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BACKGROUND: Despite being among the most abundant biological entities on earth, bacteriophage (phage) remain an understudied component of host-associated systems. One limitation to studying host-associated phage is the lack of consensus on methods for sampling phage communities. Here, we compare paired total metagenomes and viral size fraction metagenomes (viromes) as methods for investigating the dsDNA viral communities associated with the GI tract of two bee species: the European honey bee Apis mellifera and the eastern bumble bee Bombus impatiens. RESULTS: We find that viromes successfully enriched for phage, thereby increasing phage recovery, but only in honey bees. In contrast, for bumble bees, total metagenomes recovered greater phage diversity. Across both bee species, viromes better sampled low occupancy phage, while total metagenomes were biased towards sampling temperate phage. Additionally, many of the phage captured by total metagenomes were absent altogether from viromes. Comparing between bees, we show that phage communities in commercially reared bumble bees are significantly reduced in diversity compared to honey bees, likely reflecting differences in bacterial titer and diversity. In a broader context, these results highlight the complementary nature of total metagenomes and targeted viromes, especially when applied to host-associated environments. CONCLUSIONS: Overall, we suggest that studies interested in assessing total communities of host-associated phage should consider using both approaches. However, given the constraints of virome sampling, total metagenomes may serve to sample phage communities with the understanding that they will preferentially sample dominant and temperate phage. Video Abstract.

When proteins evolve new activity, a concomitant decrease in stability is often observed because the mutations that confer new activity can destabilize the native fold. In the conventional model of protein evolution, reduced stability is considered a purely deleterious cost of molecular innovation because unstable proteins are prone to aggregation and are sensitive to environmental stressors. However, recent work has revealed that nonnative, often unstable protein conformations play an important role in mediating evolutionary transitions, raising the question of whether instability can itself potentiate the evolution of new activity. We explored this question in a bacteriophage receptor-binding protein during host-range evolution. We studied the properties of the receptor-binding protein of bacteriophage λ before and after host-range evolution and demonstrated that the evolved protein is relatively unstable and may exist in multiple conformations with unique receptor preferences. Through a combination of structural modeling and in vitro oligomeric state analysis, we found that the instability arises from mutations that interfere with trimer formation. This study raises the intriguing possibility that protein instability might play a previously unrecognized role in mediating host-range expansions in viruses.

Background: Xanthomonas vasicola pv. musacearum is responsible for the widespread Banana Xanthomonas Wilt in banana cultivation regions across the globe. Biocontrol measures for disease management remain limited amidst increasing antimicrobial resistance and unsustainable conventional agricultural practices. The purpose of this study is to explore a viable alternative or adjunct strategy through the use of bacteriophages for disease management. Results: Kintu was isolated from sewage and displayed clear and circular plaques measuring 3 mm. Based on transmission electron microscopy, Kintu displays siphovirus characteristics, including an icosahedral head and a non-contractile tail. Kintu infects 78% (22 out of 28) Ugandan Xvm strains, has an optimal multiplicity of infection of 1, a 10 min adsorption and latent period, a 35 min burst period, and a burst size of 15 particles per bacterium. Phage titers remain stable for two and half months (75 days) in SM buffer at -20 oC and − 40 oC but decrease significantly (p ≤ 0.0001) at 4 oC. Kintu is active at pH 3 and 11, maintains viability at temperatures between 25 oC and 120 oC and tolerates UV irradiation for up to 2 min and 20 s. Kintu inhibits Xvm growth at MOI ratios of 0.1, 1 and 10. The genome is a double stranded DNA molecule that consists of 48,985 base pairs and a G + C content of 51.71%. Antibiotic resistance genes or genes associated with a lysogenic life cycle are absent. There is limited sequence similarity of Kintu with other phages, making it a novel phage belonging to an unclassified genus of the class Caudoviricetes. Conclusion: Kintu is a novel bacteriophage that infects and lyses Xanthomonas vasicola pv. musacearum, the causative agent for Banana Xanthomonas Wilt. Its stability across diverse temperatures and pH conditions highlights its potential as a biocontrol agent for managing the disease. [ABSTRACT FROM AUTHOR]

Antibiotic use can lead to the expansion of multi-drug-resistant pathobionts within the gut microbiome that can cause life-threatening infections. Selective alternatives to conventional antibiotics are in dire need. Here, we describe a Klebsiella PhageBank for the tailored design of bacteriophage cocktails to treat multi-drug-resistant Klebsiella pneumoniae. Using a transposon library in carbapenem-resistant K. pneumoniae , we identify host factors required for phage infection in major Klebsiella phage families. Leveraging the diversity of the PhageBank, we formulate phage combinations that eliminate K. pneumoniae with minimal phage resistance. Optimized cocktails selectively suppress the burden of K. pneumoniae in the mouse gut and drive the loss of key virulence factors that act as phage receptors. Phage-mediated diversification of bacterial populations in the gut leads to co-evolution of phage variants with higher virulence and broader host range. Altogether, the Klebsiella PhageBank charts a roadmap for phage therapy against a critical multidrug-resistant human pathogen. [Display omitted] • A diverse Klebsiella PhageBank is used to construct cocktails that prevent resistance • Host factors required by the taxonomically diverse Klebsiella phage are mapped • Phage cocktails suppress gut K. pneumoniae in mice and select for capsule loss • Antagonistic co-evolution drives phage and bacterial diversification in the gut Leveraging a well-characterized library of bacteriophages, Rotman et al. demonstrate that cocktails of phages can suppress the burden of carbapenem-resistant Klebsiella pneumoniae in the mouse gut microbiome. In the gut, phage-bacterial interactions select for bacterial mutants with lessened virulence and phage variants with broadened host range. [ABSTRACT FROM AUTHOR]

The microbes, nutrients and toxins that we are exposed to can have a profound effect on the composition and function of the gut microbiome. Thousands of peer-reviewed publications link microbiome composition and function to health from the moment of birth, right through to centenarians, generating a tantalizing glimpse of what might be possible if we could intervene rationally. Nevertheless, there remain relatively few real-world examples where successful microbiome engineering leads to beneficial health effects. Here we aim to provide a framework for the progress needed to turn gut microbiome engineering from a trial-and-error approach to a rational medical intervention. The workflow starts with truly understanding and accurately diagnosing the problems that we are trying to fix, before moving on to developing technologies that can achieve the desired changes. In this review, E. Slack and colleagues discuss the potential and challenges of engineering the gut microbiota for medical intervention. [ABSTRACT FROM AUTHOR]

Background: Researchers gradually focus on the relationship between phage and cancer. Objective: To summarize the research hotspots and trends in the field of bacteriophage and cancer. Methods: The downloaded articles were searched from the Web of Science Core Collection database from January 2008 to June 2023. Bibliometric analysis was carried out through CiteSpace, including the analysis of cooperative networks (country/region, institution, and author), co-citations of references, and key words.Visual analysis of three topics, including gut phage, phage and bacteria, and phage and tumor, was conducted. Results: Overall, the United States and China have the most phage-related research. In terms of gut phage, the future research directions are "gut microbiome", "database" and "microbiota". The bursting citations explored the phage-dominated viral genome to discover its diversity and individual specificity and investigated associations among bacteriome, metabolome, and virome. In terms of phage and bacteria, "lipopolysaccharide" and "microbiota" are future research directions. Future research hotspots should mainly concentrate on the further exploration and application of phage properties. As for phages and tumors, the future research directions should be "colorectal cancer", "protein" and "phage therapy". Future directions are likely to focus on the research on phages in cancer mechanisms, cancer diagnosis, and cancer treatment combined with genetic engineering techniques. Conclusion: Phage therapy would become a hot spot and research direction of tumor and phage research, and the relationship between phage and tumor, especially colorectal cancer (CRC), is expected to be further explored. [ABSTRACT FROM AUTHOR]

