Bacteriophages as a potential substitute for antibiotics: A comprehensive review.

Over the years, the administration of antibiotics for the purpose of addressing bacterial infections has become increasingly challenging due to the increased prevalence of antimicrobial resistance exhibited by various strains of bacteria. Multidrug-resistant (MDR) bacterial species are rising due to the unavailability of novel antibiotics, leading to higher mortality rates. With these conditions, there is a need for alternatives in which phage therapy has made promising results. Phage-derived endolysins, phage cocktails, and bioengineered phages are effective and have antimicrobial properties against MDR and extensively drug-resistant strains. Despite these, it has been observed that phages can give antimicrobial activity to more than one bacterial species. Thus, phage cocktail against resistant strains provides broad spectrum treatment and magnitude of effectivity, which is many folds higher than antibiotics. Many commercially available endolysins such as Staphefekt SA.100, Exebacase (CF-301), and N-Rephasin®SAL200 are used in biofilm penetration and treating plant diseases. The role of CMP1 phage endolysin in transgenic tomato plants in preventing Clavibacter michiganensis infection and the effectiveness of phage in protecting Atlantic salmon from vibriosis have been reported. Furthermore, phage-derived endolysin therapy, such as TSPphg phage exogenous treatment, can aid in disrupting cell walls, leading to bacterial cell lysis. As animals in aquaculture and slaughterhouses are highly susceptible to bacterial infections, effective phage therapy instead of antibiotics can help treat poultry animals, preserve them, and facilitate disease-free trade. Using bioengineered phages and phage cocktails enhances the effectiveness by providing a broad spectrum of phages and target specificity. Research is currently being conducted on clinical trials to confirm the efficacy of engineered phages and phage cocktails in humans. Although obtaining commercial approval may be time-consuming, it will be beneficial in the postantibiotic era. This review provides an overview of the significance of phage therapy as a potential alternative to antibiotics in combating resistant bacterial strains and its application to various fields and emphasizes the importance of safeguarding and ensuring treatment efficacy.

[Experience with the use of bacteriophages in patients with neurogenic lower urinary tract dysfunction, complicated by recurrent lower urinary tract infection].

Clinical cases of three patients with neurogenic lower urinary tract dysfunction, complicated by chronic urinary tract infection are presented in the article. All patients underwent clean intermittent catheterization and, in order to prevent symptomatic lower urinary tract infections, received bacteriophage therapy with a clinically proven positive effect. During 3 months follow-up, there were no episodes of urinary tract infection. A change in the concentration of uropathogens and restoration of sensitivity to a number of antimicrobial drugs were observed. Although phage therapy in urology requires further clinical research, it provides an additional strategy to treat urinary tract infections considering an increase in antibiotic resistance.

Bacteriophage EPP-1, a potential antibiotic alternative for controlling edwardsiellosis caused by Edwardsiella piscicida while mitigating drug-resistant gene dissemination.

Edwardsiella piscicida causes significant economic losses to the aquaculture industry worldwide. Phage-based biocontrol methods are experiencing a renaissance because of the spread of drug-resistant genes and bacteria resulting from the heavy use of antibiotics. Here, we showed that the novel Edwardsiella phage EPP-1 could achieve comparable efficacy to florfenicol using a zebrafish model of Edwardsiella piscicida infection and could reduce the content of the floR resistance gene in zebrafish excreta. Specifically, phage EPP-1 inhibited bacterial growth in vitro and significantly improved the zebrafish survival rate in vivo (P = 0.0035), achieving an efficacy comparable to that of florfenicol (P = 0.2304). Notably, integrating the results of 16S rRNA sequencing, metagenomic sequencing, and qPCR, although the effects of phage EPP-1 converged with those of florfenicol in terms of the community composition and potential function of the zebrafish gut microbiota, it reduced the floR gene content in zebrafish excreta and aquaculture water. Overall, our study highlights the feasibility and safety of phage therapy for edwardsiellosis control, which has profound implications for the development of antibiotic alternatives to address the antibiotic crisis.

Lytic bacteriophages induce the secretion of antiviral and proinflammatory cytokines from human respiratory epithelial cells.

