Isolation and Characterization of Bacteriophages against E.coli Urinary Tract Infection and Evaluating their Anti-biofilm Activity and Antibiotic Synergy.

Urinary tract infections (UTIs) by Uropathogenic Escherichia coli (UPEC) are a significant health concern, especially due to the increasing prevalence of antibiotic resistance. This study focuses on isolating and characterizing bacteriophages specific to UPEC strains isolated from UTI samples. The isolated phages were assessed for their ability to target and lyse UPEC in vitro, focusing on their efficacy in disrupting biofilms, a key virulence factor contributing to UTI recurrence and antibiotic resistance. The morphological structure observed by TEM belongs to Myoviridae, the phage exhibited icosahedral symmetry with a long non-constricting tail, the approximate measurement of the phage head was 39nm in diameter, and the phage tail was 105.317nm in length. One-step growth experiments showed that the latent period was approximately 20 min, followed by a rise period of 40 min, and a growth plateau was reached within 20 min and the burst size observed was 26 phages/ infected bacterial cells. These phages were capable of killing cells within the biofilms, leading to a reduction in living cell counts after a single treatment. This study highlights the potential of phages to play a significant role in disrupting, inactivating, and destroying Uropathogenic Escherichia coli (UPEC) biofilms. Such findings could be instrumental in developing treatment strategies that complement antibiotics and disinfectants. The phage-antibiotic synergistic activity was compared to have the possibility to facilitate the advancement of focused and enduring alternatives to traditional antibiotic therapies for UTIs.

A pangenome analysis of ESKAPE bacteriophages: the underrepresentation may impact machine learning models.

Bacteriophages are the most prevalent biological entities in the biosphere. However, limitations in both medical relevance and sequencing technologies have led to a systematic underestimation of the genetic diversity within phages. This underrepresentation not only creates a significant gap in our understanding of phage roles across diverse biosystems but also introduces biases in computational models reliant on these data for training and testing. In this study, we focused on publicly available genomes of bacteriophages infecting high-priority ESKAPE pathogens to show the extent and impact of this underrepresentation. First, we demonstrate a stark underrepresentation of ESKAPE phage genomes within the public genome and protein databases. Next, a pangenome analysis of these ESKAPE phages reveals extensive sharing of core genes among phages infecting the same host. Furthermore, genome analyses and clustering highlight close nucleotide-level relationships among the ESKAPE phages, raising concerns about the limited diversity within current public databases. Lastly, we uncover a scarcity of unique lytic phages and phage proteins with antimicrobial activities against ESKAPE pathogens. This comprehensive analysis of the ESKAPE phages underscores the severity of underrepresentation and its potential implications. This lack of diversity in phage genomes may restrict the resurgence of phage therapy and cause biased outcomes in data-driven computational models due to incomplete and unbalanced biological datasets.

A diverse set of Enterococcus-infecting phage provides insight into phage host-range determinants.

Enterococci are robust Gram-positive bacteria that pose a significant threat in healthcare settings due to antibiotic resistance, with vancomycin-resistant enterococci (VRE) most prominent. To tackle this issue, bacteriophages (bacterial viruses) can be exploited as they specifically and efficiently target bacteria. Here, we successfully isolated and characterised a set of novel phages: SHEF10, SHEF11, SHEF13, SHEF14, and SHEF16 which target E. faecalis (SHEF10,11,13), or E. faecium (SHEF13, SHEF14 & SHEF16) strains including a range of clinical and VRE isolates. Genomic analysis shows that all phages are strictly lytic and diverse in terms of genome size and content, quickly and effectively lysing strains at different multiplicity of infections. Detailed analysis of the broad host-range SHEF13 phage revealed the crucial role of the enterococcal polysaccharide antigen (EPA) variable region in its infection of E. faecalis V583. In parallel, the discovery of a carbohydrate-targeting domain (CBM22) found conserved within the three phage genomes indicates a role in cell surface interactions that may be important in phage-bacterial interactons. These findings advance our comprehension of phage-host interactions and pave the way for targeted therapeutic strategies against antibiotic-resistant enterococcal infections.

Characterization of bacteriophages infecting multidrug-resistant uropathogenic Escherichia coli strains.

