Genomic surveillance as a scalable framework for precision phage therapy against antibiotic-resistant pathogens.

Phage therapy is gaining increasing interest in the fight against critically antibiotic-resistant nosocomial pathogens. However, the narrow host range of bacteriophages hampers the development of broadly effective phage therapeutics and demands precision approaches. Here, we combine large-scale phylogeographic analysis with high-throughput phage typing to guide the development of precision phage cocktails targeting carbapenem-resistant Acinetobacter baumannii, a top-priority pathogen. Our analysis reveals that a few strain types dominate infections in each world region, with their geographical distribution remaining stable within 6 years. As we demonstrate in Eastern Europe, this spatiotemporal distribution enables preemptive preparation of region-specific phage collections that target most local infections. Finally, we showcase the efficacy of phage cocktails against prevalent strain types using in vitro and animal infection models. Ultimately, genomic surveillance identifies patients benefiting from the same phages across geographical scales, thus providing a scalable framework for precision phage therapy.

Discovery of phage determinants that confer sensitivity to bacterial immune systems.

Over the past few years, numerous anti-phage defense systems have been discovered in bacteria. Although the mechanism of defense for some of these systems is understood, a major unanswered question is how these systems sense phage infection. To systematically address this question, we isolated 177 phage mutants that escape 15 different defense systems. In many cases, these escaper phages were mutated in the gene sensed by the defense system, enabling us to map the phage determinants that confer sensitivity to bacterial immunity. Our data identify specificity determinants of diverse retron systems and reveal phage-encoded triggers for multiple abortive infection systems. We find general themes in phage sensing and demonstrate that mechanistically diverse systems have converged to sense either the core replication machinery of the phage, phage structural components, or host takeover mechanisms. Combining our data with previous findings, we formulate key principles on how bacterial immune systems sense phage invaders.

Episymbiotic Saccharibacteria TM7x modulates the susceptibility of its host bacteria to phage infection and promotes their coexistence.

Bacteriophages (phages) play critical roles in modulating microbial ecology. Within the human microbiome, the factors influencing the long-term coexistence of phages and bacteria remain poorly investigated. Saccharibacteria (formerly TM7) are ubiquitous members of the human oral microbiome. These ultrasmall bacteria form episymbiotic relationships with their host bacteria and impact their physiology. Here, we showed that during surface-associated growth, a human oral Saccharibacteria isolate (named TM7x) protects its host bacterium, a Schaalia odontolytica strain (named XH001) against lytic phage LC001 predation. RNA-Sequencing analysis identified in XH001 a gene cluster with predicted functions involved in the biogenesis of cell wall polysaccharides (CWP), whose expression is significantly down-regulated when forming a symbiosis with TM7x. Through genetic work, we experimentally demonstrated the impact of the expression of this CWP gene cluster on bacterial-phage interaction by affecting phage binding. In vitro coevolution experiments further showed that the heterogeneous populations of TM7x-associated and TM7x-free XH001, which display differential susceptibility to LC001 predation, promote bacteria and phage coexistence. Our study highlights the tripartite interaction between the bacterium, episymbiont, and phage. More importantly, we present a mechanism, i.e., episymbiont-mediated modulation of gene expression in host bacteria, which impacts their susceptibility to phage predation and contributes to the formation of "source-sink" dynamics between phage and bacteria in biofilm, promoting their long-term coexistence within the human microbiome.

Characterization of phage evolution and phage resistance in drug-resistant Stenotrophomonas maltophilia.

