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Bacteriophages are viruses of bacteria that are among the leading alternative antimicrobials for treatment of the multidrug resistant (MDR) and opportunistic pathogen Pseudomonas aeruginosa. Although phage therapy is now generally considered safe, access to the treatment remains limited and its efficacy remains unproven. The goal of this dissertation is to develop innovative phage processes and intravenous treatment regimens to combat MDR P. aeruginosa. In the third chapter, I evaluate how to select phages to combat P. aeruginosa and study the interactions between different phage strains during combination treatment. In two-phage cocktail treatment, I found that mixing phages with inequal activities diminishes initial treatment potency. Sequential treatment with the same phages improved potency but generated unique patterns of resistance. This study provides new insight on how to formulate and administer phage combinations based on the infectivity of each phage to its bacterial host. In the fourth chapter, I develop and optimize the standardized production of phages by introducing cross-flow filtration to improve the removal of endotoxin from phage preparations. The simultaneous washing and concentration of phage particles was found to improve endotoxin removal across multiple Gram-negative species. Leveraging the kinetics of each phage, the accumulation of phage-resistant bioburden could then be reduced in an improved purification process. In the fifth chapter, I validate the targeting and replication of clinical phages during the treatment of patients infected by multidrug resistant P. aeruginosa. Phages suitable for therapy were determined by screening across a multi-infection isolate library. The efficacy of clinical phage treatments were then validated using a combination of microbiological and metagenomic markers of failure or success. In conclusion, this thesis provides insights into current barriers for phage therapy. Identifying and characterizing individual phage properties will have important implications on how to treat and reduce the mortality of MDR P. aeruginosa infections.

Bacteriophages are the most abundant and genetically diverse life forms, even greater than their numerous bacterial prey. This observation seems to violate fundamental ecological theory that predicts higher trophic levels should be less diverse than lower trophic levels, leading to the question of what mechanisms could support greater parasite diversity than host diversity. Here we provide unexpected results from a bacteriophage λ evolution experiment where λ was continually supplied a single isogenic host, yet repeatedly evolved a diversity of genotypes that diverged in their receptor use. Measurements of negative frequency-dependent selection between the receptor specialists revealed that they evolved because of diversifying selection. Such selection in viruses usually stems from adaptations to specialize on different hosts, but only a single host was provided and replenished every eight hours. A series of experiments conducted over the 8-hour incubation period revealed that selection for the different specialist phages oscillated because gene expression of the two receptors oscillated (E. coli outer membrane proteins LamB and OmpF). The cause of the receptor expression changes was in part attributable to rapid evolution in the bacteria within the eight-hour period that was documented through both phenotypic resistance assays and population genome sequencing. Altogether this suggests that rapid host evolution on the timescales of hours can drive the emergence of multiple host genotypes and their oscillations can promote the evolution of phage diversity. This result suggests that rapid host evolution may play a role in driving viral diversity and may promote diversity in other non-microbial systems too.

Antibiotic resistance is a prominent global health challenge as more bacteria are gaining resistance, making antibiotic obsolete to a growing number of strains. To combat the antibiotic resistant microbes, bacteriophage (phage) therapy, using viruses that target bacteria, became a viable option for aiding patients fighting resistant microbes. In performing phage therapy for these patients, antibiotics are often administered with the phages to provide the best chance for the patients’ survival, but these chemical and viral interactions or even synergistic effects have not been thoroughly studied due to the difficulty and inaccessibility of the current liquid assay methods. Development of solid media assays to test for antibiotic and phage interactions on bacterial lawns allows for easier and accessible testing methods for phage-antibiotic synergy (PAS) using materials readily available in clinical labs worldwide, lowering the entry barrier in international communities to further investigate PAS or to consider synergy in phage therapy testing.

Recently, a family of bacteriophages has been found to form a nucleus-like replication compartment, called the phage nucleus, which encapsulates the phage DNA and protects it from bacterial host defense systems. Although we have discovered the general replication with the phage nucleus, it is still poorly understood at the molecular level, especially the macromolecule translocations and the key proteins that play roles in these functions. The aim of this thesis is to identify the phage nucleus and its associated proteins in greater detail to shed light specifically on how DNA and mRNA export occurs, as well as the selective protein transport into this structure.In Chapter 2, we identify a new set of phage nucleus-associated proteins through comprehensive proteomics and biochemistry. Among the identified proteins, now termed ChmB, is further investigated and found to be directly interacting with ChmA, which forms most of the phage nucleus. In addition, it is found to be directly interacting with the portal protein, suggesting that ChmB might be forming pore-like structures to accommodate DNA packaging. This study provides new insights into the composition and functions of the phage nucleus, particularly in protein-protein interactions.In Chapter 3, we focus on another phage nucleus-associated protein, ChmC. We found that ChmC has structural homology to known RNA binding proteins. We confirmed that ChmC binds to mRNA through its surface-exposed positively charged residues and that it also has phase separation properties. Investigating the samples collected during the infection confirmed the phage mRNA binding, particularly in 5’ regions of the transcripts. When ChmC was knocked down via ddCas13d system, microscopy images showed that the phage nucleus could form, but the replication was arrested at an early stage. Correspondingly, we found that the knockdowns cause a significant reduction in phage bouquet formation as well as phage titers. These results show that ChmC has a critical role in the phage nucleus system, possibly being a part of the mRNA export mechanism after the switch to non-virion RNA Polymerase for transcription.Chapter 4 describes the other key proteins for the phage nucleus system, such as the portal protein and its potential docking site of octameric assembly of ChmB. We also investigate the other phage nucleus-associated proteins we found by proximity labeling, gp63 and gp64 of phiPA3. Our observations suggest gp63 has a critical role in selective protein transport to the phage nucleus and that gp64 interacts with it to potentially form a complex. Finally, we identify phage proteins that potentially inhibit host cell division, which is required as the jumbo phages require longer time to complete their replication compared to their hosts. These encoded proteins might provide a more comprehensive understanding of the replication mechanism as it is crucial for phages' ability to propagate in bacterial hosts, while potentially providing underlying mechanisms that could be used in therapeutics and the biotech industry.In conclusion, this thesis provides important insights into the phage nucleus system and the molecular mechanisms behind it. We believe that identifying and characterizing the phage nucleus and its associated proteins can have important implications for developing host-immune system evading phage therapies and allowing broader abilities to prevent severe bacterial infections.