Streptococcus (S.) equi subspecies (subsp.) zooepidemicus is an important facultative pathogen in horses and can cause severe infections in other species including humans. Facing the post-antibiotic era, novel antimicrobials are needed for fighting bacterial infections. Bacteriophages (phages) are the natural predators of bacteria and discussed as a promising antimicrobial treatment option. The objective of this study was to isolate and characterize S. equi subsp. zooepidemicus- specific phages for the first time and to evaluate their efficacy in vitro and ex vivo. In total, 13 phages with lytic activity were isolated and host ranges were determined. Two phages with broad host ranges and high efficiency of plating (vB_SeqZP_LmqsRe26-2 (lytic activity: 30/37 bacterial isolates) and vB_SeqZP_LmqsRe26-3 (lytic activity: 29/37 bacterial isolates)) and one phage with relatively low efficiency of plating (vB_SeqZP_LmqsRe26-1) were selected for further characterization, including electron microscopy and whole genome sequencing. In in vitro planktonic killing assays at two tested multiplicities of infection (MOI 1 and MOI 10), significant bacterial growth reduction was observed when the phages vB_SeqZP_LmqsRe26-2 and vB_SeqZP_LmqsRe26-3 were added. These phages were subsequently co-incubated with clinical S. equi subsp. zooepidemicus isolates in an equine endometrial explant model but did not achieve bacterial growth reduction at MOI 1 and MOI 10. However, helium ion microscopy revealed presence of particles adherent to the bacteria on the explant after incubation (25 h), suggesting possible phage-bacteria interactions. In conclusion, phages against S. equi subsp. zooepidemicus were successfully isolated and characterized. Promising results were observed in in vitro but no significant reduction was detected in ex vivo experiments, requiring additional investigations. However, after further adaptations (e.g., optimization of MOIs and phage administration or use of phage-antibiotic combination), phages could be a potential antimicrobial tool for future therapeutic use in S. equi subsp. zooepidemicus infections, although the available results do not currently support the therapeutic usage. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii , a predominant nosocomial pathogen, represents a grave threat to public health due to its multiple antimicrobial resistance. Managing patients afflicted with severe infections caused by multiple drug-resistant A. baumannii is particularly challenging, given the associated high mortality rates and unfavorable prognoses. The diminishing efficacy of antibiotics against this superbug underscores the urgent necessity for novel treatments or strategies to address this formidable issue. Bacteriophage-derived polysaccharide depolymerase enzymes present a potential approach to combating this pathogen. These enzymes target and degrade the bacterial cell's exopolysaccharide, capsular polysaccharide, and lipopolysaccharide, thereby disrupting biofilm formation and impairing the bacteria's defense mechanisms. Nonetheless, the narrow host range of phage depolymerases limits their therapeutic efficacy. Despite the benefits of these enzymes, phage-resistant strains have been identified, highlighting the complexity of phage-host interactions and the need for further investigation. While preliminary findings are encouraging, current investigations are limited, and clinical trials are imperative to advance this treatment approach for broader clinical applications. This review explores the potential of phage-derived depolymerase enzymes against A. baumannii infections. [ABSTRACT FROM AUTHOR]

Background: Acinetobacter baumannii is a gram-negative, opportunistic pathogen, that is responsible for a wide variety of infections and is a significant cause of hospital-acquired infections. A. baumannii is listed by the World Health Organization (WHO) as a critical priority pathogen because of its high level of antibiotic resistance and the urgent need for alternative treatment solutions. To address this challenge, bacteriophages have been used to combat bacterial infections for more than a century, and phage research has regained interest in recent years due to antimicrobial resistance (AMR). However, although the vast majority of deaths from the AMR crisis will occur in developing countries in Africa and Asia, few phages' studies have been conducted in these regions. In this study, we present a comprehensive characterization of the bacteriophages vAbBal23 and vAbAbd25, actives against extremely drug-resistant (XDR) A. baumannii. Methods: Phages were isolated from environmental wastewaters in Dakar, Senegal. The host-range, thermal and pH stabilities, infection kinetics, one step growth assay, antibiofilm activity assay, sequencing, and genomic analysis, were performed to characterize the isolated phages. Results: Comparative genomic and phylogenetic analyses revealed that vAbBal23 and vAbAbd25 belong to the Caudoviricetes class, Autographiviridae family and Friunavirus genus. Both phages demonstrated activity against strains with capsular type KL230. They were stable over a wide pH range (pH 3 to 9) and at temperatures ranging from 25 °C to 40 °C. Additionally, the phages exhibited notable activity against both planktonic and biofilm cells of targeted extremely drug resistant A. baumannii. The results presented here indicate the lytic nature of vAbBal23 and vAbAbd25. This is further supported by the absence of genes encoding toxins, resistance genes and bacterial virulence factors, highlighting their potential for future phage applications. Conclusion: Phages vAbBal23 and vAbAbd25 are promising biological agents that can infect A. baumannii, making them suitable candidates for use in phage therapies. [ABSTRACT FROM AUTHOR]

Recent advances highlight the role of gut virome, particularly phageome, in metabolic disorders such as obesity, type 2 diabetes mellitus, metabolic dysfunction‐associated fatty liver disease, and cardiovascular diseases, including hypertension, stroke, coronary heart disease, and hyperlipidemia. While alterations in gut bacteria are well‐documented, emerging evidence suggests that changes in gut viruses also contribute to these disorders. Bacteriophages, the most abundant gut viruses, influence bacterial populations through their lytic and lysogenic cycles, potentially modulating the gut ecosystem and metabolic pathways. Phage therapy, previously overshadowed by antibiotics, is experiencing renewed interest due to rising antibiotic resistance. It offers a novel approach to precisely edit the gut microbiota, with promising applications in metabolic diseases. In this review, we summarize recent discoveries about gut virome in metabolic disease patients, review preclinical and clinical studies of phage therapy on metabolic diseases as well as the breakthroughs and currently faced problems and concerns. [ABSTRACT FROM AUTHOR]

Various directed evolution methods exist that seek to procure bacteriophages with expanded host ranges, typically targeting phage-resistant or non-permissive bacterial hosts. The general premise of these methods involves propagating phage(s) on multiple bacterial hosts, pooling the lysate, and repeating this process until phage(s) can form plaques on the target host(s). In theory, this produces a lysate containing input phages and their evolved phage progeny. However, in practice, this lysate can also include prophages originating from bacterial hosts. Here, we describe our experience implementing one directed evolution method, the Appelmans protocol, to study phage evolution in the Pseudomonas aeruginosa phage–host system, where we observed rapid host-range expansion of the phage cocktail. Further experimentation and sequencing revealed that the observed host-range expansion was due to a Casadabanvirus prophage originating from a lysogenic host that was only included in the first three rounds of the experiment. This prophage could infect five of eight bacterial hosts initially used, allowing it to persist and proliferate until the termination of the experiment. This prophage was represented in half of the sequenced phage samples isolated from the Appelmans experiment, but despite being subjected to directed evolution conditions, it does not appear to have evolved. This work highlights the impact of prophages in directed evolution experiments and the importance of genetically verifying output phages, particularly for those attempting to procure phages intended for phage therapy applications. This study also notes the usefulness of intraspecies antagonism assays between bacterial host strains to establish a baseline for inhibitory activity and determine the presence of prophage. IMPORTANCE Directed evolution is a common strategy for evolving phages to expand the host range, often targeting pathogenic strains of bacteria. In this study, we investigated phage host-range expansion using directed evolution in the Pseudomonas aeruginosa system. We show that prophages are active players in directed evolution and can contribute to observation of host-range expansion. Since prophages are prevalent in bacterial hosts, particularly pathogenic strains of bacteria, and all directed evolution approaches involve iteratively propagating phage on one or more bacterial hosts, the presence of prophage in phage preparations is a factor that needs to be considered in experimental design and interpretation of results. These results highlight the importance of screening for prophages either genetically or through intraspecies antagonism assays during selection of bacterial strains and will contribute to improving the experimental design of future directed evolution studies. [ABSTRACT FROM AUTHOR]

In 1952, Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Over 70 years later, our understanding of bacteriophage structure has grown dramatically, mainly thanks to the cryogenic electron microscopy revolution. In stark contrast, phage genome delivery in prokaryotes remains poorly understood, mainly due to the inherent challenge of studying such a transient and complex process. Here, we review the current literature on viral genome delivery across bacterial cell surfaces. We focus on icosahedral bacterial viruses that we arbitrarily sort into three groups based on the presence and size of a tail apparatus. We inventory the building blocks implicated in genome delivery and critically analyze putative mechanisms of genome ejection. Bacteriophage genome delivery into bacteria is a topic of growing interest, given the renaissance of phage therapy in Western medicine as a therapeutic alternative to face the antibiotic resistance crisis. [ABSTRACT FROM AUTHOR]

Background: Periprosthetic Joint Infection (PJI) following hip and knee arthroplasty is a catastrophic complication in orthopaedic surgery. It has long been a key focus for orthopaedic surgeons in terms of prevention and management. With the increasing incidence of antibiotic resistance in recent years, finding more targeted treatment methods has become an increasingly urgent issue. Bacteriophage Therapy (BT) has emerged as a promising adjunctive treatment for bone and joint infections in recent years. It not only effectively kills bacteria but also demonstrates significant anti-biofilm activity, garnering substantial clinical interest due to its demonstrated efficacy and relatively low incidence of adverse effects. Purpose: This review aims to systematically evaluate the efficacy and safety of bacteriophage therapy in treating PJI following hip and knee arthroplasty, providing additional reference for its future clinical application. Methods: Following predefined inclusion and exclusion criteria, our team conducted a systematic literature search across seven databases (PubMed, Embase, Web of Science, Cochrane Library, ClinicalTrials.gov, CNKI, and WanFang Database). The search was conducted up to May 2024 and included multiple clinical studies on the use of bacteriophage therapy for treating PJI after hip and knee arthroplasty to assess its efficacy and safety. Results: This systematic review included 16 clinical studies after screening, consisting of 15 case reports and one prospective controlled clinical trial, involving a total of 42 patients with PJI treated with bacteriophage therapy. The average patient age was 62.86 years, and 43 joints were treated, with patients undergoing an average of 5.25 surgeries. The most common pathogen in these infections was Staphylococcus aureus, accounting for 18 cases. 33 patients received cocktail therapy, while nine were treated with a single bacteriophage preparation. Additionally, all patients underwent suppressive antibiotic therapy (SAT) postoperatively. All patients were followed up for an average of 13.55 months. There were two cases of recurrence, one of which resulted in amputation one year postoperatively. The remaining patients showed good recovery outcomes. Overall, the results from the included studies indicate that bacteriophage therapy effectively eradicates infectious strains in various cases of PJI, with minimal side effects, demonstrating promising clinical efficacy. Conclusion: In the treatment of PJI following hip and knee arthroplasty, bacteriophages, whether used alone or in combination as cocktail therapy, have shown therapeutic potential. However, thorough preoperative evaluation is essential, and appropriate bacteriophage types and treatment regimens must be selected based on bacteriological evidence. Future large-scale, randomized controlled, and prospective trials are necessary to validate the efficacy and safety of this therapy. [ABSTRACT FROM AUTHOR]