Phage therapy has become a viable antimicrobial treatment as an alternative to antibiotic treatment, with an increase in antibiotic resistance. Phage resistance is a major limitation in the therapeutic application of phages, and the lack of understanding of the dynamic changes between bacteria and phages constrains our response strategies to phage resistance. In this study, we investigated the changing trends of mutual resistance between Stenotrophomonas maltophilia (S. maltophilia) and its lytic phage, BUCT603. Our results revealed that S. maltophilia resisted phage infection through mutations in the cell membrane proteins, while the evolved phage re-infected the resistant strain primarily through mutations in structure-related proteins. Compared with the wild-type strain (SMA118), the evolved phage-resistant strain (R118-2) showed reduced virulence, weakened biofilm formation ability, and reduced resistance to aminoglycosides. In addition, the evolved phage BUCT603B1 in combination with kanamycin could inhibit the development of phage-resistant S. maltophilia in vitro and significantly improve the survival rate of S. maltophilia-infected mice. Altogether, these results suggest that in vitro characterization of bacteria-phage co-evolutionary relationships is a useful research tool to optimize phages for the treatment of drug-resistant bacterial infections.IMPORTANCEPhage therapy is a promising approach to treat infections caused by drug-resistant Stenotrophomonas maltophilia (S. maltophilia). However, the rapid development of phage resistance has hindered the therapeutic application of phages. In vitro evolutionary studies of bacteria-phage co-cultures can elucidate the mechanism of resistance development between phage and its host. In this study, we investigated the resistance trends between S. maltophilia and its phage and found that inhibition of phage adsorption is the primary strategy by which bacteria resist phage infection in vitro, while phages can re-infect bacterial cells by identifying other adsorption receptors. Although the final bacterial mutants were no longer infected by phages, they incurred a fitness cost that resulted in a significant reduction in virulence. In addition, the combination treatment with phage and aminoglycoside antibiotics could prevent the development of phage resistance in S. maltophilia in vitro. These findings contribute to increasing the understanding of the co-evolutionary relationships between phages and S. maltophilia.

Conserved domains can be found across distinct phage defence systems.

Bacteria are continuously exposed to predation from bacteriophages (phages) and, in response, have evolved a broad range of defence systems. These systems can prevent the replication of phages and other mobile genetic elements (MGE). Defence systems are often encoded together in genomic loci defined as "defence islands", a tendency that has been extensively exploited to identify novel antiphage systems. In the last few years, >100 new antiphage systems have been discovered, and some display homology to components of the immune systems of plants and animals. In many instances, prediction tools have found domains with similar predicted functions present as different combinations within distinct antiphage systems. In this Perspective Article, we review recent reports describing the discovery and the predicted domain composition of several novel antiphage systems. We discuss several examples of similar protein domains adopted by different antiphage systems, including domains of unknown function (DUFs), domains involved in nucleic acid recognition and degradation, and domains involved in NAD+ depletion. We further discuss the potential evolutionary advantages that could have driven the independent acquisition of these domains by different antiphage systems.

fENko-Kae01 is a flagellum-specific jumbo phage infecting Klebsiella aerogenes.

BACKGROUND: Klebsiella aerogenes is an opportunistic pathogen that causes a wide variety of infections. Due to the rising problem of antibiotic resistance, novel antibiotics and strategies to combat bacterial infections are needed. Host-specific bacteriophages are natural enemies of bacteria and can be used in phage therapy as an alternative form of treatment against bacterial infections. Jumbo phages are defined as phages with genomes larger than 200 kb. Relatively few studies have been done on jumbo phages compared to smaller phages. RESULTS: A novel phage, fENko-Kae01, was isolated from a commercial phage cocktail. Genomic analysis revealed that fENko-Kae01 is a lytic jumbo phage with a 360 kb genome encoding 578 predicted genes. No highly similar phage genomes were identified and fENko-Kae01 may be a completely new genus representative. No known genes associated with lysogenic life cycle, bacterial virulence, or antibiotic resistance were identified. The phage had myovirus morphology and a narrow host range. Phage resistant bacterial mutants emerged under phage selection. Whole genome sequencing revealed that the biogenesis of the flagellum was affected in four mutants and the lack of functional flagellum was confirmed in motility assays. Furthermore, phage fENKo-Kae01 failed to adsorb on the non-motile mutants indicating that the bacterial flagellum is the phage-binding receptor. CONCLUSIONS: fENko-Kae01 is a novel jumbo bacteriophage that is considered safe for phage therapy. fENko-Kae01 uses the flagellum as the phage-binding receptor and may represent a completely novel genus.