The ΦKZ-like family jumbo bacteriophage ΦPA3 infects the pathogenic bacterium Pseudomonas aeruginosa. Upon infection, the bacteriophage forms a dynamic, bipolar spindle within the host using a divergent tubulin family protein known as phage tubulin/FtsZ or PhuZ. This spindle is important for infectivity and centers the phage DNA. The phage DNA, along with the DNA replication and transcription machinery, is found inside of a proteinaceous shell, while the translation and nucleotide synthesis machinery is excluded. This partitioning of proteins according to function suggests a mechanism of selectivity, akin to that of the eukaryotic nucleus. This similarity inspired the naming of this proteinaceous, DNA-containing structure the phage nucleus. The phage nucleus is irregularly shaped and grows over the course of infection, reaching up to half the host cell size or about 0.5 μm in diameter. The dramatic phage nucleus growth is likely driven by DNA replication with which the protein shell must keep up by incorporating new subunits. The proteinaceous exterior of the ΦPA3 phage nucleus is constructed using the most highly expressed, non-virion protein, Gp53, which we name phage nuclear enclosure protein or PhuN. At the beginning of this research, PhuN had no previously characterized homologues. Since then, PhuN has been studied and observed in a series of jumbo phages spanning bacteria ranging from Escherichia coli to Serratia. Using an in vitro approach coupled with direct visualization using electron microscopy, the work presented in this dissertation begins to uncover the principles and mechanisms underlying the remarkable formation, growth, and function of the phage nucleus. In a series of collaborative work, we show that the phage nucleus shields the bacteriophage DNA from host defense systems, we utilize cryo-EM to show to 3.9 Å that the phage nucleus is enclosed in a proteinaceous 2D crystal, and, finally, I share unpublished observations worthy of further exploration. This work lays the foundations for future biochemical and structural investigation probing jumbo phage mechanisms including but not limited to phage nucleus subunit addition, selectivity, and capsid packaging.

Під час розвитку маститу у корів формування біоплівки збудником захворювання є ефективним способом його збереження в мікрооточенні молочної залози. Біоплівкові інфекції важко піддаються лікуванню антимікробними засобами, порівняно з тим, що спостерігається при вирощуванні в планктонних умовах. Мета роботи – визначити та порівняти вплив антимікробних препаратів та бактеріофагу Phage SAvB14 при знищенні біоплівок сформованих S. aureus var. bovis. За результатами дослідження встановлено, що антибіотики згубно впливали на кількість бактерій у складі біоплівки, проте знищували її, в середньому, на 60%. 100% ефективність проявляв лише один антибіотик фторхінолонового ряду – енрофлоксацин - ймовірно, через його низьку молекулярну масу. При дослідженні впливу бактеріофагу Phage SAvB14 на життєздатність S. aureus var. bovis у складі біоплівки встановлено, що фаг впродовж 24 годин повністю руйнував сформовану біоплвку. Отже, отримані результати вказують на перспективність ефективного використання стафілококового бактеріофагу Phage SAvB14 для руйнування біоплівки, сформованої S. aureus var. bovis – при маститі корів.

Chronic wounds represent a large and growing disease burden worldwide. Infection, biofilm formation, and associated pathological inflammation are some of the leading impediments to healing, suggesting an important role for the microbiome of these wounds. Studies of the bacterial fraction suggest that community composition, and its temporal variance, may be associated with healing outcomes, yet the forces that drive these dynamics are not well understood. The viral fraction of the microbiome, called the virome, may be a major contributing factor; other human viromes are dominated by bacteriophages, which not only infect and lyse bacteria, but can have profound impacts on host functionality. Despite its potential, the wound virome has not yet been described, largely due to the challenges associated with preparing and analyzing low-biomass clinical samples, like those obtained from skin and wounds. To facilitate the study of skin and wound viromes, we developed an improved sample processing method for obtaining both viral-enriched and bacterial DNA from a single swab sample, resulting in higher yields and viral purity when compared to traditional methods. The improved swab processing protocol was then employed in a small-cohort metagenomic survey of the skin and chronic wound microbiomes of 20 volunteers at an outpatient wound care clinic. We report taxonomic composition and diversity of the bacterial and viral fractions, and their associations to clinical features. Bacterial genera and viral species, as well as their respective oxygen requirements and auxiliary genes, were associated with both positive and negative healing outcomes. Additionally, we assessed how the microbiome is immediately impacted by sharp debridement, a standard of care procedure that physically removes necrotic tissue. There were no significant differences between microbiomes before and after a single debridement, suggesting that changes in microbial community structure are longitudinal and derived from repeated treatments. This work establishes novel methodology for studying the wound microbiome and virome, confirms previous findings in the field, presents the first chronic wound virome findings, and identifies correlations and associations to healing outcomes that causation studies may investigate in the future.

Escherichia albertii is an emerging zoonotic foodborne pathogen. Several outbreaks of E. albertii have occurred particularly in Japan. Although birds have been considered as one of the most important reservoirs of this bacterium, information regarding the prevalence in birds is still scanty. We performed a survey of E. albertii in wild birds in Japan, and examined characteristics of the isolates. E. albertii specific gene was detected in 5 cloacal swabs out of 156 birds by PCR. Four E. albertii were isolated from a swallow with 2 different E. albertii strains and 2 pigeons in a flock by XRM-MacConkey agar. These isolates were assigned to biogroup 3, shown no resistance to any antimicrobials tested, and classified into 2 EAO-genotypes (EAOg2 and EAOg33) and untypable. Similar to clinical E. albertii strains, these isolates carried virulence genes including eae (n=4), paa (n=4), Eccdt-I (n=2) and stx2f (n=1) in addition to Eacdt. Interestingly, stx2f genes in a strain were located on an inducible bacteriophage, which can confer the ability to produce Stx2f to E. coli. In conclusion, Japanese wild birds carried E. albertii at the similar levels to the reported prevalence in birds. These isolates may have a potential to cause gastroenteritis in humans.