Ensuring food safety measures are essential to minimize the risk of foodborne diseases linked to raw food products. Here, we investigated the efficacy of an innovative approach for the control of Salmonella Typhimurium found in fresh produces. Plasma activated water (PAW) and bacteriophages are emerging effective and valuable alternative methods for microbiological decontamination. The efficacy of PAW and a lytic bacteriophage (109 PFU/mL), both separately and sequentially, against S. Typhimurium in fresh produce was investigated. S. Typhimurium (105–107 CFU/g) were inoculated on lettuce leaves and treated with PAW, S. Typhimurium phage SK-T2 or their combination. PAW or bacteriophage inactivated S. Typhimurium, on lettuce leaves at different initial populations, by 2.90–3.46 or 1.45–3.25 log CFU/g, respectively. After sequential treatments of PAW and bacteriophage, S. Typhimurium populations, initially applied at ~ 105 CFU/g reduced by 4.47 log CFU/g, but when the order of application was changed (i.e., bacteriophage followed by PAW), the combination synergistically decreased the Salmonella numbers below the detection limit of the method used for the enumeration (i.e., < 101 CFU/g). At the high-level inoculum (~ 7 log CFU/g), consecutive treatments of PAW and phage decreased the S. Typhimurium population by 3.28 log CFU/g, and a reduction of 6.20 log CFU/g was achieved after reversing the order of treatment. Regardless of the bacterial inoculum level, sequential applications of bacteriophage and PAW resulted in a higher level of inactivation. This study proved that the bacteriophage–PAW combination constitutes a promising alternative approach to the conventional washing process in fresh produce wash waters in the food industry. [ABSTRACT FROM AUTHOR]

Bacterial cells often exist in the form of sessile aggregates known as biofilms, which are polymicrobial in nature and can produce slimy Extracellular Polymeric Substances (EPS). EPS is often referred to as a biofilm matrix and is a heterogeneous mixture of various biomolecules such as polysaccharides, proteins, and extracellular DNA/RNA (eDNA/RNA). In addition, bacteriophage (phage) was also found to be an integral component of the matrix and can serve as a protective barrier. In recent years, the roles of proteins, polysaccharides, and phages in the virulence of biofilms have been well studied. However, a mechanistic understanding of the release of such biomolecules and their interactions with antimicrobials requires a thorough review. Therefore, this article critically reviews the various mechanisms of release of matrix polymers. In addition, this article also provides a contemporary understanding of interactions between various biomolecules to protect biofilms against antimicrobials. In summary, this article will provide a thorough understanding of the functions of various biofilm matrix molecules. [ABSTRACT FROM AUTHOR]

Background/Objectives: Avian pathogenic Escherichia coli (APEC) infection is a significant problem for the global chicken industry, as it decreases animal welfare and is associated with substantial economic losses. Traditionally, APEC infections have been controlled through the use of antibiotics, which has led to an increased prevalence of antibiotic-resistant E. coli. Therefore, developing alternative treatments for APEC infection is crucial. Methods: In this study, an Escherichia phage specific to multidrug-resistant (MDR) APEC, designated as phage vB_EcoP_PW8 (phage vECPW8), was isolated. The morphology, phage adsorption to host cells, one-step growth curve, thermal stability, pH stability, whole-genome sequencing, antibacterial ability, and antibiofilm efficacy of phage vECPW8 were evaluated. Results: The results demonstrated that phage vECPW8 has a Podoviridae morphology and is effective at lysing bacteria. Phage vECPW8 exhibited a high absorption rate to bacterial cells (more than 85% within 10 min) and had a latent period of 20 min, with a burst size of 143 plaque-forming units per cell. Additionally, phage vECPW8 showed good temperature and pH stability. The phage displayed strong antibacterial activity in vitro, and its efficacy in controlling bacteria was confirmed through scanning electron microscopy. Whole-genome sequencing revealed that the phage has a linear genome with 69,579 base pairs. The genome analysis supported the safety of the phage, as no toxin, virulence, or resistance-related genes were detected. Phage vECPW8 was identified as a novel lytic phage in the Gamaleyavirus genus and Schitoviridae family. The phage also demonstrated antibiofilm efficacy by reducing and preventing biofilm formation, as evidenced by biofilm biomass and bacterial cell viability measurements. Conclusions: These results indicate that phage vECPW8 is a promising candidate for the effective treatment of MDR APEC infections in poultry. [ABSTRACT FROM AUTHOR]

Burkholderia cepacia complex is a cause of serious lung infections in people with cystic fibrosis, exhibiting extremely high levels of antimicrobial resistance. These infections are difficult to treat and are associated with high morbidity and mortality. With a notable lack of new antibiotic classes currently in development, exploring alternative antimicrobial strategies for Burkholderia cepacia complex is crucial. One potential alternative seeing renewed interest is the use of bacteriophage (phage) therapy. This review summarises what is currently known about Burkholderia cepacia complex in cystic fibrosis, as well as challenges and insights for using phages to treat Burkholderia cepacia complex lung infections. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is a prevalent endocrine disorder characterized by chronic hyperglycemia. Chronic wounds in diabetic patients are a major concern, with high morbidity and mortality rates due to lower-extremity amputations and death. Impaired wound healing in diabetes is a complex process because of various factors like elevated oxidative stress, prolonged inflammation, and high bacterial burden. Understanding the differences in molecular and immunological responses between normal and diabetic wound healing is vital. Hence, this review highlights the impact of hyperglycemia and the polyol pathway on mitochondrial dysfunction in diabetic wounds. It explores the potential of various nanoparticles such as inorganic, organic, biomaterials, and hydrogels as a novel therapeutic strategy for diabetic wound healing through improved its efficacy. In addition, it focuses on the mechanisms by which nanoparticles can influence molecular and pathophysiological changes to promote healing. It emphasizes the growing interest in advanced approaches like hydrogels and liposome-encapsulated bacteriophages or peptides for future applications. Overall, this review examines the advancements in nanoparticle-based therapies specifically designed to revolutionize diabetic wound healing in patients. [ABSTRACT FROM AUTHOR]

Bacillus tropicus is a recently identified subspecies of the Bacillus cereus group of bacteria that have been shown to possess genes associated with antimicrobial resistance (AMR) and identified as the causative agent for anthrax-like disease in Chinese soft-shelled turtles. In addition, B. tropicus has demonstrated great potential in the fields of bioremediation and bioconversion. This article describes the comparative genomics of a Bacillus phage vB_Btc-RBClinn15 (referred to as RBClin15) infecting the recently identified B. tropicus AOA-CPS1. RBClin15 is a temperate phage with a putative parABS partitioning system as well as an arbitrium system, which are presumed to enable extrachromosomal genome maintenance and regulate the lysis/lysogeny switch, respectively. The temperate phage RBClin15 has been sequenced however, was erroneously deposited as a plasmid in the NCBI GenBank database. A BLASTn search against the GenBank database using the whole genome sequence of RBClin15 revealed seven other putative temperate phages that were also deposited as plasmids in the database. Comparative genomic analyses shows that RBClin15 shares between 87 and 92% average nucleotide identity (ANI) with the seven temperate phages from the GenBank database. All together RBClin15 and the seven putative temperate phages share common genome arrangements and < 29% protein homologs with the closest phages, including 0105phi7-2. A phylogenomic tree and proteome-based phylogenetic tree analysis showed that RBClin15 and the seven temperate phages formed a separate branch from the closest phage, 0105phi7-2. In addition, the intergenomic similarity between RBClin15 and its closely related phages ranged between 0.3 and 47.7%. Collectively, based on the phylogenetic, and comparative genomic analyses, we propose three new species which will include RBClin15 and the seven temperate phages in the newly proposed genus Theosmithvirus under Caudoviricetes. [ABSTRACT FROM AUTHOR]