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infections, and strains that are resistant to antibiotics are a major problem in treating these infections. Phage therapy is a promising alternative approach that can be used to treat infections caused by polyresistant bacterial strains. In the present study, 16 bacteriophages isolated from sewage and surface water were investigated. Phage host specificity was tested on a collection of 77 UPEC strains. The phages infected 2-44 strains, and 80% of the strains were infected by at least one phage. The susceptible E. coli strains belonged predominantly to the B2 phylogenetic group, including strains of two clones, CC131 and CC73, that have a worldwide distribution. All of the phages belonged to class Caudoviricetes and were identified as members of the families Straboviridae, Autographiviridae, and Drexlerviridae and the genera Kagunavirus, Justusliebigvirus, and Murrayvirus. A phage cocktail composed of six phages - four members of the family Straboviridae and two members of the family Autographiviridae - was prepared, and its antibacterial activity was tested in liquid medium. Complete suppression of bacterial growth was observed after 5-22 hours of cultivation, followed by partial regrowth. At 24 hours postinfection, the cocktail suppressed bacterial growth to 43-92% of control values. Similar results were obtained when testing the activity of the phage cocktail in LB and in artificial urine medium. The results indicate that our phage cocktail has potential to inhibit bacterial growth during infection, and they will therefore be preserved in the national phage bank, serving as valuable resources for therapeutic applications.

Driving through stop signs: predicting stop codon reassignment improves functional annotation of bacteriophages.

The majority of bacteriophage diversity remains uncharacterized, and new intriguing mechanisms of their biology are being continually described. Members of some phage lineages, such as the Crassvirales, repurpose stop codons to encode an amino acid by using alternate genetic codes. Here, we investigated the prevalence of stop codon reassignment in phage genomes and its subsequent impacts on functional annotation. We predicted 76 genomes within INPHARED and 712 vOTUs from the Unified Human Gut Virome Catalogue (UHGV) that repurpose a stop codon to encode an amino acid. We re-annotated these sequences with modified versions of Pharokka and Prokka, called Pharokka-gv and Prokka-gv, to automatically predict stop codon reassignment prior to annotation. Both tools significantly improved the quality of annotations, with Pharokka-gv performing best. For sequences predicted to repurpose TAG to glutamine (translation table 15), Pharokka-gv increased the median gene length (median of per genome median) from 287 to 481 bp for UHGV sequences (67.8% increase) and from 318 to 550 bp for INPHARED sequences (72.9% increase). The re-annotation increased median coding capacity from 66.8% to 90.0% and from 69.0% to 89.8% for UHGV and INPHARED sequences predicted to use translation table 15. Furthermore, the proportion of genes that could be assigned functional annotation increased, including an increase in the number of major capsid proteins that could be identified. We propose that automatic prediction of stop codon reassignment before annotation is beneficial to downstream viral genomic and metagenomic analyses.

Characterization of Salmonella phage of the genus Kayfunavirus isolated from sewage infecting clinical strains of Salmonella enterica.

The emergence of multi-drug resistance in Salmonella, causing food-borne infections, is a significant issue. With over 2,600 serovars in in Salmonella sp., it is crucial to identify specific solutions for each serovar. Phage therapy serves as an alternate treatment option. In this study, vB_SalP_792 phage was obtained from sewage, forming plaques in eight out of 13 tested clinical S. enterica isolates. Transmission electron microscopy (TEM) examination revealed a T7-like morphotype. The phage was characterized by its stability, life cycle, antibiofilm, and lytic ability in food sources. The phage remains stable throughout a range of temperatures (-20 to 70°C), pH levels (3-11), and in chloroform and ether. It also exhibited lytic activity within a range of MOIs from 0.0001 to 100. The life cycle revealed that 95% of the phages attached to their host within 3 min, followed by a 5-min latent period, resulting in a 50 PFU/cell burst size. The vB_SalP_792 phage genome has a dsDNA with a length of 37,281 bp and a GC content of 51%. There are 42 coding sequences (CDS), with 24 having putative functions and no resistance or virulence-related genes. The vB_SalP_792 phage significantly reduced the bacterial load in the established biofilms and also in egg whites. Thus, vB_SalP_792 phage can serve as an effective biocontrol agent for preventing Salmonella infections in food, and its potent lytic activity against the clinical isolates of S. enterica, sets out vB_SalP_792 phage as a successful candidate for future in vivo studies and therapeutical application against drug-resistant Salmonella infections.