Antibacterial efficacy of mycobacteriophages against virulent Mycobacterium tuberculosis.

Tuberculosis (TB) remains a major global health concern, with drug-resistant strains posing a significant challenge to effective treatment. Bacteriophage (phage) therapy has emerged as a potential alternative to combat antibiotic resistance. In this study, we investigated the efficacy of widely used mycobacteriophages (D29, TM4, DS6A) against Mycobacterium tuberculosis (M. tuberculosis) under pathophysiological conditions associated with TB, such as low pH and hypoxia. We found that even at low multiplicity of infection (MOI), mycobacteriophages effectively infected M. tuberculosis, got rapidly amplified, and lysed M. tuberculosis, demonstrating their potential as therapeutic agents. Furthermore, we observed a novel phage tolerance mechanism with bacteria forming aggregates after several days of phage treatment. These aggregates were enriched with biofilm components and metabolically active bacteria. However, no phage tolerance was observed upon treatment with the three-phage mixture, highlighting the dynamic interplay between phages and bacteria and emphasizing the importance of phage cocktails. We also observed that phages were effective in lysing bacteria even under low pH and low oxygen concentrations as well as antibiotic-resistant bacteria. Our results provide key insights into phage infection of slow-growing bacteria and suggest that mycobacteriophages can effectively eliminate M. tuberculosis in complex pathophysiological environments like hypoxia and acidic pH. These results can aid in developing targeted phage-based therapies to combat antibiotic-resistant mycobacterial infections.

Salmonella enteritidis acquires phage resistance through a point mutation in rfbD but loses some of its environmental adaptability.

Phage therapy holds promise as an alternative to antibiotics for combating multidrug-resistant bacteria. However, host bacteria can quickly produce progeny that are resistant to phage infection. In this study, we investigated the mechanisms of bacterial resistance to phage infection. We found that Rsm1, a mutant strain of Salmonella enteritidis (S. enteritidis) sm140, exhibited resistance to phage Psm140, which was originally capable of lysing its host at sm140. Whole genome sequencing analysis revealed a single nucleotide mutation at position 520 (C → T) in the rfbD gene of Rsm1, resulting in broken lipopolysaccharides (LPS), which is caused by the replacement of CAG coding glutamine with a stop codon TAG. The knockout of rfbD in the sm140ΔrfbD strain caused a subsequent loss of sensitivity toward phages. Furthermore, the reintroduction of rfbD in Rsm1 restored phage sensitivity. Moreover, polymerase chain reaction (PCR) amplification of rfbD in 25 resistant strains revealed a high percentage mutation rate of 64% within the rfbD locus. We assessed the fitness of four bacteria strains and found that the acquisition of phage resistance resulted in slower bacterial growth, faster sedimentation velocity, and increased environmental sensitivity (pH, temperature, and antibiotic sensitivity). In short, bacteria mutants lose some of their abilities while gaining resistance to phage infection, which may be a general survival strategy of bacteria against phages. This study is the first to report phage resistance caused by rfbD mutation, providing a new perspective for the research on phage therapy and drug-resistant mechanisms.

Screening of genes involved in phage-resistance of Escherichia coli and effects of substances interacting with primosomal protein A on the resistant bacteria.

AIMS: The study was to identify the genes involved in phage resistance and to develop an effective biocontrol method to improve the lytic activity of phages against foodborne pathogens. METHODS AND RESULTS: A total of 3,909 single gene-deletion mutants of Escherichia coli BW25113 from the Keio collection were individually screened for genes involved in phage resistance. Phage S127BCL3 isolated from chicken liver, infecting both E. coli BW25113 and O157: H7, was characterized and used for screening. The 10 gene-deletion mutants showed increased susceptibility to phage S127BCL3. Among them, priA gene-deletion mutant strain showed significant susceptibility to the phages S127BCL3 and T7. Furthermore, we investigated the substances that have been reported to inhibit the function of primosomal protein A (PriA) and were used to confirm increased phage susceptibility in E. coli BW25113 (Parent strain) and O157: H7. CONCLUSION: PriA inhibitors at a low concentration showed combined effects with phage against E. coli O157: H7 and delayed the regrowth rate of phage-resistant cells.