Despite the intensive research on gut microbiome-associated diseases over the past 20 years, pharmacological methods for effectively eliminating pathobionts remain unsatisfactory. This study investigated the therapeutic potential of bacteriophages against Enterococcus faecalis, in which bacterial tyrosine decarboxylase (TDC) converts orally administered levodopa (L-DOPA) to dopamine, in an MPTP mouse model of Parkinson's disease (PD). E. faecalis bacteriophages PBEF62, PBEF66, and PBEF67 (4 × 1010 PFU total/200 µl/day), and E. faecalis cells (2 × 109 CFU/200 µl/day) were orally administered at 2-h intervals before every MPTP (i.p.) and/or L-DOPA (p.o.) treatments for 13 days. The relative abundances of E. faecalis cells and bacteriophages in the feces peaked at 4 and 12 h after administration and gradually decreased by 12 and 48 h, respectively. While the administration of E. faecalis cells eliminated the beneficial effect of L-DOPA on MPTP-induced behavioral deficits, as assessed by cylinder and rotarod tests, the co-administration of bacteriophages with bacterial cells restored this effect. The modulating effects of L-DOPA, E. faecalis, and bacteriophages on PD behavior were closely associated with choline acetyltransferase expression levels in the striatum but not with tyrosine hydroxylase in the substantia nigra of each group. Recurrence and extinction of PD behaviors following treatment with E. faecalis and/or bacteriophages were also coincident with the dopamine levels in the blood and brain tissues of PD mice. The effectiveness of L-DOPA was restored after the three types of E. faecalis bacteriophages selectively eliminated E. faecalis cells, along with the TDC gene copies and transcripts responsible for converting L-DOPA to dopamine in the gastrointestinal tract. In conclusion, a combination of bacteriophages PBEF62, PBEF66, and PBEF67 targeting E. faecalis demonstrates potential as a valuable supplement to L-DOPA therapy for PD. [ABSTRACT FROM AUTHOR]

Background: The extensively drug-resistant (XDR) strains of Acinetobacter baumannii have become a major cause of nosocomial infections, increasing morbidity and mortality worldwide. Many different treatments, including phage therapy, are attractive ways to overcome the challenges of antibiotic resistance. Methods: This study investigates the biofilm formation ability of 30 XDR A. baumannii isolates and the efficacy of a cocktail of four tempetate bacteriophages (SA1, Eve, Ftm, and Gln) and different antibiotics (ampicillin/sulbactam, meropenem, and colistin) in inhibiting and degrading the biofilms of these strains. Results: The majority (83.3%) of the strains exhibited strong biofilm formation. The bacteriophage cocktail showed varying degrees of effectiveness against A. baumannii biofilms, with higher concentrations generally leading to more significant inhibition and degradation rates. The antibiotics-bacteriophage cocktail combinations also enhanced the inhibition and degradation of biofilms. Conclusion: The findings suggested that the bacteriophage cocktail is an effective tool in combating A. baumannii biofilms, with its efficacy depending on the concentration. Combining antibiotics with the bacteriophage cocktail improved the inhibition and removal of biofilms, indicating a promising strategy for managing A. baumannii infections. These results contribute to our understanding of biofilm dynamics and the potential of bacteriophage cocktails as a novel therapeutic approach to combat antibiotic-resistant bacteria. [ABSTRACT FROM AUTHOR]

Dental infections, such as apical Periodontitis, periodontitis, and peri-implantitis (PI), are closely associated with specific bacterial species, including Streptococcus mutans (S. mutans), Porphyromonas gingivalis (P. gingivalis), and Fusobacterium nucleatum (F. nucleatum), among others. Antibiotics are extensively utilized for prophylactic and therapeutic purposes in the treatment of dental infections and other dental-related issues. Unfortunately, the rapid emergence of antimicrobial resistance has accompanied the increased use of antibiotics in recent years. Specific bacterial pathogens have reached a critical stage of antibiotic resistance, characterized by the proliferation of pan-resistant strains and the scarcity of viable therapeutic alternatives. Therapeutic use of particular bacteriophage (phage) particles that target bacterial pathogens is one potential alternative to antibiotics that are now being seriously considered for treating bacterial illnesses. A kind of virus known as a phage is capable of infecting and eliminating bacteria. Because they can't infect cells in plants and animals, phages might be a harmless substitute for antibiotics. To control oral disorders including periodontitis and dental caries, several research have been conducted in this area to study and identify phages from human saliva and dental plaque. The capacity of these agents to disturb biofilms expands their effectiveness against dental plaque biofilms and oral pathogens in cases of periodontitis, PI, and apical periodontitis. This review summarizes the current antibacterial properties of phages used to treat a variety of dental infections, such as periodontitis, peri-implantitis, infected dentin, and apical periodontitis. [ABSTRACT FROM AUTHOR]

Phage display is widely used in biomedical research. One of the great advantages of phage display is the specificity of the connection of a foreign peptide exposed outside the capsid to the intended target. Secondary detection systems, which are often laborious and costly, are required to identify and quantify the peptide/target interaction. In this study, we generated a novel dual-display phage to facilitate the detection and quantification of the peptide/target interaction. First, we generated a biotin-tagged phage by adding a small biotin-accepting peptide (sBT) to gene-3 of the M13K07 helper phage. Subsequently, we enhanced the M13K07 biotin-tagged phage by incorporating a selective peptide on gene-8, which is then exposed to the phage capsid. The exposed peptide acts as a probe to bind to a selective molecular target, whose interaction can be readily visualized thanks to the biotinylated phage. Our versatile dual-display phage exhibits high flexibility; by swapping the displayed peptide/probe, one can change the phage target while retaining the sBT gene in-frame with the pIII. We expect the generated biotin-tagged dual phages to be used as a multifunctional probe to couple with several streptavidin-biotin-based systems. [ABSTRACT FROM AUTHOR]

Background: Increased prevalence of Enterobacter cloacae within food products underscores food as an underexplored reservoir for antibiotic resistance, thus requiring particular intervention. Bacteriophages have been explored as a promising approach for controlling bacterial growth in different matrices. Moreover, their specific interaction and self-replication, put them apart from traditional methods for controlling bacteria in different matrices. Methods: Sixteen Enterobacter cloacae strains were recovered from raw chicken. These strains were used to isolate bacteriophages using enrichment protocol. The broad-spectrum bacteriophage was evaluated in terms of thermal, pH, shearing stress and storge. Moreover, its infection kinetics, in vitro antibacterial activity, cytotoxicity were also assessed. Genomic sequencing was performed to exclude any potential virulence or resistance genes. Finally, the capability of the isolated phages to control bacterial growth in different chicken samples was assessed alone and in combination with sodium nitrite. Results: The lytic bacteriophage vB_EclM_HK6 was isolated and showed the broadest spectrum being able to infect 8/16 E. cloacae strains with a lytic activity against its host strain, E. cloacae EC21, as low as MOI of 10–6. The phage displays a latent period of 10 min and burst size of 115 ± 44 and resistance frequency of 5.7 × 10–4 ± 3.0 × 10–4. Stability assessment revealed a thermal tolerance up to 60 ˚C, wide range pH stability (3–10) and the ability to withstand shearing stress up to 250 rpm. HK6 shows no cytotoxicity against oral epithelial cells up to 1012 PFU/ml. Genomic analysis revealed a Strabovirus with total size of 177,845 bp that is free from known resistance and virulence genes. Finally, HK6 pretreatment of raw chicken, chicken nuggets and ready-made cheese salad shows a reduced bacterial count up to 4.6, 2.96 and 2.81 log-units, respectively. Moreover, combing HK6 with sodium nitrite further improved the antibacterial activity in both raw chicken and chicken nuggets without significant enhancement in case of cheese salad. Conclusion: Enterobacter bacteriophage vB_EclM_HK6 presents a safe and effective approach for controlling E. cloacae contaminating stored chicken food samples. Moreover, they could be combined with a reduced concentrations of sodium nitrite to improve the killing capacity. [ABSTRACT FROM AUTHOR]