Abstract: Bacteriophages are considered as a potential alternative to fight pathogenic bacteria during the antibiotic resistance era. With their high specificity, they are widely used in various applications: medicine, food industry, agriculture, animal farms, biotechnology, diagnosis, etc. Many techniques have been designed by different researchers for phage isolation, purification, and amplification, each of which has strengths and weaknesses. However, all aim at having a reasonably pure phage sample that can be further characterized. Phages can be characterized based on their physiological, morphological or inactivation tests. Microscopy, in particular, opened a wide gate, not only for visualizing phage morphological structure, but also for monitoring biochemistry and behavior. Meanwhile, computational analysis of phage genomes provides more details about phage history, lifestyle, and the potential for toxigenic or lysogenic conversion, which translate to safety in biocontrol and phage therapy applications. This review article summarizes phage application pipelines at different levels, and addresses specific restrictions and knowledge gaps in the field. Recently developed computational approaches, which are used in phage genome analysis, are critically assessed. We hope that this assessment provides researchers with useful insights for the selection of suitable approaches for phage-related research aims and applications.

This study aimed to evaluate lytic bacteriophage supplementation in pre-weaned dairy calves over neonatal calf diarrhea and respiratory diseases occurrence, performance and biochemical parameters. Also, to determine bacterial agents causing NCD. Two hundred Holstein×Gyr crossbred female calves were divided into two groups: Control (CON, n = 100), no supplementation; and Bacteriophage (PHAGE, n = 100) bacteriophage supplementation (1 g/day) from d 3 until d 70 of life. Calves were monitored daily for respiratory disease and diarrhea, as for age at the first diarrheic episode and its duration. Fecal samples were cultured for isolation of Escherichia coli and Salmonella spp. colonies and PCR was performed to identify E. coli virulence genes and to confirm Salmonella spp. Performance outcomes were evaluated up to 80 d of age. Blood samples were collected to determine serum levels of total proteins, albumin, cholesterol, γ-glutamyl transferase and urea. PHAGE group had fewer days in diarrhea and duration of the first episode was lower, compared to CON group. Fecal samples of three animals in PHAGE and nine in CON were positive for E. coli in PCR. Thoracic perimeter tended to be higher in supplemented animals. Average daily gain mean of PHAGE was higher in the first 30 d of life, at the beginning of step-down weaning (up to 42 d) and after weaning (up to 80 d). PHAGE mean was lower for albumin and higher for urea. Therefore, phage therapy during the pre-weaned period reduced the duration of neonatal diarrhea, providing greater weight gain for calves.

Resistance to antibiotics is one of the most critical health problems in the world. Therefore, finding new treatment methods to be used as alternatives to antibiotics has become a priority for researchers. Similar to phages, certain products containing antimicrobial components, such as molasses, are widely used to eliminate resistant bacteria. Molasses has a strong antimicrobial effect on bacterial cells, and this effect is thought to be due to the breakdown of the cytoplasmic cell membrane and cell proteins of the polyphenols in molasses. In the present study, phage–molasses interactions were investigated to examine the effects of concomitant use. It was found that molasses samples increased the size of phage plaques by up to 3-fold, and MIC and 1/2 x MIC concentrations of molasses increased the burst size of phages. Although no synergistic effect was found between the phage and molasses, the antimicrobial activities of the components and the effect of molasses on phage activity were demonstrated. © 2022, Canadian Science Publishing. All rights reserved. Emine Ku€bra Tayyarcan is supported by the 100/ 2000 doctoral scholarship provided by the Council of Higher Education, Turkey.

YdaT is a functional equivalent of the CII repressor in certain lambdoid phages and prophages. YdaT from the cryptic prophage CP-933P in the genome of Escherichia coli O157:H7 is functional as a DNA-binding protein and recognizes a 5′-TTGATTN6AATCAA-3′ inverted repeat. The DNA-binding domain is a helix–turn–helix (HTH)-containing POU domain and is followed by a long α-helix (α6) that forms an antiparallel four-helix bundle, creating a tetramer. The loop between helix α2 and the recognition helix α3 in the HTH motif is unusually long compared with typical HTH motifs, and is highly variable in sequence and length within the YdaT family. The POU domains have a large degree of freedom to move relative to the helix bundle in the free structure, but their orientation becomes fixed upon DNA binding.

Aims To isolate and characterise phage which could lyse P. acnes and to formulate the phage into a delivery form for potential application in topical treatment of acne infection. Methods and Results Using standard phage isolation techniques, ten phage capable of lysing P. acnes were isolated from human skin microflora. Their genomes showed high homology to previously reported P. acnes phage. These phage were formulated into cetomacrogol cream aqueous at a concentration of 2.5x108 PFU per gram, and shown to lyse underlying P. acnes cells grown as lawn cultures. These phage formulations remained active for at least 90 days when stored at four degrees Celsius in a light protected container. Conclusions P. acnes phage formulated into cetomacrogol cream aqueous will lyse surrounding and underlying P. acnes bacteria, and are effective for at least 90 days if stored appropriately. Significance and Impact of the Study There are few reports of phage formulation into semi solid preparations for application as phage therapy. The formulation method described here could potentially be applied topically to treat human acne infections. The potential exists for this model to be extended to other phage applied to treat other bacterial skin infections.

Aim To isolate and characterize bacteriophage lytic for the opportunistic pathogen Klebsiella oxytoca and their formulation into a range of solid dosage forms for in-vitro testing. Methods and results We report the isolation, genomic and functional characterization of a novel bacteriophage lytic for Klebsiella oxytoca, which does not infect the closely related Klebsiella pneumoniae. This bacteriophage was formulated into suppositories and troches and shown to be released and lyse underlying Klebsiella oxytoca bacteria in an in-vitro model. These bacteriophage formulations were stable for at least 49 days at 4°C. Conclusions The successful in-vitro assay of these formulations here suggests that they could potentially be tested in-vivo to determine whether such a therapeutic approach could modulate the gut microbiome, and control Klebsiella oxytoca overgrowth, during antibiotic therapy regimes. Significance and impact of the study This study reports a novel bacteriophage specific for Klebsiella oxytoca which can be formulated into solid dosage forms appropriate for potential delivery in testing as a therapy to modulate gut microbiome during antibiotic therapies.