K1 capsule-dependent phage-driven evolution in Escherichia coli leading to phage resistance and biofilm production.

AIMS: Understanding bacterial phage resistance mechanisms has implications for developing phage-based therapies. This study aimed to explore the development of phage resistance in Escherichia coli K1 isolates' to K1-ULINTec4, a K1-dependent bacteriophage. METHODS AND RESULTS: Resistant colonies were isolated from two different strains (APEC 45 and C5), both previously exposed to K1-ULINTec4. Genome analysis and several parameters were assessed, including growth capacity, phage adsorption, phenotypic impact at capsular level, biofilm production, and virulence in the in vivo Galleria mellonella larvae model. One out of the six resistant isolates exhibited a significantly slower growth rate, suggesting the presence of a resistance mechanism altering its fitness. Comparative genomic analysis revealed insertion sequences in the region 2 of the kps gene cluster involved in the capsule biosynthesis. In addition, an immunoassay targeting the K1 capsule showed a very low positive reaction compared to the control. Nevertheless, microscopic images of resistant strains revealed the presence of capsules with a clustered organization of bacterial cells and biofilm assessment showed an increased biofilm production compared to the sensitive strains. In the G. mellonella model, larvae infected with phage-resistant isolates showed better survival rates than larvae infected with phage-sensitive strains. CONCLUSIONS: A phage resistance mechanism was identified at the genomic level and had a negative impact on the K1 capsule production. The resistant isolates showed an increased biofilm production and a decreased virulence in vivo.

Correlation between the development of phage resistance and the original antibiotic resistance of host bacteria under the co-exposure of antibiotic and bacteriophage.

Bacteriophages (phages) are viruses capable of regulating the proliferation of antibiotic resistant bacteria (ARB). However, phages that directly cause host lethality may quickly select for phage resistant bacteria, and the co-evolutionary trade-offs under varying environmental conditions, including the presence of antibiotics, remains unclear as to their impact on phage and antibiotic resistance. Here, we report the emergence of phage resistance in three distinct E. coli strains with varying resistance to ß-lactam antibiotics, treated with different ampicillin (AMP) concentrations. Hosts exhibiting stronger antibiotic resistance demonstrated a higher propensity to develop and maintain stable phage resistance. When exposed to polyvalent phage KNT-1, the growth of AMP-sensitive E. coli K12 was nearly suppressed within 18 h, while the exponential growth of AMP-resistant E. coli TEM and super-resistant E. coli NDM-1 was delayed by 12 h and 8 h, respectively. The mutation frequency and mutated colony count of E. coli NDM-1 were almost unaffected by co-existing AMP, whereas for E. coli TEM and K12, these metrics significantly decreased with increasing AMP concentration from 8 to 50 µg/mL, becoming unquantifiable at 100 µg/mL. Furthermore, the fitness costs of phage resistance mutation and its impact on initial antibiotic resistance in bacteria were further examined, through analyzing AMP susceptibility, biofilm formation and EPS secretion of the isolated phage resistant mutants. The results indicated that acquiring phage resistance could decrease antibiotic resistance, particularly for hosts lacking strong antibiotic resistance. The ability of mutants to form biofilm contributes to antibiotic resistance, but the correlation is not entirely positive, while the secretion of extracellular polymeric substance (EPS), especially the protein content, plays a crucial role in protecting the bacteria from both antibiotic and phage exposure. This study explores phage resistance development in hosts with different antibiotic resistance and helps to understand the limitations and possible solutions of phage-based technologies.

Quorum Sensing Controls Adaptive Immunity through the Regulation of Multiple CRISPR-Cas Systems.