Background: Foodborne disease and food spoilage are the prime public health issue and food security round the globe. Significant disease outbreaks mostly linked to the existence of pathogenic bacteria that extremely challenging due to the persistence of biofilm-forming. Proteins and bacterial metabolites have been shown to have good antibacterial activity and effectively removal bacterial biofilm. Recently, bacteriophage and their encoded lytic proteins such as lysin have attracted attention as potential alternative agent to control undesirable pathogens in human body infection, increasing food safety as advance preservations and medical treatment such as phage therapy. For these reasons, the efficacy of bacteriophage and their potential in combination with bacterial metabolites from Phyllosphere and Actinomycetes bacteria (Pseudomonas fluorescens JB3B and Streptomyces thermocarboxydus 18PM crude extracts) was the aim of this present study. Results: In this study, bacteriophage BC-VP (1.28 ± 0.29 × 1011 PFU/ml) and ETEC-phage-TG (8.9 ± 2.19 × 108 PFU/ml) isolated from artificial lake water from previous study showed potential activity to control Bacillus cereus (BC) and Enterotoxigenic Escherichia coli (ETEC) population. The combination of BC-VP with metabolite (P. fluorescens JB3B and S. thermocarboxydus 18PM) which were known from previous study had antibiofilm activities were able to inhibit (86.1%; 83.3%) and destruct (41%; 45.5%) biofilm formation of B. cereus respectively. Likewise, the synergy of bacteriophage ETEC-phage-TG with the same crude extract also showed promising activity against biofilm of ETEC with percentage of inhibition (81.9%; 76.4%) and percentage of destruction (54.1%; 44.4%). Application in various food, combination of BC-VP and bacterial metabolite extract (P. fluorescens JB3B; S. thermocarboxydus 18PM) were able to reduce Bacillus cereus population in mashed potato (99.6%; 99.4%) at cold temperature (4 °C) and (68.9%; 56.6%) at room temperature (28 °C), boiled pasta (99.5%; 99.4%) and (84.7%; 75.7%), also soymilk (96.9%; 96.7%) and (42.4%; 39.4%) respectively. Likewise, combination of ETEC-phage-TG and bacterial metabolite (P. fluorescens JB3B; S. thermocarboxydus 18PM) potentially reduced ETEC population after two different temperatures (4 °C and 28 °C) incubation in bean sprouts (TFTC; TFTC) and (47.5%; 49.1%), chicken meat (TFTC; TFTC) and (58.1%; 54%), also minced beef (99.5%; 99.4%) and (41.1%; 28%). GC-MS determination performed, oxalic acid, phenol, phenylethyl alcohol, N-hexadecanoic acid, and pyrolol[1,2-a]pyrazine-1,4-dione, hexadro-3-92-methylpropyl was the most active compound in P. fluorescens JB3B. 2,4-Di-tert-butylphenol, phenyl acetic acid, N-Hexadecanoic acid, pyrolol[1,2-a]pyrazine-1,4-dione, hexadro-3-92-methylpropyl, and Bis(2-ethylhexyl) phthalate was most active compound in the S. thermocarboxydus 18PM isolates. Conclusions: The combination of isolated bacteriophages and bacterial metabolite showed promising results to be used as biocontrol candidate to overcome biofilm formed by foodborne and food spoilage bacteria using their ability to produce antibiofilm compounds and lytic activity. In addition, this combination also potentially reduces the use or replace the drawbacks of common application such as antibiotic treatment. [ABSTRACT FROM AUTHOR]

Intratumoral microbiota (IM) has emerged as a significant component of the previously thought sterile tumor microenvironment (TME), exerting diverse functions in tumorigenesis and immune modulation. This review outlines the historical background, classification, and diversity of IM, elucidating its pivotal roles in oncogenicity, cancer development, and progression, alongside its influence on anti-tumor immunity. The signaling pathways through which IM impacts tumorigenesis and immunity, including reactive oxygen species (ROS), β-catenin, stimulator of interferon genes (STING), and other pathways [NF-κB, Toll-like receptor (TLR), complement, RhoA/ROCK, PKR-like ER kinase (PERK)], are discussed comprehensively. Furthermore, we briefly introduce the clinical implications of IM, emphasizing its potential as a target for novel cancer therapies, diagnostic biomarkers, and prognostic indicators. Notably, microbe-based therapeutic strategies such as fecal microbiome transplantation (FMT), probiotics regulation, bacteriotherapy, bacteriophage therapy, and oncolytic virotherapy are highlighted. These strategies hold promise for enhancing the efficacy of current cancer treatments and warrant further exploration in clinical settings. [ABSTRACT FROM AUTHOR]

Background/Objectives: The World Health Organization has selected enterococci as one of the priority multidrug-resistant microorganisms for the development of new antibacterial drugs. Bacteriophages are promising antibacterial agents, but the biology of bacteriophages requires deeper understanding. Methods: The vB_EfS_SE phage which is capable of infecting four species of the genus Enterococci was isolated from sewage plant. The complete genome of the vB_EfS_SE phage was sequenced using illumina technology. The endolysin gene was cloned into pBAD18 expression vector. Two chimeric endolysins were engineered using the vB_EfS_SE carbohydrate-binding domain (CBD) and replacing its enzymatically active domain (EAD). Results: The bacteriophage exhibits promising lytic properties and persists at temperatures of 40 °C and below, and under pH conditions ranging from 5 to 11. The genome sequence is 57,904 bp in length. The vB_EfS_SE endolysin PlySE and chimeric endolysins PlyIME-SE and PlySheep-SE were found to have the same range of specificity, but different thermostability properties and a different pH range for enzyme activity. Conclusions: Taking together the results obtained in this work and other published studies, we can highly appreciate the potential of Saphexavirus phages and their endolysins as novel antibacterial compounds. [ABSTRACT FROM AUTHOR]

Agricultural crop yield losses and food destruction due to infections by phytopathogenic bacteria such as Burkholderia gladioli, which causes devastating diseases in onion, mushroom, corn, and rice crops, pose major threats to worldwide food security and cause enormous damage to the global economy. Biocontrol using bacteriophages has emerged as a promising strategy against a number of phytopathogenic species but has never been attempted against B. gladioli due to a lack of quantitative infection models and a scarcity of phages targeting this specific pathogen. In this study, we present a novel, procedurally straightforward, and highly generalizable fully quantitative ex planta maceration model and an accompanying quantitative metric, the ex planta maceration index (xPMI). In utilizing this model to test the ex planta virulence of a panel of 12 strains of B. gladioli in Allium cepa and Agaricus bisporus, we uncover substantial temperature-, host-, and strain-dependent diversity in the virulence of this fascinating pathogenic species. Crucially, we demonstrate that Burkholderia phages KS12 and AH2, respectively, prevent and reduce infection-associated onion tissue destruction, measured through significant (P < 0.0001) reductions in xPMI, by phytopathogenic strains of B. gladioli, thereby demonstrating the potential of agricultural phage biocontrol targeting this problematic microorganism. IMPORTANCE Agricultural crop destruction is increasing due to infections caused by bacteria such as Burkholderia gladioli, which causes plant tissue diseases in onion, mushroom, corn, and rice crops. These bacteria pose a major threat to worldwide food production, which, in turn, damages the global economy. One potential solution being investigated to prevent bacterial infections of plants is "biocontrol" using bacteriophages (or phages), which are bacterial viruses that readily infect and destroy bacterial cells. In this article, we demonstrate that Burkholderia phages KS12 and AH2 prevent or reduce infection-associated plant tissue destruction caused by strains of B. gladioli, thereby demonstrating the inherent potential of agricultural phage biocontrol. [ABSTRACT FROM AUTHOR]

Despite many efforts to understand and leverage the functional potential of environmental viromes, most bacteriophage genes are largely uncharacterized. To explore novel biology from uncultivated microbes like phages, metagenomics has emerged as a powerful tool to directly mine new genes without the need to culture the diverse microbiota and the viruses within. When a pure computational approach cannot infer gene function, it may be necessary to create a DNA library from environmental genomic DNA, followed by the screening of that library for a particular function. However, these screens are often initiated without a metagenomic analysis of the completed DNA library being reported. Here, we describe the construction and characterization of DNA libraries from a single cultured phage (FT4), five cultured Escherichia coli phages, and three metagenomic viral sets built from freshwater, seawater, and wastewater samples. Through next-generation sequencing of five independent samplings of the libraries, we found a consistent number of recovered genes per replicate for each library, with many genes classifiable via the KEGG and Pharokka databases. By characterizing the size of the genes and inserts, we found that our libraries contain a median of one to two genes per contig with a median gene length of 303-381 bp for all libraries, reflective of the small genomes of viruses. The environmental libraries were genetically diverse compared to the single phage and multi-phage libraries. Additionally, we found reduced coverage of individual genomes when five phages were used as opposed to one. Taken together, this work provides a comprehensive analysis of the DNA libraries from phage genomes that can be used for metagenomic exploration and functional screens to infer and identify new biology. IMPORTANCE Functional metagenomics is an approach that aims to characterize the putative biological function of genes in the microbial world. This includes an examination of the sequencing data collected from a pooled source of diverse microbes and inference of gene function by comparison to annotated and studied genes from public databases. At times, DNA libraries are made from these genes, and the library is screened for a specific function. Hits are validated using a combination of biological, computational, and structural analysis. Left unresolved is a detailed characterization of the library, both its diversity and content, for the purposes of imputing function entirely by computational means, a process that may yield findings that aid in designing useful screens to identify novel gene functions. In this study, we constructed libraries from cultured phages and uncultured viromes from the environment and characterized some important parameters, such as gene number, genes per contig, ratio of hypothetical to known proteins, total genomic coverage and recovery, and the effect of pooling genetic information from multiple sources, to provide a better understanding of the nature of these libraries. This work will aid the design and implementation of future screens of pooled DNA libraries to discover and isolate viral genes with novel biology across various biomes. [ABSTRACT FROM AUTHOR]