Detecting DNA termini, such as ends of linear extrachromosomal DNA, plays an essential role in understanding the structure and functions of DNA molecules. Here we describe an approach combining direct and indirect computational methods to detect DNA termini from next-generation short-read sequencing. While a direct inference of ends can come from mapping the specific capture points of DNA fragments, this approach is insufficient for analytical pipelines where the DNA termini are not captured. Thus, we add an indirect detection of ends based on strand bias, the difference in sequence representation between the plus and minus strands of DNA in a dataset. Termini are reflected by a strong strand bias, with inward-facing reads greatly enriched over outward-facing reads in the immediate proximity of any end. Applying this analysis to negative control regions (where DNA is continuous and with no known termini), we observe no strong end capture peaks or strand bias. Applying to positive control regions where known DNA termini are present yields strong strand bias signals even in cases where blocked termini prevent end capture (for a protein-blocked adenovirus), or where ends are not explicitly captured (tagmentation of restriction digested lambda DNA). Analysis of a more complex situation (HIV replication) produces a picture that includes both the known termini of the reverse-transcribed genome (the PBS [primer binding site] on the negative strand and the PPT [3’ polypurine tract] on the positive strand) as well as a signal corresponding to a previously described additional initiation site for second strand synthesis (cPPT [central polypurine tract]). These results confirm the ability to detect DNA structural discontinuities in a pooled sample where high throughput shotgun sequence data is available. In addition to the known initiation sequence in the HIV genome, we detect a signal of positive strand DNA termini at several positions on the plus strand sequence. These sites share several characteristics with the previously characterized second strand initiation sites (the cPPT and 3’ PPT sites): (i) observed spike in directly captured cDNA ends, (ii) an indirect terminus signal evident in localized strand bias, (iii) a strong preference for forward-facing termini, (iv) an upstream purine-rich motif, and (v) a decrease in terminus signal at late time points after infection. These characteristics are consistent in duplicate samples in two different genotypes (wild type and integrase-lacking HIV). The observation of distinct internal termini associated with multiple purine rich regions suggests the possibility that multiple internal initiations of second strand synthesis might contribute to HIV replication through acceleration of second strand synthesis and/or strand displacement at the HIV 3’ end.

Cellular lysates capable of transcription and translation have become valuable tools for prototyping genetic circuits, screening engineered functional parts, and producing biological components. Here we report that lysates derived from Yersinia pestis CO92- are functional and can utilize both the E. coli σ70 and the bacteriophage T7 promoter systems to produce green fluorescent protein (GFP). Because of the natural lifestyle of Y. pestis, lysates were produced from cultures grown at 21 °C, 26 °C, and 37 °C to mimic the infection cycle. Regardless of the promoter system the GFP production from 37 °C was the most productive and the 26 °C lysate was the least. When reactions are initiated with 5 nM of DNA, the GFP output of the 37 °C lysate is comparable with the productivity of other non-E. coli systems. The data we present demonstrate that, without genetic modification to enhance productivity, cell-free extracts from Y. pestis are functional and dependent on the temperature at which the bacterium was grown.

Although skin infections are usually uncomplicated, it may indicate systemic disease or lead to life-threatening systemic infections. Staphylococcus aureus is the most common cause of skin infectio...

Erwinia tracheiphila, the causal pathogen of bacterial wilt of cucurbit crops, is disseminated by cucumber beetles. A bacteriophage, designated FBB1 (Fu-Beattie-Beetle-1), was isolated from spotted cucumber beetles (Diabrotica undecimpunctata) that were collected from a field in which E. tracheiphila is endemic. FBB1 was classified into the Myoviridae family based on its morphology, which includes an elongated icosahedral head (106 × 82 nm) and a putatively contractile tail (120 nm). FBB1 infected all 62 E. tracheiphila strains examined and three Pantoea spp. strains. FBB1 virions were stable at 55°C for 1 h and tolerated a pH range from 3 to 12. FBB1 has a genome of 175,994 bp with 316 predicted coding sequences and a GC content of 36.5%. The genome contains genes for a major bacterial outer-membrane protein, a putative exopolysaccharide depolymerase, and 22 predicted transfer RNAs. The morphology and genome indicate that FBB1 is a T4-like virus and thus in the Tevenvirinae subfamily. FBB1 is the first virulent phage of E. tracheiphila to be reported and, to date, is one of only two bacteriophages to be isolated from insect vectors of phytopathogens. Collectively, the results support FBB1 as a promising candidate for biocontrol of E. tracheiphila based on its virulent (lytic) rather than lysogenic lifestyle, its infection of all E. tracheiphila strains examined to date, and its infection of a few nonpathogenic bacteria that could be used to support phage populations when pathogen numbers are low.

The number of sequenced bacteriophage genomes is growing at an exponential rate. The majority of sequenced bacteriophage genomes are annotated by one or more of several freely available gene identification programs (Glimmer, GeneMark, RAST, Prodigal, etc.). No program has been shown to consistently outperform the others; thus, the choice of which program to use is not obvious. We present the Phage Commander application for rapid identification of bacteriophage genes using multiple gene identification programs. Phage Commander runs a bacteriophage genome sequence through nine gene identification programs (and an additional program for identification of tRNAs) and integrates the results within a single output table. Phage Commander also generates formatted output files for direct export to National Center for Biotechnology Information GenBank or genome visualization programs such as DNA Master. Users can select the threshold for which genes to export (genes identified by at least one program, genes identified by at least two programs, etc.). Phage Commander was benchmarked using eight high-quality bacteriophage genomes whose genes are backed by experimental data. Our results show that the most accurate annotations are obtained by exporting genes identified by at least two or three programs. Many groups opt to manually curate the annotations obtained from gene identification programs, and Phage Commander was designed to facilitate manual curation of genome annotations. Our benchmarking results show that manual curation does indeed produce more accurate annotations than any individual gene identification program. The authors thus recommend manually curating the output of Phage Commander to generate maximally accurate annotations. Phage Commander is currently being used in the corresponding author's bacteriophage genome annotation class and has reduced the labor cost and improved the quality of genome annotations.

Bacteriophages or Phages are viruses that specifically infect and kill bacteria, and they have been shown to be effective against antibiotic-resistant bacteria in various studies. Phages are also able to evolve and adapt to the bacteria they infect, allowing them to overcome bacterial resistance. During this process, the phage can exchange genetic material with the bacteria, potentially giving rise to new phage strains that are better able to infect and kill the bacteria. This process of evolution and adaptation allows phages to keep up with the evolution of antibiotic-resistant bacteria. One concern is that the use of phages could lead to the evolution of phage-resistant bacteria. However, this risk can be mitigated by using a cocktail of different phages that target the same bacteria, as this reduces the likelihood of the bacteria developing resistance to all of the phages.