Biological Characterization and Genomic Analysis of Three Novel Serratia- and Enterobacter-Specific Virulent Phages.

Due to the high microbiological contamination of raw food materials and the increase in the incidence of multidrug-resistant bacteria, new methods of ensuring microbiological food safety are being sought. One solution may be to use bacteriophages (so-called phages) as natural bacterial enemies. Therefore, the aim of this study was the biological and genomic characterization of three newly isolated Serratia- and Enterobacter-specific virulent bacteriophages as potential candidates for food biocontrol. Serratia phage KKP_3708 (vB_Sli-IAFB_3708), Serratia phage KKP_3709 (vB_Sma-IAFB_3709), and Enterobacter phage KKP_3711 (vB_Ecl-IAFB_3711) were isolated from municipal sewage against Serratia liquefaciens strain KKP 3654, Serratia marcescens strain KKP 3687, and Enterobacter cloacae strain KKP 3684, respectively. The effect of phage addition at different multiplicity of infection (MOI) rates on the growth kinetics of the bacterial hosts was determined using a Bioscreen C Pro growth analyzer. The phages retained high activity in a wide temperature range (from -20 °C to 60 °C) and active acidity values (pH from 3 to 12). Based on transmission electron microscopy (TEM) imaging and whole-genome sequencing (WGS), the isolated bacteriophages belong to the tailed bacteriophages from the Caudoviricetes class. Genomic analysis revealed that the phages have linear double-stranded DNA of size 40,461 bp (Serratia phage KKP_3708), 67,890 bp (Serratia phage KKP_3709), and 113,711 bp (Enterobacter phage KKP_3711). No virulence, toxins, or antibiotic resistance genes were detected in the phage genomes. The lack of lysogenic markers indicates that all three bacteriophages may be potential candidates for food biocontrol.

Optimizing bacteriophage treatment of resistant Pseudomonas.

Phage therapy is increasing in relevance as an alternative treatment to combat antibiotic resistant bacteria. Phage cocktails are the state-of-the-art method of administering phages in clinical settings, preferred over monophage treatment because of their ability to eliminate multiple bacterial strains and reduce resistance formation. In our study, we compare monophage applications and phage cocktails to our chosen method of phage sequential treatments. To do so, we isolated four novel bacteriophages capable of infecting Pseudomonas alcaligenes T3, a close relative of P. aeruginosa, and characterized them using sequencing and transmission electron microscopy. While investigating monophage treatments, we observed that different phage concentrations had a strong impact on the timing and amount of resistance formation. When using phage cocktails, we observed that P. alcaligenes were capable of forming resistance in the same timespan it took them to become resistant to single phages. We isolated mutants resistant to each single phage as well as mutants exposed to phage cocktails, resulting in bacteria resistant to all four phages at once. Sequencing these mutants showed that different treatments yielded unique single nucleotide polymorphism mutation patterns. In order to combat resistance formation, we added phages one by one in intervals of 24 h, thus managing to delay resistance development and keeping bacterial growth significantly lower compared to phage cocktails.IMPORTANCEWHO declared antimicrobial resistance a top threat to global health; while antibiotics have stood at the forefront in the fight against bacterial infection, the increasing number of multidrug-resistant bacteria highlights a need to branch out in order to address the threat of antimicrobial resistance. Bacteriophages, viruses solely infecting bacteria, could present a solution due to their abundance, versatility, and adaptability. For this study, we isolated new phages infecting a fast-mutating Pseudomonas alcaligenes strain capable of forming resistance within 30 h. By using a sequential treatment approach of adding one phage after another, we were able to curb bacterial growth significantly more compared to state-of-the-art phage cocktails.

Antibiotic Resistance Profile and Bio-Control of Multidrug-Resistant Escherichia coli Isolated from Raw Milk in Vietnam Using Bacteriophages.