Background: Infections following shoulder surgery, particularly periprosthetic joint infection (PJI), are challenging to treat. Cutibacterium acnes is the causative pathogen in 39% to 76% of these cases. This study explores the efficacy of bacteriophage therapy as an alternative to conventional antibiotics for treating such infections. Methods: Nine phages with lytic activity were isolated from the skin of humans using C. acnes ATCC 6919 as the indicator host. These phages were tested individually or in combination to assess host range and antibiofilm activity against clinical strains of C. acnes associated with PJIs. The phage cocktail was optimized for broad-spectrum activity and tested in vitro against biofilms formed on titanium discs to mimic the prosthetic environment. Results: The isolated phages displayed lytic activity against a range of C. acnes clinical isolates. The phage cocktail significantly reduced the bacterial load of C. acnes strains 183, 184, and GG2A, as compared with untreated controls (p < 0.05). Individual phages, particularly CaJIE7 and CaJIE3, also demonstrated significant reductions in bacterial load with respect to specific strains. Moreover, phages notably disrupted the biofilm structure and reduced biofilm biomass, confirming the potential of phage therapy in targeting biofilm-associated infections. Conclusions: Our preclinical findings support the potential of phage therapy as a viable adjunct to traditional antibiotics for treating C. acnes infections in orthopedic device-related infections. The ability of phages to disrupt biofilms may be particularly beneficial for managing infections associated with prosthetic implants. [ABSTRACT FROM AUTHOR]

The poultry industry is facing challenges from multiple drug-resistant pathogens such as Cam‐ pylobacter, Salmonella, Escherichia coli, and Clostridium perfringens. Excessive use of antibiotics disrupts the balance of the normal microbial community in the intestines, leading to dysbiosis, immune suppression, and secondary infections. Bacteriophages are viruses that can specifically infect and kill bacteria. The use of bacteriophage preparations to control bacterial diseases in poultry is of great significance for ensuring poultry health and improving farming efficiency. Therefore, this review focuses on the current applications of bacteriophages against poultry pathogens, aiming to provide a comprehensive understanding of the potential of bacteriophages in poultry farming and to serve as an important reference for future research and applications. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the effects of dietary bacteriophage administration on diarrhea and intestinal impairment induced by enterotoxigenic Escherichia coli (ETEC) in a newly weaned mouse model. Forty-four newly weaned C57BL/6 mice were divided into four treatment groups, where they were provided either the control diet or the bacteriophage-supplemented diet, with or without ETEC infection. The results show that the bacteriophage administration resulted in increased body weight, decreased diarrhea score, and improved jejunal histopathology in ETEC-infected mice. The bacteriophage administration enhanced the intestinal barrier function of the ETEC-infected mice, as indicated by the reduced serum DAO level and the increased expression of Claudin-1, Occludin, and ZO-1 at both the mRNA and protein levels in the jejunum. Also, the bacteriophage administration resulted in a decrease in serum TNF-α and IL-1β levels, a down-regulation of TNF-α and IL-6 mRNA levels in the jejunum, and the inhibition of jejunal TLR-4/NF-κB pathway activation induced by ETEC infection. Moreover, the bacteriophage administration increased the levels of acetic acid, propionic acid, butyric acid, and total short-chain fatty acids in the caecum content. The bacteriophage administration increased the Shannon index, increased the abundance of Bacteroidota and Muribaculaceae, and decreased the abundance of Verrucomicrobiota and Akkermansiaceae in the colon contents of the ETEC-infected mice. Spearman's correlation analysis indicates that the protective effects of bacteriophage on ETEC-induced intestinal impairment, inflammation, and intestinal barrier function are associated with regulating the abundance of Bacteroidota and Muribaculaceae in the colon contents of mice. Collectively, bacteriophage administration alleviates ETEC-induced diarrhea and intestinal impairment through regulating intestinal inflammation and gut microbiota in newly weaned mice. [ABSTRACT FROM AUTHOR]

Tailocins are naturally occurring bacteriocins that are promising alternative antimicrobials, yet the diversity of naturally occurring tailocins is limited. As tailocins structurally and functionally resemble phage tails, phages may be used as scaffolds for developing novel antimicrobial tailocins. The abundance and diversity of tailed phages will provide a much broader repertoire of tailocins to target any required bacteria. Tailocins may be produced by disrupting the capsid–tail junction of phage particles or by preventing capsid assembly during phage infection. Novel technologies for phage engineering and synthetic biology can be utilized to produce tailocins using synthetic biology approaches. Tailocins are high-molecular-weight bacteriocins produced by bacteria to kill related environmental competitors by binding and puncturing their target. Tailocins are promising alternative antimicrobials, yet the diversity of naturally occurring tailocins is limited. The structural similarities between phage tails and tailocins advocate using phages as scaffolds for developing new tailocins. This article reviews three strategies for producing tailocins: disrupting the capsid–tail junction of phage particles, blocking capsid assembly during phage propagation, and creating headless phage particles synthetically. Particularly appealing is the production of tailocins through synthetic biology using phages with contractile tails as scaffolds to unlock the antimicrobial potential of tailocins. [ABSTRACT FROM AUTHOR]

There is evidence of bacteriophages driving microbial community composition from natural examples, such as cholera outbreaks, during phage therapy, experiments, and mathematical models. However, many studies suggest little or no role of phages in shaping community dynamics, suggesting that viruses may instead follow microbial population dynamics. Evidence from coevolutionary studies suggests that phages may have a greater role in shaping composition at the strain rather than the species level (where evolutionary mechanisms predominate). Phages are most likely to affect community composition when encounter rates with susceptible hosts is high, over short timescales with novel phage, and in relatively simple communities. The ubiquity of bacteriophages (phages) and the major evolutionary and ecological impacts they can have on their microbial hosts has resulted in phages often cited as key drivers shaping microbial community composition (the relative abundances of species). However, the evidence for the importance of phages is mixed. Here, we critically review the theory and data exploring the role of phages in communities, identifying the conditions when phages are likely to be important drivers of community composition. At ecological scales, we conclude that phages are often followers rather than drivers of microbial population and community dynamics. While phages can affect strain diversity within species, there is yet to be strong evidence suggesting that fluctuations in species' strains affects community composition. [ABSTRACT FROM AUTHOR]

The list of standard abbreviations for JDS is available at adsa.org/jds-abbreviations-24. Nonstandard abbreviations are available in the Notes. Traditional fermented milks are produced through an inoculation process that involves the deliberate introduction of microorganisms that have been adapted and perpetuated across successive generations. However, the changes in the microbiota of traditional fermented milk during long-term inoculation fermentation in a laboratory environment remain unclear. In this study, we collected 5 samples of traditional fermented milk samples from 5 different counties in Tibet (3 kurut products) and Xinjiang (2 tarag products) of China, which served as starter cultures for a 9-mo continuous inoculation fermentation experiment. We analyzed the inter- and intrapopulation variations in the microbial communities of the collected samples, representing their macrodiversity and microdiversity, using shotgun metagenomic sequencing. Across all samples, we obtained a total of 186 high-quality metagenomic-assembled genomes, including 7 genera and 13 species with a relative abundance of more than 1%. The majority of these genomes were annotated as Lactobacillus helveticus (60.46%), Enterococcus durans (9.52%), and Limosilactobacillus fermentum (6.23%). We observed significant differences in species composition and abundance among the 5 initial inoculants. During the long-term inoculation fermentation, we found an overall increasing trend in species diversity, composition, and abundances of carbohydrate metabolism module-encoding genes in the fermented milk bacterial metagenome, while the fermented milk virome exhibited a relatively narrow range of variation. Lactobacillus helveticus, a dominant species in traditional fermented milk, displayed high stability during the long-term inoculation fermentation. Our study provides valuable insights for the industrial production of traditional fermented milk. [ABSTRACT FROM AUTHOR]

This research aimed to address the potential bacterial contamination risks in developmental engineering (DE) using bacteriophages. To compare and contrast the exemplar Escherichia coli T4 and M13 bacteriophages, human dermal fibroblasts cultivated on culture plates, natural cellulosic scaffolds, and poly(methyl methacrylate) (PMMA) particles were utilized as two‐dimensional (2D) cell, three‐dimensional (3D) tissue, and modular tissue culture models, respectively. When directly introduced into these distinct culture systems, both phages survived, exhibited no significant effects on the cultured cells or tissues, yet displayed their potentials to alleviate the infections caused by corresponding bacterial host cells. Apart from direct addition into the culture medium, both phages were also coated on PMMA, polystyrene, poly(lactic acid) particles with different diameters (5, 10, 30, and 100 µm) and cellulosic scaffolds. The coated phages endured the coating processes and demonstrated their viabilities in plaque assays. Further testing indicated that the phages coated on the PMMA particles tolerated multiple deliberate rinses and centrifugations, but not thermal treatment at 60–80°C. In summary, T4 and M13 bacteriophages not only manifested their antibacterial functions in diverse 2D cell, 3D tissue, and modular tissue culture systems, but also demonstrated their potentials of coating modular scaffolds to alleviate the bacterial contamination risks in DE. [ABSTRACT FROM AUTHOR]

