based sequence prediction indicated cyanophages, largely myoviruses, accounted for almost half the reads and provided insights into environmental infection events. T4-phage core-gene phylogenetic reconstruction revealed unique diversity among uncultured cyanophages and reference isolates resulting in identification of a new phylogenetic cluster. Genomic comparison of reference cyanophage strains S-SM2 and Syn1 with putative homologous contigs recovered from metagenomes provided evidence that gene insertion, deletion and recombination have occurred among, and are likely important for diversification of, natural populations. Identification of putative genetic exchange between cyanophage and non-cyanophage viruses, i.e. Micromonas virus and Pelagibacter phage, supports hypotheses related to a significant role for viruses in mediating transfer of genetic material between taxonomically diverse organisms with overlapping ecological niches.

Overuse of antibiotics in clinical medicine has contributed to the global spread of multidrug-resistant bacterial pathogens, including Acinetobacter baumannii. We present a case of an 88-year-old Chinese man who developed hospital-acquired pneumonia caused by carbapenem-resistant A. baumannii (CRAB). A personalized lytic pathogen-specific single-phage preparation was nebulized to the patient continuously for 16 days in combination with tigecycline and polymyxin E. The treatment was well tolerated and resulted in clearance of the pathogen and clinical improvement of the patient's lung function. [ABSTRACT FROM AUTHOR]

Vibrio is one of the most detrimental agents of shrimp premature death syndrome. Phage therapy for prevention and treatment of Vibrio infections has attracted increasing attentions due to the emergence of antibiotic-resistant bacterial variants. Here, we describe a workflow of preparing a phage cocktail against Vibrio infections for practical applications. Twenty Vibrio strains were isolated from the gut of diseased shrimp and aquaculture wastewater, and five of them were identified as pathogens causing shrimp vibriosis. Twenty-two lytic phages were then isolated using the above five pathogens as hosts, and five of them showed broad host ranges and high lytic capability against the Vibrio strains. Whole genomic sequencing and phylogenetic analysis of the five phages indicated that they are novel and belong to the Siphoviridae family. The phage cocktail consisting of these five phages showed higher efficiency in inhibiting the growth of pathogenic Vibrio sp. Va-F3 than any single phage in vitro. We then evaluated the performance of the phage cocktail in protecting shrimp against Vibrio sp. Va-F3 infections in situ. The results showed that shrimp survival rates could reach 91.4 and 91.6% in 7 days, for the cocktail-treated and the antibiotic-treated groups, respectively. By contrast, the shrimp survival rate of the group without any treatment was only 20.0%. Overall, this study describes a general workflow of how to prepare a phage cocktail and apply it in controlling bacterial infections in the shrimp aquaculture. Knowledge gained from this study will not only help fight against the shrimp vibriosis in practical but also facilitate the design of phage cocktails with a satisfying performance in controlling other animal diseases in aquaculture. [ABSTRACT FROM AUTHOR]

Salmonella phages SenALZ1 and SenASZ3, two novel phages infecting Salmonella enterica, were isolated and analyzed. The genomes of these two phages consist of 154,811 and 157,630 base pairs (bp), with G+C contents of 44.56% and 44.74%, respectively. Fifty-nine of 199 open reading frames (ORFs) in the SenALZ1 genome, and 60 of the 204 in the SenASZ3 genome show similarity to reference sequences in the NCBI nr database that encode putative phage proteins with predicted functions. Based on the results of transmission electron microscopy (TEM) examination, complete genome sequence alignment, phylogenetic analysis, and gene annotation, we propose that these two phages are representative isolates of two new species of the genus Cba120virus, subfamily Cvivirinae, family Ackermannviridae. [ABSTRACT FROM AUTHOR]