E. coli is an important zoonotic pathogen capable of causing foodborne illness and bovine mastitis. Bacteriophages have been increasingly considered a promising tool to control unwanted bacteria. The aim of this study is to determine the antibiotic resistance profile of E. coli isolated from raw milk and the efficacy of phage in controlling multidrug-resistant E. coli in raw milk. Antibiotic susceptibility testing showed the highest resistance rates of E. coli isolates to co-trime (27.34%) and ampicillin (27.34%), followed by streptomycin (25.18%), tetracycline (23.02%), and the lowest resistance rates to ciprofloxacin, gentamycin, and ceftazidime, all at a rate of 2.16%. All isolates were susceptible to meropenem. Of the 139 E. coli isolates, 57 (41.01%) were resistant to at least one antibiotic, and 35 (25.18%) were classified as MDR strains. Molecular characterization indicated that 5 (3.6%) out of the 139 isolates were STEC strains carrying stx1 gene. Seven (5.04%) isolates were phenotypically identified as ESBLEC, and four isolates (2.88%) were resistant to colistin. The results of the genotypic test revealed that four out of seven ESBLEC strains carried both blaTEM and blaCTX-M-1, two harbored blaTEM, and one possessed blaCTX-M-1, while mcr-1 was detected in all four colistin-resistant E. coli isolates. In particular, one isolated E. coli strain (EM148) was determined to be a multidrug-resistant strain simultaneously carrying blaTEM, blaCTX-M-1, and mcr-1. A total of eight phages were successfully recovered from raw milk. The application of phage PEM3 significantly reduced viable counts of multidrug-resistant host EM148 in raw milk by at least 2.31 log CFU/mL at both 24 °C and 4 °C.

Characterization and Abundance of Plasmid-Dependent Alphatectivirus Bacteriophages.

Antimicrobial resistance (AMR) is a major public health threat, exacerbated by the ability of bacteria to rapidly disseminate antimicrobial resistance genes (ARG). Since conjugative plasmids of the incompatibility group P (IncP) are ubiquitous mobile genetic elements that often carry ARG and are broad-host-range, they are important targets to prevent the dissemination of AMR. Plasmid-dependent phages infect plasmid-carrying bacteria by recognizing components of the conjugative secretion system as receptors. We sought to isolate plasmid-dependent phages from wastewater using an avirulent strain of Salmonella enterica carrying the conjugative IncP plasmid pKJK5. Irrespective of the site, we only obtained bacteriophages belonging to the genus Alphatectivirus. Eleven isolates were sequenced, their genomes analyzed, and their host range established using S. enterica, Escherichia coli, and Pseudomonas putida carrying diverse conjugative plasmids. We confirmed that Alphatectivirus are abundant in domestic and hospital wastewater using culture-dependent and culture-independent approaches. However, these results are not consistent with their low or undetectable occurrence in metagenomes. Therefore, overall, our results emphasize the importance of performing phage isolation to uncover diversity, especially considering the potential of plasmid-dependent phages to reduce the spread of ARG carried by conjugative plasmids, and to help combat the AMR crisis.

Comprehensive Analysis of Antiphage Defense Mechanisms: Serovar-Specific Patterns.

Leptospirosis is a major zoonotic disease caused by pathogenic spirochetes in the genus Leptospira, affecting over a million people annually and causing approximately 60,000 deaths. Leptospira interrogans, a key causative agent, likely possesses defense systems against bacteriophages (leptophages), yet these systems are not well understood. We analyzed 402 genomes of L. interrogans using the DefenseFinder tool to identify and characterize the antiphage defense systems. We detected 24 unique systems, with CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins), PrrC, Borvo, and Restriction-Modification (R-M) being the most prevalent. Notably, Cas were identified in all strains, indicating their central role in phage defense. Furthermore, there were variations in the antiphage system distribution across different serovars, suggesting unique evolutionary adaptations. For instance, Retron was found exclusively in the Canicola serovar, while prokaryotic Argonaute proteins (pAgo) were only detected in the Grippotyphosa serovar. These findings significantly enhance our understanding of Leptospira's antiphage defense mechanisms. They reveal the potential for the development of serovar-specific phage-based therapies and underscore the importance of further exploring these defense systems.

A Novel Bacteriophage Infecting Multi-Drug- and Extended-Drug-Resistant Pseudomonas aeruginosa Strains.