Bacteria commonly exist in high cell density populations, making them prone to viral predation and horizontal gene transfer (HGT) through transformation and conjugation. To combat these invaders, bacteria possess an arsenal of defenses, such as CRISPR-Cas adaptive immunity. Many bacterial populations coordinate their behavior as cell density increases, using quorum sensing (QS) signaling. In this study, we demonstrate that QS regulation results in increased expression of the type I-E, I-F, and III-A CRISPR-Cas systems in Serratia cells in high-density populations. Strains unable to communicate via QS were less effective at defending against invaders targeted by any of the three CRISPR-Cas systems. Additionally, the acquisition of immunity by the type I-E and I-F systems was impaired in the absence of QS signaling. We propose that bacteria can use chemical communication to modulate the balance between community-level defense requirements in high cell density populations and host fitness costs of basal CRISPR-Cas activity.

Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation.

Prokaryotes use a mechanism called priming to update their CRISPR immunological memory to rapidly counter revisiting, mutated viruses, and plasmids. Here we have determined how new spacers are produced and selected for integration into the CRISPR array during priming. We show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30-100 nt enriched for thymine-stretches in their 3' ends. The Cas1-2 complex further processes these fragments and integrates them sequence-specifically into CRISPR repeats by coupling of a 3' cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers.

Correlation of Pseudomonas aeruginosa Phage Resistance with the Numbers and Types of Antiphage Systems.

Phage therapeutics offer a potentially powerful approach for combating multidrug-resistant bacterial infections. However, to be effective, phage therapy must overcome existing and developing phage resistance. While phage cocktails can reduce this risk by targeting multiple receptors in a single therapeutic, bacteria have mechanisms of resistance beyond receptor modification. A rapidly growing body of knowledge describes a broad and varied arsenal of antiphage systems encoded by bacteria to counter phage infection. We sought to understand the types and frequencies of antiphage systems present in a highly diverse panel of Pseudomonas aeruginosa clinical isolates utilized to characterize novel antibacterials. Using the web-server tool PADLOC (prokaryotic antiviral defense locator), putative antiphage systems were identified in these P. aeruginosa clinical isolates based on sequence homology to a validated and curated catalog of known defense systems. Coupling this host bacterium sequence analysis with host range data for 70 phages, we observed a correlation between existing phage resistance and the presence of higher numbers of antiphage systems in bacterial genomes. We were also able to identify antiphage systems that were more prevalent in highly phage-resistant P. aeruginosa strains, suggesting their importance in conferring resistance.

Phage Mediated Biocontrol: A Promising Green Solution for Sustainable Agriculture.

In the current scenario of growing world population, limited cultivable land resources, plant diseases, and pandemics are some of the major factors responsible for declining global food security. Along with meeting the food demand, the maintenance of food quality is also required to ensure healthy consumption and marketing. In agricultural fields, pest infestations and bacterial diseases are common causes of crop damage, leading to massive yield losses. Conventionally, antibiotics and several pesticides have been used to manage and control these plant pathogens. However, the overuse of antibiotics and pesticides has led to the emergence of resistant strains of pathogenic bacteria. The bacteriophages are the natural predators of bacteria and are host-specific in their action. Therefore, the use of bacteriophages for the biocontrol of pathogenic bacteria is serving as a sustainable and green solution in crop protection and production. In this review, we have discussed the important plant pathogens and their impact on plant health and yield loss. Further, we have abridged the role of bacteriophages in the protection of crops from bacterial disease by discussing various greenhouse and field trials. Finally, we have discussed the impact of bacteriophages on the plant microbiome, phage resistance, and legal challenges in the registration and commercial production of bacteriophage-based biopesticides. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-024-01204-x.

Ciprofloxacin in combination with bacteriophage cocktails against multi-drug resistant Pseudomonas aeruginosa in ex vivo simulated endocardial vegetation models.