Maximal standard-of-care (SOC) management could not stop the life-threatening progression of a necrotizing fasciitis induced by Panton-Valentine Leukocidin-producing Methicillin-Resistant Staphylococcus aureus (MRSA) in a 12-year-old boy. Multi-route phage therapy was initiated along with antibiotics against Staphylococcus aureus, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, eventually leading to full recovery with no reported adverse events. [ABSTRACT FROM AUTHOR]

Background: Proteus mirabilis is a Gram-negative, rod-shaped bacterium widely found in natural environments. It is known for causing a range of severe illnesses in mammals, particularly urinary tract infections (UTIs). This study evaluates the therapeutic efficacy of phage P2-71 against Proteus mirabilis in vivo and in vitro environments. Methods: The in vitro therapeutic potential of bacteriophage P2-71 was assessed through the ability of phage to kill Proteus mirabilis by using a plate counting assay, and biofilm inhibition and biofilm lysis assays using a microtitre plate method. Additionally, an in vivo UTI model in C57BL/6Jmice was developed via urethral inoculation of the bacterium. Phage therapy was administered through urethral injection over a period of 5 days. Therapeutic outcomes were measured by analyzing bacterial load, phage titer, inflammatory markers, and histopathological changes in the urine, urogenital tissues, and spleen. Results: In vitro, bacteriophage P2-71 achieved significant reductions in P. mirabilis concentrations, with log reductions of 1.537 and 0.7009 CFU/mL in laboratory and urine environments, respectively (p < 0.001). The phage also decreased biofilm formation by 34-49% and lysed 15-25% of mature biofilms at various multiplicities of infection (MOIs) (p < 0.001). In vivo, phage treatment significantly lowered bacterial concentrations in the urine on Days 1 and 3 (p < 0.0001), achieving a maximum reduction of 4.602 log10 CFU/mL; however, its effectiveness diminished by Day 5 (p > 0.05). Concurrently, phage titers decreased over time. Importantly, phage treatment notably reduced bacterial load in the bladder, kidneys, and spleen (p < 0.001). Inflammatory markers such as IL-6, IL-1β, and TNF-α were significantly lower in the treatment group, especially in the bladder (p < 0.0001), indicating an effective reduction in inflammation. Histopathological analysis showed significant mitigation of tissue damage. Conclusion: The results demonstrated that bacteriophage P2-71 is a promising alternative therapy for UTIs caused by MDR Proteus mirabilis. This bacteriophage therapy offers a viable strategy for managing infections where traditional antimicrobials fail, highlighting its potential in clinical applications. [ABSTRACT FROM AUTHOR]

Nocardia spp., which belongs to one of the Nocardio-form filamentous bacteria, is usually surface hydrophobic and when overproduced attaches to the surface of bubbles under the action of surfactants, allowing the stable presence of foam on the surface of aeration tanks, leading to the occurrence of sludge-foaming events. Two novel phages, P69 and KYD2, were isolated from the environment, and their hosts were Nocardia transvalensis and Nocardia carnea, respectively. These two phages are Siphophages-like with long tails. An aeration tank pilot plant was constructed in the laboratory to simulate sludge foaming, and these two strains of phage were applied. Compared with the reactor not dosed with phage, the application of phage could reduce the host level in the reactor, resulting in the highest decrease in turbidity by more than 68% and sludge volume index by more than 25%. The time for surface foam disappearance was 9 h earlier than that of the control group (the group with the same concentration of Nocardia carnea but no bacteriophage applied), significantly improving water quality. The phage can effectively inhibit the propagation of Nocardia in the actual sludge-foaming event, control the sludge foaming, and improve the effluent quality. It provides a novel and relatively economical solution for controlling sludge foaming in sewage treatment plants in the future, shows that the phages have potential application value in the prevention and control of Nocardia, and provides another way to control the sludge-foaming event caused by the excessive reproduction of Nocardia in the future. [ABSTRACT FROM AUTHOR]

The pathogenic strains of Escherichia coli (E. coli) are frequent cause of urinary tract infections including catheter-associated, soft tissue infections and sepsis. The growing antibiotic resistance in E. coli is a major health concern. Bacteriophages are specific for their bacterial host, thus providing a novel and effective alternatives. This study focuses on isolation of bacteriophages from urban sewage treatment plants. Initially 50 different bacteriophages have been isolated against non-resistant reference E. coli strain and fifty multidrug resistant clinical isolates of extraintestinal infections. Out of which only thirty-one lytic phages which gave clear plaques were further analysed for different physico-chemical aspects such as thermal inactivation, pH, effect of organic solvents and detergents. Two bacteriophages, ASEC2201 and ASEC2202, were selected for their ability to withstand temperature fluctuation from −20 to 62 °C and a pH range from 4 to 10. They also showed good survival (40–94%) in the presence of organic solvents like ethanol, acetone, DMSO and chloroform or ability to form plaques even after the treatment with detergents like SDS, CTAB and sarkosyl. Both efficiently killed reference strain and 40–44% of multidrug resistant clinical isolates of E. coli. Later ASEC2201 and ASEC2202 were subjected to morphological characterisation through transmission electron microscopy, which revealed them to be tailed phages. The genomic analysis confirmed them to be Escherichia phages which belonged to family Drexlerviridae of Caudovirales. [ABSTRACT FROM AUTHOR]

The rapid rise of multidrug-resistant (MDR) organisms has created a critical need for alternative treatment options. Phage therapy is gaining attention as an effective way to fight bacterial infections by using lytic bacteriophages to specifically target and kill harmful bacteria. This review discusses several phage therapeutic options and emphasizes new developments in phage biology. Phage treatment has proven to be successful against MDR bacteria, as evidenced by multiple human clinical trials that indicate favorable results in treating a range of diseases caused by these pathogens. Despite these promising results, challenges such as phage resistance, regulatory hurdles, and the need for standardized treatment protocols remain. To effectively combat MDR bacterial infections, future research must focus on enhancing phage effectiveness, guaranteeing safety for human usage and incorporating phage therapy into clinical practice. [ABSTRACT FROM AUTHOR]

The demand for highly sensitive methods of pathogen detection drives the development of new diagnostic assays. While nucleic acid amplification methods such as PCR are very sensitive and remain widely used, they may be limited in complex sample matrices due to the presence of polymerase inhibitors. On the other hand, the direct detection of nucleic acids by DNA hybridization assays is simple but typically less sensitive. This work combines a branched DNA (bDNA) hybridization assay with upconversion nanoparticle (UCNP) labels to enhance the sensitivity of DNA detection. The anti‐Stokes emission of UCNP labels enables measurements without optical background interference. The bDNA assay relies on a series of oligonucleotide probes creating a branched structure with several binding sites for biotinylated amplification probes and streptavidin‐conjugated UCNPs. Several configurations of the bDNA assay are investigated to achieve the highest signal amplification and the lowest background signal. The optimal configuration of bDNA assay yields a limit of detection (LOD) of 5.9 × 104 cfu mL−1 for the target DNA of the bacteriophage M13. The upconversion‐linked bDNA assay is easily adaptable to other target DNAs by adjusting the oligonucleotide probes. [ABSTRACT FROM AUTHOR]

Lytic phages control the timepoint of host cell lysis by timing the holin-mediated release of cell wall-degrading endolysins. In phage T4, the antiholin RI inhibits the holin T, thereby preventing the early release of the T4 endolysin and lysis. The antiholin achieves lysis inhibition (LIN) in response to phage superinfections, thereby increasing the chance for lysis in an environment with a lower phage concentration. The holin T consists of a small N-terminal cytoplasmic domain, a transmembrane helix, and a periplasmic C-terminal domain. The antiholin is targeted to the periplasm by a cleavable signal peptide. Recently, the periplasmic soluble domains of the holin and the antiholin were found to form T2/RI2 tetramers in crystals. To investigate the functional relevance of this complex, we reconstituted LIN in a phage-free system, using only RI, T, and endolysin, and combined targeted mutagenesis with functional analyses. Inactivation of the RI signal peptide cleavage site did not abolish LIN, indicating that RI can function in a membrane-bound state, which argued against the tetramer. This led to analyses showing that only one of the two T/RI interfaces in the tetramer is physiologically relevant, which is also the only interaction site predicted by AlphaFold2. Some holin mutations at this interaction site prevented lysis, suggesting that the RI interaction likely acts by blocking the holin oligomerization required for hole formation. We conclude that LIN is mediated by a dimeric T/RI complex that, unlike the tetramer, can be easily formed when both partners are membrane-anchored. [ABSTRACT FROM AUTHOR]