Phage therapy is a therapeutic approach to treat multidrug-resistant (MDR) infections that employs lytic bacteriophages (phages) to eliminate bacteria. Despite the abundant evidence for its success as an antimicrobial in Eastern Europe, there is scarce data regarding its effects on the human host. Here, we aimed to understand how lytic phages interact with cells of the airway epithelium, the tissue site that is colonized by bacterial biofilms in numerous chronic respiratory disorders. Using a panel of Pseudomonas aeruginosa phages and human airway epithelial cells (AECs) derived from a person with cystic fibrosis (CF), we determined that interactions between phages and epithelial cells depend on specific phage properties as well as physiochemical features of the microenvironment. Although poor at internalizing phages, the airway epithelium responds to phage exposure by changing its transcriptional profile and secreting antiviral and proinflammatory cytokines that correlate with specific phage families. Overall, our findings indicate that mammalian responses to phages are heterogenous and could potentially alter the way that respiratory local defenses aid in bacterial clearance during phage therapy. Thus, besides phage receptor specificity in a particular bacterial isolate, the criteria to select lytic phages for therapy should be expanded to include mammalian cell responses.

The Medicinal Phage-Regulatory Roadmap for Phage Therapy under EU Pharmaceutical Legislation.

Bacteriophage therapy is a promising approach to treating bacterial infections. Research and development of bacteriophage therapy is intensifying due to the increase in antibiotic resistance and the faltering development of new antibiotics. Bacteriophage therapy uses bacteriophages (phages), i.e., prokaryotic viruses, to specifically target and kill pathogenic bacteria. The legal handling of this type of therapy raises several questions. These include whether phage therapeutics belong to a specially regulated class of medicinal products, and which legal framework should be followed with regard to the various technical ways in which phage therapeutics can be manufactured and administered. The article shows to which class of medicinal products phage therapeutics from wild type phages and from genetically modified (designer) phages do or do not belong. Furthermore, the article explains which legal framework is relevant for the manufacture and administration of phage therapeutics, which are manufactured in advance in a uniform, patient-independent manner, and for tailor-made patient-specific phage therapeutics. For the systematically coherent, successful translation of phage therapy, the article considers pharmaceutical law and related legal areas, such as genetic engineering law. Finally, the article shows how the planned legislative revisions of Directive 2001/83/EC and Regulation (EC) No 726/2004 may affect the legal future of phage therapy.

Bacteriophage DNA induces an interrupted immune response during phage therapy in a chicken model.

One of the hopes for overcoming the antibiotic resistance crisis is the use of bacteriophages to combat bacterial infections, the so-called phage therapy. This therapeutic approach is generally believed to be safe for humans and animals as phages should infect only prokaryotic cells. Nevertheless, recent studies suggested that bacteriophages might be recognized by eukaryotic cells, inducing specific cellular responses. Here we show that in chickens infected with Salmonella enterica and treated with a phage cocktail, bacteriophages are initially recognized by animal cells as viruses, however, the cGAS-STING pathway (one of two major pathways of the innate antiviral response) is blocked at the stage of the IRF3 transcription factor phosphorylation. This inhibition is due to the inability of RNA polymerase III to recognize phage DNA and to produce dsRNA molecules which are necessary to stimulate a large protein complex indispensable for IRF3 phosphorylation, indicating the mechanism of the antiviral response impairment.

Phage-layer interferometry: a companion diagnostic for phage therapy and a bacterial testing platform.

The continuing and rapid emergence of antibiotic resistance (AMR) calls for innovations in antimicrobial therapies. A promising, 're-emerging' approach is the application of bacteriophage viruses to selectively infect and kill pathogenic bacteria, referred to as phage therapy. In practice, phage therapy is personalized and requires companion diagnostics to identify efficacious phages, which are then formulated into a therapeutic cocktail. The predominant means for phage screening involves optical-based assays, but these methods cannot be carried out in complex media, such as colored solutions, inhomogeneous mixtures, or high-viscosity samples, which are often conditions encountered in vivo. Moreover, these assays cannot distinguish phage binding and lysis parameters, which are important for standardizing phage cocktail formulation. To address these challenges, we developed Phage-layer Interferometry (PLI) as a companion diagnostic. Herein, PLI is assessed as a quantitative phage screening method and prototyped as a bacterial detection platform. Importantly, PLI is amenable to automation and is functional in complex, opaque media, such as baby formula. Due to these newfound capabilities, we foresee immediate and broad impact of PLI for combating AMR and protecting against foodborne illnesses.