The rise in multi-drug resistant bacteria and the inability to develop novel antibacterial agents limits our arsenal against infectious diseases. Antibiotic resistance is a global issue requiring an immediate solution, including the development of new antibiotic molecules and other alternative modes of therapy. This article highlights the mechanism of bacteriophage treatment that makes it a real solution for multidrug-resistant infectious diseases. Several case reports identified phage therapy as a potential solution to the emerging challenge of multi-drug resistance. Bacteriophages, unlike antibiotics, have special features, such as host specificity and do not impact other commensals. A new outlook has also arisen with recent advancements in the understanding of phage immunobiology, where phages are repurposed against both bacterial and viral infections. Thus, the potential possibility of phages in COVID-19 patients with secondary bacterial infections has been briefly elucidated. However, significant obstacles that need to be addressed are to design better clinical studies that may contribute to the widespread use of bacteriophage therapy against multi-drug resistant pathogens. In conclusion, antibacterial agents can be used with bacteriophages, i.e. bacteriophage-antibiotic combination therapy, or they can be administered alone in cases when antibiotics are ineffective. Key points • AMR, a consequence of antibiotic generated menace globally, has led to the resurgence of phage therapy as an effective and sustainable solution without any side effects and high specificity against refractory MDR bacterial infections. • Bacteriophages have fewer adverse reactions and can thus be used as monotherapy as well as in conjunction with antibiotics. • In the context of the COVID-19 pandemic, phage therapy may be a viable option.

Ring ATPases perform a variety of tasks in the cell. Their function involves complex communication and coordination among the often identical subunits. Translocases in this group are of particular interest as they involve both chemical and mechanical actions in their operation. We study the DNA packaging motor of bacteriophage φ29, and using single-molecule optical tweezers and single-particle cryo-electron microscopy, have discovered a novel translocation mechanism for a molecular motor.

Salmonella contamination is a critical problem in poultry farms, with serious consequences for both animals and food products. The aim of this study is to investigate the use of phage cocktails to reduce Salmonella contamination in poultry farms. Within the scope of the study, Salmonella phages were isolated from chicken stool. After the host range of phages was determined, morphological characterization was performed through transmission electron microscopy analysis. Then, replication parameters and adsorption rates were determined by one-step growth curves. After that, phage cocktail was prepared, and its effectiveness was tested in three environments, which were drinking water, shavings, and plastic surfaces. The results obtained have demonstrated that the phage cocktail can reduce Salmonella count up to 2.80 log10 units in drinking water, up to 2.30 log10 units on shavings, and 2.31 log10 units on plastic surfaces. It has been determined that phage cocktails could be a successful alternative in reducing Salmonella contamination in poultry environment. This work is the first study to investigate the use of phage cocktails for reducing Salmonella contamination in poultry water and on shavings, and it is presumed that the results obtained will contribute to the fight against pathogens by making them applicable to poultry farms.

The CRISPR-Cas genome editing system is an intrinsic property of a bacteria-based immune system. This employs a guide RNA to detect and cleave the PAM-associated target DNA or RNA in subsequent infections, by the invasion of a similar bacteriophage. The discovery of Cas systems has paved the way to overcome the limitations of existing genome editing tools. In this review, we focus on Cas proteins that are available for gene modifications among which Cas9, Cas12a, and Cas13 have been widely used in the areas of medicine, research, and diagnostics. Since CRISPR has been already proven for its potential research applications, the next milestone for CRISPR will be proving its efficacy and safety. In this connection, we systematically review recent advances in exploring multiple variants of Cas proteins and their modifications for therapeutic applications.

Interest in phage-based therapeutics is increasing, at least in part due to the need for new treatment options for infections caused by antibiotic-resistant bacteria. It is possible to use wild-type (WT) phages to treat bacterial infections, but it is also possible to modify WT phages to generate therapeutics with improved features. Here, we will discuss features of Phico Therapeutics’ SASPject technology, which modifies phages for use as targetable nano-delivery vehicles (NDV), to introduce antibacterial Small Acid Soluble Spore Protein (SASP) genes into specific target bacteria.

The present review encompasses a patent landscape on bacteriophage as an antimicrobial agent and one of the alternatives to combat antibiotic resistance in bacteria. This study gives a perspective on use of bacteriophages in various industries such as healthcare, food safety and animal and plant protection. Patenting activity was noted for all the antibiotic-resistant bacterial pathogens listed in the ‘critical’ category by the WHO. Broadly, claims of the analyzed patents were directed toward bacteriophage, composition/formulation containing phage, phage proteins and various methods of using or producing phage. The challenges to approval of phage therapy in clinical use may be overcome with the help of focused research and modification of the regulatory guidelines for phage therapy.

Cronobacter sakazakii is an opportunistic pathogenic bacterium found in powdered infant formula and is fatal to neonates. Antibiotic resistance has emerged owing to overuse of antibiotics. Therefore, demand for high-yield bacteriophages as an alternative to antibiotics has increased. Accordingly, we developed a modified mass-production method for bacteriophages by introducing a two-stage self-cycling (TSSC) process, which yielded high-concentration bacteriophage solutions by replenishing the nutritional medium at the beginning of each process, without additional challenge. pH of the culture medium was monitored in real-time during C. sakazakii growth and bacteriophage CS01 propagation, and the changes in various parameters were assessed. The pH of the culture medium dropped to 5.8 when the host bacteria reached the early log phase (OD540 = 0.3). After challenge, it decreased to 4.65 and then recovered to 4.94; therefore, we set the optimum pH to challenge the phage at 5.8 and that to harvest the phage at 4.94. We then compared phage production during the TSSC process in jar-type bioreactors and the batch culture process in shaker flasks. In the same volume of LB medium, the concentration of the phage titer solution obtained with the TSSC process was 24 times higher than that obtained with the batch culture process. Moreover, we stably obtained high concentrations of bacteriophage solutions for three cycles with the TSSC process. Overall, this modified TSSC process could simplify large-scale production of bacteriophage CS01 and reduce the unit cost of phage titer solution. These results could contribute to curing infants infected with antibiotic-resistant C. sakazakii.