Introduction: Phages are viruses that infect prokaryotes and can shape microbial communities by lysis, thus offering applications in various fields. However, challenges exist in sampling, isolation and accurate prediction of the host specificity of phages as well as in the identification of newly replicated virions in response to environmental challenges. Methods: A new workflow using biorthogonal non-canonical amino acid tagging (BONCAT) and click chemistry (CC) allowed the combined analysis of phages and their hosts, the identification of newly replicated virions, and the specific tagging of phages with biotin for affinity chromatography. Results: Replication of phage λ in Escherichia coli was selected as a model for workflow development. Specific labeling of phage λ proteins with the non-canonical amino acid 4-azido-L-homoalanine (AHA) during phage development in E. coli was confirmed by LC-MS/MS. Subsequent tagging of AHA with fluorescent dyes via CC allowed the visualization of phages adsorbed to the cell surface by fluorescence microscopy. Flow cytometry enabled the automated detection of these fluorescent phage-host complexes. Alternatively, AHA-labeled phages were tagged with biotin for purification by affinity chromatography. Despite biotinylation the tagged phages could be purified and were infectious after purification. Discussion: Applying this approach to environmental samples would enable host screening without cultivation. A flexible and powerful workflow for the detection and enrichment of phages and their hosts in pure cultures has been established. The developed method lays the groundwork for future workflows that could enable the isolation of phage-host complexes from diverse complex microbial communities using fluorescence-activated cell sorting or biotin purification. The ability to expand and customize the workflow through the growing range of compounds for CC offers the potential to develop a versatile toolbox in phage research. This work provides a starting point for these further studies by providing a comprehensive standard operating procedure. [ABSTRACT FROM AUTHOR]

Background: The infant gut microbiome's establishment is pivotal for health and immune development. Understanding it unveils insights into growth, development, and maternal microbial interactions. Research often emphasizes gut bacteria, neglecting the phageome. Methods: To investigate the influence of geographic or maternal factors (mode of delivery, mode of breastfeeding, gestational diabetesmellitus) on the gut microbiota and phages of newborns, we collected fecal samples from 34 pairs of mothers and their infants within 24 hours of delivery from three regions (9 pairs from Enshi, 7 pairs from Hohhot, and 18 pairs from Hulunbuir) using sterile containers. Gut microbiota analysis by Shotgun sequencing was subsequently performed. Results: Our results showed that geographic location affects maternal gut microbiology (P < 0.05), while the effect on infant gut microbiology was not significant (P = 0.184). Among the maternal factors, mode of delivery had a significant (P < 0.05) effect on the newborn. Specific bacteria (e.g., Bacteroides, Escherichia spp., Phocaeicola vulgatus, Escherichia coli, Staphylococcus hominis, Veillonella spp.), predicted active metabolites, and bacteriophage vOTUs varied with delivery mode. Phocaeicola vulgatus significantly correlated with some metabolites and bacteriophages in the early infant gut (P < 0.05). In the GD group, a strong negative correlation of phage diversity between mother and infants was observed (R = -0.58, P=0.04). Conclusion: In conclusion, neonatal early gut microbiome (including bacteria and bacteriophages) colonization is profoundly affected by the mode of delivery, and maternal gestational diabetes mellitus. The key bacteria may interact with bacteriophages to influence the levels of specific metabolites. Our study provides new evidence for the study of the infant microbiome, fills a gap in the analysis of the infant gut microbiota regarding the virome, and emphasizes the importance of maternal health for the infant initial gut virome. [ABSTRACT FROM AUTHOR]

This study shows how bacterial viruses (bacteriophages, phages) interact with calcium carbonate during precipitation from aqueous solution. Using electron microscopy, epifluorescence microscopy, X-ray diffraction, and image analysis, we demonstrate that bacteriophages can strongly influence the formation of the vaterite phase. Importantly, bacteriophages may selectively bind both amorphous calcium carbonate (ACC) and vaterite, and indirectly affect the formation of structural defects in calcite crystallites. Consequently, the surface properties of calcium carbonate phases precipitating in the presence of viruses may exhibit different characteristics. These findings may have significant implications in determining the role of bacterial viruses in modern microbially-rich carbonate sedimentary environments, as well as in biomedical technologies. Finally, the phage-vaterite system, as a biocompatible material, may serve as a basis for the development of promising drug delivery carriers. [ABSTRACT FROM AUTHOR]

Postoperative endophthalmitis is a serious complication of cataract surgery. It may leads to vision loss. The most common organism cause endophthalmitis is gram-positive bacteria, mainly Staphylococcus aureus (S. aureus). To prevent postoperative endophthalmitis, eye drops or intracameral administration of antibiotic agents are universally used. In recent years, the trend of endophthalmitis treatment has grown rapidly. Administration of bacteriophage is a subject of research for the treatment and prophylaxis of postoperative endophthalmitis. This literature review investigates the potential of bacteriophage to provide a rapid, effective alternative to antibiotic treatments for postoperative endophthalmitis caused by S. aureus. [ABSTRACT FROM AUTHOR]

Background and Aim: The dearth of new antibiotics necessitates alternative approaches for managing infections caused by resistant superbugs. This study aimed to evaluate the lytic potential of the purified bacteriophage PKp-V1 against extendedspectrum β-lactamase (ESBL) harboring hypervirulent Klebsiella pneumoniae (hvKp)-K1 recovered from veterinary specimens. Materials and Methods: A total of 50 samples were collected from various veterinary specimens to isolate K. pneumoniae, followed by antimicrobial susceptibility testing and molecular detection of various virulence and ESBL genes. Multilocus sequence typing of the isolates was performed to identify prevalent sequence types. The bacteriophages were isolated using the double-agar overlay method and characterized using transmission electron microscopy, spot tests, plaque assays, stability tests, and one-step growth curve assays. Results: Among 17 (34%) confirmed K. pneumoniae isolates, 6 (35%) were hvKp, whereas 13 (76%) isolates belonging to the K1 type were positive for the wzy (K1) virulence gene. All (100%) hvKp isolates exhibited the allelic profile of ST258. Overall, PKp-V1 exhibited an 88 % (15/17; (p ≤ 0.05) host range, among which all (100 %; p ≤ 0.01) hvKp isolates were susceptible to PKp-V1. PKp-V1 exhibited a lytic phage titer of 2.4 × 108 plaque forming unit (PFU)/mL at temperatures ranging from 25°C to 37°C. The lytic phage titers of PKp-V1 at pH = 8 and 0.5% chloroform were 2.1 × 108 PFU/mL and 7.2 × 109 PFU/mL, respectively. Conclusion: Although the incidence of ESBL-infected K. pneumoniae in veterinary settings is worrisome, PKp-V1 phages showed considerable lytic action against the host bacterium, indicating the potential of PKp-V1 as a possible alternative therapeutic option against MDR K. pneumoniae. [ABSTRACT FROM AUTHOR]

Bacteriophages are the most numerous entities on earth and are found everywhere their bacterial hosts live. As natural bacteria killers, phages are extensively investigated as a potential cure for bacterial infections. Neisseria gonorrhoeae (the gonococcus) is the etiologic agent of a sexually transmitted disease: gonorrhea. The rapid increase of resistance of N. gonorrhoeae to antibiotics urges scientists to look for alternative treatments to combat gonococcal infections. Phage therapy has not been tested as an anti-gonococcal therapy so far. To date, no lytic phage has been discovered against N. gonorrhoeae. Nevertheless, gonococcal genomes contain both dsDNA and ssDNA prophages, and viral particle induction has been documented. In this review, we consider literature data about the attempts of hunting for a bacteriophage specific for gonococci – the gonophage. We also discuss the potential application of prophage elements in the fight against N. gonorrhoeae. Temperate phages may be useful in preventing and treating gonorrhea as a scaffold for anti-gonococcal vaccine development and as a source of lytic enzymes with anti-gonococcal activity. [ABSTRACT FROM AUTHOR]

Food-contaminating bacteria pose a threat to food safety and the economy by causing foodborne illnesses and spoilage. Bacteriophages, a group of viruses that infect only bacteria, have the potential to control bacteria throughout the "farm-to-fork continuum". Phage application offers several advantages, including targeted action against specific bacterial strains and minimal impact on the natural microflora of food. This review covers multiple aspects of bacteriophages applications in the food industry, including their use as biocontrol and biopreservation agents to fight over 20 different genera of food-contaminating bacteria, reduce cross-contamination and the risk of foodborne diseases, and also to prolong shelf life and preserve freshness. The review also highlights the benefits of using bacteriophages in bioprocesses to selectively inhibit undesirable bacteria, such as substrate competitors and toxin producers, which is particularly valuable in complex microbial bioprocesses where physical or chemical methods become inadequate. Furthermore, the review briefly discusses other uses of bacteriophages in the food industry, such as sanitizing food processing environments and detecting specific bacteria in food products. The review also explores strategies to enhance the effectiveness of phages, such as employing multi-phage cocktails, encapsulated phages, phage products, and synergistic hurdle approaches by combining them with antimicrobials. [ABSTRACT FROM AUTHOR]