The prevalence of carbapenem-resistant P. aeruginosa has dramatically increased over the last decade, and antibiotics alone are not enough to eradicate infections caused by this opportunistic pathogen. Phage therapy is a fresh treatment that can be administered under compassionate use, particularly against chronic cases. However, it is necessary to thoroughly characterize the virus before therapeutic application. Our work describes the discovery of the novel sequenced bacteriophage, vB_PaeP-F1Pa, containing an integrase, performs a phylogenetical analysis, describes its stability at a physiological pH and temperature, latent period (40 min), and burst size (394 ± 166 particles per bacterial cell), and demonstrates its ability to infect MDR and XDR P. aeruginosa strains. Moreover, this novel bacteriophage was able to inhibit the growth of bacteria inside preformed biofilms. The present study offers a road map to analyze essential areas for successful phage therapy against MDR and XDR P. aeruginosa infections, and shows that a phage containing an integrase is also able to show good in vitro results, indicating that it is very important to perform a genomic analysis before any clinical use, in order to prevent adverse effects in patients.

Alleviation of lipid metabolic dysfunction through regulation of intestinal bacteriophages and bacteria by green tea polyphenols in Ob/Ob mice.

Green tea polyphenols (GTP) have been shown to ameliorate lipid metabolic disorders by regulating intestinal bacteria. Given the significant role of intestinal bacteriophages in shaping the gut microbiota, this study investigates GTP's influence on gut bacteriophage-bacteria interactions and lipid metabolism using metagenomics and metabonomics. The research results indicated that GTP significantly reduced body weight, serum triglycerides, leptin, insulin resistance, interleukin-6, and TNF-α levels while increasing adiponectin in ob/ob mice fed high-fat diet, aiding intestinal repair. GTP improved gut health by decreasing Enterobacter, Siphoviridae and Enterobacteria_phage_sfv, increasing Bifidobacterium and intestinal metabolites SCFA and hippuric acid. Correlation analysis showed negative correlations between Enterobacter sp. 50,588,862 and Enterobacteria_phages, Shigella_phages with 4-hydroxyphenylpyruvate and hippuric acid. Bifidobacterium choerinum and Bifidobacterium sp. AGR2158 were positively correlated with fatty acids and bile acids. In conclusion, GTP reduced fat accumulation and inflammation, enhanced gut barrier function in obese mice, closely associated with changes in the gut bacteriophage community.

Novel phages of Pseudomonas syringae unveil numerous potential auxiliary metabolic genes.

Relatively few phages that infect plant pathogens have been isolated and investigated. The Pseudomonas syringae species complex is present in various environments, including plants. It can cause major crop diseases, such as bacterial canker on apricot trees. This study presents a collection of 25 unique phage genomes that infect P. syringae. These phages were isolated from apricot orchards with bacterial canker symptoms after enrichment with 21 strains of P. syringae. This collection comprises mostly virulent phages, with only three being temperate. They belong to 14 genera, 11 of which are newly discovered, and 18 new species, revealing great genetic diversity within this collection. Novel DNA packaging systems have been identified bioinformatically in one of the new phage species, but experimental confirmation is required to define the precise mechanism. Additionally, many phage genomes contain numerous potential auxiliary metabolic genes with diversified putative functions. At least three phages encode genes involved in bacterial tellurite resistance, a toxic metalloid. This suggests that viruses could play a role in bacterial stress tolerance. This research emphasizes the significance of continuing the search for new phages in the agricultural ecosystem to unravel novel ecological diversity and new gene functions. This work contributes to the foundation for future fundamental and applied research on phages infecting phytopathogenic bacteria.

Genomic and taxonomic evaluation of 38 Treponema prophage sequences.

BACKGROUND: Despite Spirochetales being a ubiquitous and medically important order of bacteria infecting both humans and animals, there is extremely limited information regarding their bacteriophages. Of the genus Treponema, there is just a single reported characterised prophage. RESULTS: We applied a bioinformatic approach on 24 previously published Treponema genomes to identify and characterise putative treponemal prophages. Thirteen of the genomes did not contain any detectable prophage regions. The remaining eleven contained 38 prophage sequences, with between one and eight putative prophages in each bacterial genome. The prophage regions ranged from 12.4 to 75.1 kb, with between 27 and 171 protein coding sequences. Phylogenetic analysis revealed that 24 of the prophages formed three distinct sequence clusters, identifying putative myoviral and siphoviral morphology. ViPTree analysis demonstrated that the identified sequences were novel when compared to known double stranded DNA bacteriophage genomes. CONCLUSIONS: In this study, we have started to address the knowledge gap on treponeme bacteriophages by characterising 38 prophage sequences in 24 treponeme genomes. Using bioinformatic approaches, we have been able to identify and compare the prophage-like elements with respect to other bacteriophages, their gene content, and their potential to be a functional and inducible bacteriophage, which in turn can help focus our attention on specific prophages to investigate further.