The widespread existence of bacteriophage has been of great interest to the biological research community and ongoing investigations continue to explore their diversity and role. They have also attracted attention and in-depth research in connection to fermented food processing, in particular from the dairy and wine industries. Bacteriophage, mostly oenophage, may in fact be a 'double edged sword' for winemakers: whilst they have been implicated as a causal agent of difficulties with malolactic fermentation (although not proven), they are also beginning to be considered as alternatives to using sulphur dioxide to prevent wine spoilage. Investigation and characterisation of oenophage of Oenococcus oeni, the main species used in winemaking, are still limited compared to lactococcal bacteriophage of Lactococcus lactis and Lactiplantibacillus plantarum (formally Lactobacillus plantarum), the drivers of most fermented dairy products. Interestingly, these strains are also being used or considered for use in winemaking. In this review, the genetic diversity and life cycle of phage, together with the debate on the consequent impact of phage predation in wine, and potential control strategies are discussed. KEY POINTS: • Bacteriophage detected in wine are diverse. • Many lysogenic bacteriophage are found in wine bacteria. • Phage impact on winemaking can depend on the stage of the winemaking process. • Bacteriophage as potential antimicrobial agents against spoilage organisms.

The work is devoted to the organization of postcovid rehabilitation by developing a strategy of adative phage therapy, which is the production and application of a complex of bacteriophages for a specific medical institution/department based on an up-to-date collection of hospital bacterial strains isolated from the biomaterial of patients of the same institution. Bacteriophages were actively used in the world in the 2040s of the twentieth century in various fields of medicine, but the rapid discovery of antibiotics and the lack of understanding of bacteriophage biology limited their use. The use of complex preparations of bacteriophages from the collection pure lines of bacteriophages of the SPC "MicroMir" a set of various phages aimed at restoring the human microbiome after a covid infection allowed to sharply reduce the number of antibiotics used in intensive care units and reduce antibiotic resistance with proven safety of phage therapy.

Food contamination caused by foodborne pathogens is one of the most significant concerns in public health worldwide, and accounts for a significant portion of food loss every year. The emergence of antimicrobial resistant bacteria has turned the researchers' attention back to the potential of bacteriophages as antibacterial agents, which have been attempted in various pre-and post-harvest food production settings. While the application of phage-based antibacterial products has achieved considerable success, a number of technical, environmental and administrative challenges remain unaddressed. In this review, we summarized the current status of bacteriophage application in food industry. More importantly, we discussed the obstacles facing the further development of phage-based antibacterial products from the aspects of technology, environmental safety, and administrative policy. We also put forward some possible solutions to these challenges, serving as reference information for future studies. This article is protected by copyright. All rights reserved.

Introduction: Streptococcus pneumoniae is a gram-positive bacterium, which is the leading cause of death for young children, elderly population, and immunocompromised patients. Its ability to mutate and become resistant to some of the strongest antibiotics makes them difficult to treat and increases the risk of disease spread. Although the development of stronger antibiotics to treat such microbes may be an option, they potentially pose a dangerous threat to the body. As such, a viable treatment option to fight against antimicrobial resistance has yet been found. Methods: The study focuses on utilizing a bi-therapy system to target S. pneumoniae in biofilm, which is the site of emerging antibiotic resistant mutants, by creating levofloxacin-liposomes carrying phages and testing them both in vitro and in vivo. Anticipated results: Using bacteriophage therapy and applying bacteriophage-antibiotic synergy, it is hoped to augment the potency of the treatment while lowering its side-effects. The Cp-1 bacteriophage-liposomes complexes are expected to be specific to the S. pneumoniae to carry antibiotics to sites of infection. Discussion: The therapy could ensure targeted bacterial lysis and site-directed delivery of low-dose drugs to decrease the toxicity effect of the antibiotics. Once the efficacy is established and is proven to be significant, its potency can be tested in BALB/cByJ mice models before bringing this therapy to animal trials then human clinical trials. Conclusion: Bacteriophages are very attractive therapeutic agents that effectively target pathogenic bacteria, safe for the human body, and highly modifiable to combat newly emerging bacterial threats. In addition to its many benefits, the use of bacteriophages could significantly reduce healthcare costs. The potential use of bacteriophages-liposomes complexes could be translated to treat respiratory infections in humans after confirming its efficacy in vitro and in vivo studies.

The Supporting Information is available free of charge at: https://pubs.acs.org/doi/10.1021/acsinfecdis.1c00366, Klebsiella pneumoniae is a Gram-negative bacterium that has become one of the leading causes of life-threatening healthcare-associated infections (HAIs), including pneumonia and sepsis. Moreover, due to its increasingly antibiotic resistance, K. pneumoniae has been declared a global top priority concern. The problem of K. pneumoniae infections is due, in part, to the inability to detect this pathogen rapidly and accurately and thus to treat patients within the early stages of infections. The success in bacterial detection is greatly dictated by the biorecognition molecule used, with the current diagnostic tools relying on expensive probes often lacking specificity and/or sensitivity. (Bacterio)phage receptor-binding proteins (RBPs) are responsible for the recognition and adsorption of phages to specific bacterial host receptors and thus present high potential as biorecognition molecules. In this study, we report the identification and characterization of a novel RBP from the K. pneumoniae phage KpnM6E1 that presents high specificity against the target bacteria and high sensitivity (80\%) to recognize K. pneumoniae strains. Moreover, adsorption studies validated the role of gp86 in the attachment to bacterial receptors, as it highly inhibits (86\%) phage adsorption to its Klebsiella host. Overall, in this study, we unravel the role and potential of a novel Klebsiella phage RBP as a powerful tool to be used coupled with analytical techniques or biosensing platforms for the diagnosis of K. pneumoniae infections., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the project “Phages-on-chip” PTDC/BTM-SAL/32442/2017 (POCI-01-0145-FEDER-032442) and the strategic funding of the research units CEB (UIDB/04469/2020) and INESC MN (UID/05367/2020) through pluriannual BASE and PRO GRAMATICO financing and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. Catarina Nogueira was supported by the FCT grant PD/BD/143037/2018. The authors acknowledge Hospital of Braga and Centro Clínico Académico (2CA-Braga) for the clinical bacterial isolates., info:eu-repo/semantics/publishedVersion