Pseudomonas aeruginosa-associated infective endocarditis represents difficult-to-treat, deep-seated infections. Phage-antibiotic combinations have shown to eradicate multi-drug resistant (MDR) P. aeruginosa, limit the development of phage resistance, and restore antibiotic sensitivity. The objective of this study was to evaluate the activity of phage-ciprofloxacin (CIP) combinations in 4-day ex vivo simulated endocardial vegetation (SEV) models against drug-resistant P. aeruginosa isolates. Two P. aeruginosa isolates, extensively drug-resistant AR351 and MDR I0003-1, were selected for their drug resistance and sensitivity to phage. Three phages [LL-5504721-AH (LL), E2005-C (EC), and 109] and CIP were evaluated alone and in combination for their activity and influence on drug and phage resistance using 24-h time-kill analysis. The three-phage cocktail (q24h) in combination with CIP (400 mg q12h) was then tested in dynamic 4-day ex vivo SEV models, with reduction of log10 CFU/mL compared using ANOVA with Bonferroni analysis. Compared to other combinations, CIP-LL-EC-109 demonstrated synergistic and bactericidal activity from starting CFU/g against AR351 and I0003-1 (-Δ5.65 and 6.60 log10 CFU/g, respectively; P < 0.001). Additionally, CIP-LL-EC-109 mitigated phage resistance, while all other therapies had a high degree of resistance to >1 phages, and all phage-containing regimens prevented CIP mean inhibitory concentration increases compared to CIP alone for both AR351 and I0003-1 at 96 h.

Back to the Roots: Agrobacterium-Specific Phages Show Potential to Disinfect Nutrient Solution from Hydroponic Greenhouses.

Agrobacterium biovar 1 is a soilborne plant pathogen with the ability to colonize the irrigation system of greenhouses, causing hairy root disease (HRD). Currently, management focuses on using hydrogen peroxide to disinfect the nutrient solution, but due to the emergence of resistant strains, its efficacy and sustainability are questioned. Using a relevant collection of pathogenic Agrobacterium biovar 1 strains, OLIVR1 to 6, six phages specific to this pathogen and belonging to three different genera were isolated from Agrobacterium biovar 1-infected greenhouses. All phages were named OLIVR, referring to their location of isolation, Onze-Lieve-Vrouwe-Waver, and were characterized by whole-genome analysis, confirming their strictly lytic lifestyle. They remained stable under greenhouse-relevant conditions. To assess the efficacy of the phages, their ability to disinfect greenhouse nutrient solution inoculated with agrobacteria was tested. Each of the phages infected their host, but their ability to decrease the bacterial concentration differed. For instance, OLIVR1 reduced the bacterial concentration with 4 log units without phage resistance emerging. While OLIVR4 and OLIVR5 were also infectious in nutrient solution, they did not always decrease the bacterial load below the limit of detection, and phage resistance emerged. Finally, the mutations causing phage resistance by receptor modification were identified. For OLIVR4-resistant Agrobacterium isolates, but not for OLIVR5-resistant isolates, motility decreased. Together, these data show the potential of some of these phages as disinfectant of nutrient solution, and they might be a valuable tool to tackle HRD. IMPORTANCE Hairy root disease, caused by rhizogenic Agrobacterium biovar 1 is a rapidly emerging bacterial disease worldwide. It affects tomatoes, cucumbers, eggplant, and bell pepper, causing high yield losses in hydroponic greenhouses. Recent findings suggest that the current management practices, mainly focusing on UV-C and hydrogen peroxide to disinfect contaminated water, have a questionable efficacy. Hence, we investigate the potential of phages as a biological means of preventing this disease. Using a diverse collection of Agrobacterium biovar 1, we isolated three different phage species that together infect 75% of the collection. Since these phages are strictly lytic, while remaining both stable and infectious under greenhouse-relevant conditions, they might be suitable candidates for biological control.

Phage-bacterial evolutionary interactions: experimental models and complications.