"Tear down that wall"-a critical evaluation of bioinformatic resources available for lysin researchers.

Lytic enzymes, or lysins for short, break down peptidoglycan and interrupt the continuity of the cell wall, which, in turn, causes osmotic lysis of the bacterium. Their ability to destroy bacteria from within makes them promising antimicrobial agents that can be used as alternatives or supplements to antibiotics. In this paper, we briefly summarize basic terms and concepts used to describe lysin sequences and delineate major lysin groups. More importantly, we describe the domain repertoire found in lysins and critically review bioinformatic tools or databases which are used in studies of these enzymes (with particular emphasis on the repositories of Hidden Markov models). Finally, we present a novel comprehensive, meticulously curated set of lysin-related family and domain models, sort them into clusters that reflect major families, and demonstrate that the selected models can be used to efficiently search for new lysins.

Dynamics of the viral community on the surface of a French smear-ripened cheese during maturation and persistence across production years.

The surface of smear-ripened cheeses constitutes a dynamic microbial ecosystem resulting from the successive development of different microbial groups such as lactic acid bacteria, fungi, and ripening bacteria. Recent studies indicate that a viral community, mainly composed of bacteriophages, also represents a common and substantial part of the cheese microbiome. However, the composition of this community, its temporal variations, and associations between bacteriophages and their hosts remain poorly characterized. Here, we studied a French smear-ripened cheese by both viral metagenomics and 16S metabarcoding approaches to assess both the succession of phages and bacterial communities on the cheese surface during cheese ripening and their temporal variations in ready-to-eat cheeses over the years of production. We observed a clear transition of the phage community structure during ripening with a decreased relative abundance of viral species (vOTUs) associated with Lactococcus phages, which were replaced by vOTUs associated with phages infecting ripening bacteria such as Brevibacterium, Glutamicibacter, Pseudoalteromonas, and Vibrio. The dynamics of the phage community was strongly associated with bacterial successions observed on the cheese surface. Finally, while some variations in the distribution of phages were observed in ready-to-eat cheeses produced at different dates spanning more than 4 years of production, the most abundant phages were detected throughout. This result revealed the long-term persistence of the dominant phages in the cheese production environment. Together, these findings offer novel perspectives on the ecology of bacteriophages in smear-ripened cheese and emphasize the significance of incorporating bacteriophages in the microbial ecology studies of fermented foods.IMPORTANCEThe succession of diverse microbial populations is critical for ensuring the production of high-quality cheese. We observed a temporal succession of phages on the surface of a smear-ripened cheese, with new phage communities showing up when ripening bacteria start covering this surface. Interestingly, the final phage community of this cheese is also consistent over large periods of time, as the same bacteriophages were found in cheese products from the same manufacturer made over 4 years. This research highlights the importance of considering these bacteriophages when studying the microbial life of fermented foods like cheese.

Draft genome sequences of two wide-host-range phages of Listeria monocytogenes from food processing environments in the United States.

Listeria monocytogenes is notorious for persistence in food facilities. Phages can significantly impact the ecology of Listeria, but there is a dearth of genome sequence data for Listeria phages from food processing ecosystems. We report the genome sequences of two Listeria phages from turkey processing facilities in the USA.

Complete genome sequence of a Pseudomonas fluorescens bacteriophage UNO-G1W1 isolated from freshwater ice in Nebraska.

We provide the complete genome sequence for a novel Pseudomonas fluorescens bacteriophage named UNO-G1W1. This phage was isolated from a single ice cover sampling. The genome was sequenced on the Nanopore MinION, generated with the direct terminal repeat-phage-pipeline and polished with Illumina short reads. Sequence identity classifies the phage as an otagovirus.