Bacterial chromosome replication is mainly catalyzed by DNA polymerase III, whose beta subunits enable rapid processive DNA replication. Enabled by the clamp-loading complex, the two beta subunits form a ring-like clamp around DNA and keep the polymerase sliding along. Given the essential role of β-clamp, its inhibitors have been explored for antibacterial purposes. Similarly, β-clamp is an ideal target for bacteriophages to shut off host DNA synthesis during host takeover. The Gp168 protein of phage Twort is such an example, which binds to the β-clamp of Staphylococcus aureus and prevents it from loading onto DNA causing replication arrest. Here, we report a cryo-EM structure of the clamp–Gp168 complex at 3.2-Å resolution. In the structure of the complex, the Gp168 dimer occupies the DNA sliding channel of β-clamp and blocks its loading onto DNA, which represents a new inhibitory mechanism against β-clamp function. Interestingly, the key residues responsible for this interaction on the β-clamp are well conserved among bacteria. We therefore demonstrate that Gp168 is potentially a cross-species β-clamp inhibitor, as it forms complex with the Bacillus subtilis β-clamp. Our findings reveal an alternative mechanism for bacteriophages to inhibit β-clamp and provide a new strategy to combat bacterial drug resistance.

The growing human population is currently facing an unprecedented challenge on global food production and sustainability. Despite recognizing poultry as one of the most successful and rapidly growing food industries to address this challenge; poultry health and safety remain major issues that entail immediate attention. Bacterial diseases including colibacillosis, salmonellosis, and necrotic enteritis have become increasingly prevalent during poultry production. Likewise, outbreaks caused by consumption of undercooked poultry products contaminated with zoonotic bacterial pathogens such as Salmonella, Campylobacter and Listeria, are a serious public health concern. With antimicrobial resistance problem and restricted use of antibiotics in food producing animals, bacteriophages are increasingly recognized as an attractive natural antibacterial alternative. Bacteriophages have recently shown promising results to treat diseases in poultry, reduce contamination of carcasses, and enhance the safety of poultry products. Omics technologies have been successfully employed to accurately characterize bacteriophages and their genes/proteins important for interaction with bacterial hosts. In this review, the potential of using lytic bacteriophages to mitigate the risk of major poultry-associated bacterial pathogens are explored. This study also explores challenges associated with the adoption of this technology by industries. Furthermore, the impact of omics approaches on studying bacteriophages, their host interaction and applications is discussed.

Viral infections caused by bacteriophages, i.e., viruses that kill bacteria are one of the most dangerous and common threats for bacteria-based bioreactors. More than 70% of biotechnology companies have admitted to encountering this problem. Despite phage infections being such a dangerous and widespread risk, there are no effective methods to avoid them to date. Herein, we present a novel technology based on nanoparticles that irreversibly deactivates bacteriophages and is safe for bacteria. Our method allows for the unsupervised protection of bacterial processes in the biotechnology industry. Gold nanoparticles coated with a mixture of negatively charged 11-mercapto 1-undecanesulfonic acid (MUS) and hydrophobic 1-octanethiol (OT) ligands are effective at deactivating various types of Escherichia coli-selective phages: T1, T4, and T7. The nanoparticles can lower the titer of phages up to 2 and 5 logs in 6 and 24 h at 50 °C, respectively. A comparative analysis of nanoparticles with different ligand shells illustrates the importance of the combination of negatively charged and hydrophobic ligands that is the key to achieving a good inhibitory concentration (EC50 ≤ 1 μg mL−1) for all tested phages. We show that the nanoparticles are harmless for the commonly used bacteria in industry Escherichia coli and are effective under conditions simulating the environment of bioreactors., Gold nanoparticles allow for the unsupervised protection against bacteriophage infections of bacterial processes in the biotechnology industry. Nanoparticles are harmless for bacteria and are effective in the environment of the bioreactor.

The emergence of antibiotic resistance has been raised due to the inappropriate use of antibiotics that might be the urgent health crisis. Antibiotics in animals and food chain have been widely used as growth promoters, prophylaxis, and treatment of infectious diseases, which provides the selective pressure on foodborne pathogens. It is essential to develop novel therapeutic weapon for controlling the appearance of resistance and inhibiting imminent threat of pandemics possible by means of multidrug resistant (MDR) pathogens. Nowadays, the control of MDR pathogens has been improved with the development of bacteriophage (phage)-based approaches such as single phage therapy, synergistic approach, phage cocktail, engineered phage, and anti-clustered regularly interspaced short palindromic repeats (anti-CRISPR) system. This review overwhelmingly focuses on slash the emergence of food-borne pathogens and its bacterial resistances using phage components and also discusses on the impact of phage components to diminish the outbreak of viral infections.

Yajun Zhang,1,* Biao Meng,2,3,* Xiao Wei,2,* Yan Li,2 Xiaohui Wang,1 Yan Zheng,1 Changjun Wang,2,3 Lihong Cui,1 Xiangna Zhao2,3 1Department of Gastroenterology, The Sixth Medical Center of PLA General Hospital, Beijing, People’s Republic of China; 2Centre for Disease Control and Prevention of China PLA, Beijing, People’s Republic of China; 3Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University, Hefei, People’s Republic of China*These authors contributed equally to this workCorrespondence: Lihong Cui; Xiangna Zhao Tel +86-10-66948393Fax +86-10-66948304Email luckycui861@sina.com; xnazhao@163.comBackground: Pseudomonas aeruginosa is an important nosocomial infectious bacterium, more and more multidrug resistant P. aeruginosa have been isolated and posed severe challenges to clinical antibiotic treatment, bringing additional morbidity, mortality, and economic burden. Bacteriophages can lyse bacteria specificity and are feasible alternatives to antibiotics.Methods: A Pseudomonas aeruginosa-infecting phage vB_PaeP_PA01EW was isolated. Phage plaque assays, transmission electron microscopy, host-range determination, infection assay analyses, whole-genome sequencing and annotation were performed for the phage. Mice pneumonia model using liquid aerosol-exposure Pseudomonas aeruginosa was established, and phage therapy was evaluated.Results: vB_PaeP_PA01EW belongs to the family Podoviridae according to transmission electron microscopy and was identified as a Luz24likevirus according to the genome analysis. For the phage therapy, compared with the bacteria-infected group, the phage-rescue group has some characteristics. First, adventitial edema and diffuse infiltration of inflammatory cells in tissues were alleviated, Second, bronchial epithelial cell proliferation was reduced. Third, the bacterial burden was significantly decreased.Conclusion: This study provided data support and theoretical basis for the clinical application of bacteriophages. It has important guiding significance and reference value for the application of bacteriophage therapy of other pathogenic bacteria.Keywords: P. aeruginosa, bacteriophage, vB_PaeP_PA01EW, aerosol-exposure