Phage treatment of bacterial infections has offered some hope even as the crisis of antimicrobial resistance continues to be on the rise. However, bacterial resistance to phage is another looming challenge capable of undermining the effectiveness of phage therapy. Moreover, the consideration of including phage therapy in modern medicine calls for more careful research around every aspect of phage study. In an attempt to adequately prepare for the events of phage resistance, many studies have attempted to experimentally evolve phage resistance in different bacterial strains, as well as train phages to evolve counter-infectivity of resistant bacterial mutants, in view of answering such questions as coevolutionary dynamics between phage and bacteria, mechanisms of phage resistance, fitness costs of phage resistance on bacteria, etc. In this review, we summarised many such studies and by careful examination, highlighted critical issues to the outcome of phage therapy. We also discuss the insufficiency of many of these in vitro studies to represent actual disease conditions during phage application, alongside other complications that exist in phage-bacterial evolutionary interactions. Conclusively, we present the exploitation of phage-bacterial interactions for successful infection managements, as well as some future perspectives to direct phage research.

The current status of phage therapy and its advancement towards establishing standard antimicrobials for combating multi drug-resistant bacterial pathogens.

Phage therapy; a revived antimicrobial weapon, has great therapeutic advantages with the main ones being its ability to eradicate multidrug-resistant pathogens as well as selective toxicity, which ensures that beneficial microbiota is not harmed, unlike antibiotics. These therapeutic properties make phage therapy a novel approach for combating resistant pathogens. Since millions of people across the globe succumb to multidrug-resistant infections, the implementation of phage therapy as a standard antimicrobial could transform global medicine as it offers greater therapeutic advantages than conventional antibiotics. Although phage therapy has incomplete clinical data, such as a lack of standard dosage and the ideal mode of administration, the conducted clinical studies report its safety and efficacy in some case studies, and therefore, this could lessen the concerns of its skeptics. Since its discovery, the development of phage therapeutics has been in a smooth progression. Concerns about phage resistance in populations of pathogenic bacteria are raised when bacteria are exposed to phages. Bacteria can use restriction-modification, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) defense, or mutations in the phage receptors to prevent phage invasion. Phage resistance, however, is often costly for the bacteria and may lead to a reduction in its virulence. The ongoing competition between bacteria and phage, on the other hand, ensures the emergence of phage strains that have evolved to infect resistant bacteria. A phage can quickly adapt by altering one or more aspects of its mode of infection, evading a resistance mechanism through genetic modifications, or directly thwarting the CRISPR-Cas defense. Using phage-bacterium coevolution as a technique could be crucial in the development of phage therapy as well. Through its recent advancement, gene-editing tools such as CRISPR-Cas allow the bioengineering of phages to produce phage cocktails that have broad spectrum activities, which could maximize the treatment's efficacy. This review presents the current state of phage therapy and its progression toward establishing standard medicine for combating antibiotic resistance. Recent clinical trials of phage therapy, some important case studies, and other ongoing clinical studies of phage therapy are all presented in this review. Furthermore, the recent advancement in the development of phage therapeutics, its application in various sectors, and concerns regarding its implementation are also highlighted here. Phage therapy has great potential and could help the fight against drug-resistant bacterial pathogens.

The metastable associations of bacteriophages and Erwinia amylovora.

Bacteriophages are often considered as possible agents of biological control of unwanted bacterial populations in medicine, agriculture and food industry. Although the virulent phages can efficiently kill the infected host cells but at the population level phage attack not always leads to the host population collapse but may result in establishment of a more or less stable co-existence. The mechanism of the long-term stabilization of the mixed phage-host cultures is poorly understood. Here we describe bacteriophages VyarbaL and Hena2, the members of the Molineuxvirinae and the Ounavirinae subfamilies, respectively, that are able to form the pseudolysogenic associations (PA) with their host Erwinia amylovora 1/79Sm on solid media. These PAs were stable through multiple passages. The phenomenon of the PA formation between a bacterial culture and bacteriophages decreases the effectiveness of bacteriophage-mediated biological control agents based on lytic bacteriophages.