Bacteriophage (phage) therapy has re-gained attention lately given the ever-increasing prevalence of multi-drug resistance ‘super-bugs’. To develop therapeutic phage into clinically usable drug products, the strategy of solidifying phage formulations has been implemented to diversify the dosage forms and to overcome the storage condition limitations for liquid phage formulations. In our work, we hypothesize and tested that an advanced technology, thin film freeze-drying (TFFD), can be used to produce phage containing dry powders without significantly losing phage viability. Here we selected T7 phage as our model phage in a preliminary screening study. We found that a binary excipient matrix of sucrose and leucine at ratios of 90:10 or 75:25 by weight, protected phage from the stresses encountered during the TFFD process. In addition, we confirmed that incorporating a buffer system in the formulation significantly improved the survival of phage during the initial freezing step and subsequent sublimation step in the solidifying processes. The titer loss of phage in SM buffer (Tris/NaCl/MgSO4) containing formulation was as low as 0.19 log plaque forming units, which indicated that phage function was well preserved after the TFFD process. The presence of buffers markedly reduced the geometric particle sizes as determined by a dry dispersion method using laser diffraction, which indicated that the TFFD phage powder formulations were easily sheared into smaller powder aggregates, an ideal property for facilitating a variety of topical drug delivery routes including pulmonary delivery through dry powder inhalers, nebulization after reconstitution, and intranasal or wound therapy, etc. From these findings, we show that introducing buffer system can stabilize phage during dehydration processes, and TFFD, as a novel particle engineering method, can successfully produce phage containing powders that possess the desired properties for bioactivity and potentially for inhalation therapy.

Background Winter carbon loss in northern ecosystems is estimated to be greater than the average growing season carbon uptake and is primarily driven by microbial decomposers. Viruses modulate microbial carbon cycling via induced mortality and metabolic controls, but it is unknown whether viruses are active under winter conditions (anoxic and sub-freezing temperatures). Results We used stable isotope probing (SIP) targeted metagenomics to reveal the genomic potential of active soil microbial populations under simulated winter conditions, with an emphasis on viruses and virus-host dynamics. Arctic peat soils from the Bonanza Creek Long-Term Ecological Research site in Alaska were incubated under sub-freezing anoxic conditions with H218O or natural abundance water for 184 and 370 days. We sequenced 23 SIP-metagenomes and measured carbon dioxide (CO2) efflux throughout the experiment. We identified 46 bacterial populations (spanning 9 phyla) and 243 viral populations that actively took up 18O in soil and respired CO2 throughout the incubation. Active bacterial populations represented only a small portion of the detected microbial community and were capable of fermentation and organic matter degradation. In contrast, active viral populations represented a large portion of the detected viral community and one third were linked to active bacterial populations. We identified 86 auxiliary metabolic genes and other environmentally relevant genes. The majority of these genes were carried by active viral populations and had diverse functions such as carbon utilization and scavenging that could provide their host with a fitness advantage for utilizing much-needed carbon sources or acquiring essential nutrients. Conclusions Overall, there was a stark difference in the identity and function of the active bacterial and viral community compared to the unlabeled community that would have been overlooked with a non-targeted standard metagenomic analysis. Our results illustrate that substantial active virus-host interactions occur in sub-freezing anoxic conditions and highlight viruses as a major community-structuring agent that likely modulates carbon loss in peat soils during winter, which may be pivotal for understanding the future fate of arctic soils' vast carbon stocks.

80 Acinetobacter strains were studied. Their differentiation according to the specific belonging showed that 55 strains belonged toA. calcoaceticusvar.anitratumspecies and 25 strains toA. calcoaceticusvar.lwoffi. It was established that Acinetobacter strains are multi dry resistant. In particular they showed 100% resistance to ampicillin, carbenicillin, erythromycin, methicillin, oxacillin, tetracycline, oleandomycin, streptomycin, rondomycin, linkomycin. According to the obtained data claphoran and phortum are the most effective.

Introduction: Endolysins, antimicrobial peptides that disrupt the peptidoglycan (PG) layer of bacteria, are a strong alternative to common antibiotics and less prone to antibiotic resistance. However, endolysins are ineffective against gram-negative bacteria due to an additional outer membrane (OM) blocking the PG layer. This research protocol aims to address this caveat by developing a novel recombinant endolysin peptide, EndoT5-Ctn (15-34), comprised of phage Endolysin T5 and a Crotalicidin tag (Ctn (15-34)) capable of permeabilizing the OM. This would render the endolysin effective against gram-negative bacteria such as multidrug-resistant (MDR) Pseudomonas aeruginosa. Methods: Experimentation will begin with recombinant DNA techniques to engineer a bacterial vector which expresses the EndoT5-Ctn (15-34) peptide. Protein expression will be achieved in competent BL21 (DE3) E. coli strains before protein isolation with lysis methods and immobilized-metal affinity chromatography. Resulting sample concentrations will be assayed using spectrophotometry. Finally, in vivo minimal inhibitory and bactericidal concentration assays will be conducted on MDR P. aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA) to evaluate bactericidal efficacy. Results: A minimal bactericidal concentration of EndoT5-Ctn (15-34) recombinant endolysin is expected to yield significant bactericidal activity against the MDR P. aeruginosa strain with limited effect on the gram-positive MRSA. A higher concentration of recombinant endolysin is expected to maintain its gram-negative bactericidal efficacy with an imported gram-positive bactericidal efficacy. Discussion: The observed bactericidal efficacy on AMR and non-AMR strains on P. aeruginosa will prompt further exploration of the therapeutic feasibility of this therapy regarding factors such as cytotoxicity, immunogenicity, and thermal stability. The potential introduction of recombinant endolysins presents an alternative treatment for infections with reduced risk of inducing resistant mutations. Conclusion: The EndoT5-Ctn (15-34) recombinant endolysin is a novel anti-bacterial agent which is expected to have significant bactericidal effects on MDR strains of gram-negative bacteria which current antibiotics have been ineffective at combating.