Pseudomonas aeruginosa ventricular assist device infections: findings from ineffective phage therapies in five cases.

Left ventricular assist devices (LVAD) are increasingly used for management of heart failure; infection remains a frequent complication. Phage therapy has been successful in a variety of antibiotic refractory infections and is of interest in treating LVAD infections. We performed a retrospective review of four patients that underwent five separate courses of intravenous (IV) phage therapy with concomitant antibiotic for treatment of endovascular Pseudomonas aeruginosa LVAD infection. We assessed phage susceptibility, bacterial strain sequencing, serum neutralization, biofilm activity, and shelf-life of phage preparations. Five treatments of one to four wild-type virulent phage(s) were administered for 14-51 days after informed consent and regulatory approval. There was no successful outcome. Breakthrough bacteremia occurred in four of five treatments. Two patients died from the underlying infection. We noted a variable decline in phage susceptibility following three of five treatments, four of four tested developed serum neutralization, and prophage presence was confirmed in isolates of two tested patients. Two phage preparations showed an initial titer drop. Phage biofilm activity was confirmed in two. Phage susceptibility alone was not predictive of clinical efficacy in P. aeruginosa endovascular LVAD infection. IV phage was associated with serum neutralization in most cases though lack of clinical effect may be multifactorial including presence of multiple bacterial isolates with varying phage susceptibility, presence of prophages, decline in phage titers, and possible lack of biofilm activity. Breakthrough bacteremia occurred frequently (while the organism remained susceptible to administered phage) and is an important safety consideration.

Bacteriophage therapy for the treatment of Mycobacterium tuberculosis infections in humanized mice.

The continuing emergence of new strains of antibiotic-resistant bacteria has renewed interest in phage therapy; however, there has been limited progress in applying phage therapy to multi-drug resistant Mycobacterium tuberculosis (Mtb) infections. In this study, we show that bacteriophage strains D29 and DS6A can efficiently lyse Mtb H37Rv in 7H10 agar plates. However, only phage DS6A efficiently kills H37Rv in liquid culture and in Mtb-infected human primary macrophages. We further show in subsequent experiments that, after the humanized mice were infected with aerosolized H37Rv, then treated with DS6A intravenously, the DS6A treated mice showed increased body weight and improved pulmonary function relative to control mice. Furthermore, DS6A reduces Mtb load in mouse organs with greater efficacy in the spleen. These results demonstrate the feasibility of developing phage therapy as an effective therapeutic against Mtb infection.

Characterization and genome-informatic analysis of a novel lytic mendocina phage vB_PmeS_STP12 suitable for phage therapy pseudomonas or biocontrol.

BACKGROUND: A novel lytic bacteriophage (phage) was isolated with Pseudomonas mendocina strain STP12 (P. mendocina) from the untreated site of Sewage Treatment Plant of Lovely Professional University, India. P. mendocina is a Gram-negative, rod-shaped, aerobic bacterium belonging to the family Pseudomonadaceae and has been reported in fifteen (15) cases of economically important diseases worldwide. METHODS AND RESULTS: Here, a novel phage specifically infecting and killing P. mendocina strain STP12 was isolated from sewage sample using enrichment, spot test and double agar overlay (DAOL) method and was designated as vB_PmeS_STP12. The phage vB-PmeS-STP12 was viable at wide range of pH and temperature ranging from 4 to10 and - 20 to 70 °C respectively. Host range and efficiency of plating (EOP) analysis indicated that phage vB-PmeS-STP12 was capable of infecting and killing P. mendocina strain STP6 with EOP of 0.34. Phage vB_PmeS_STP12 was found to have a significant bacterial reduction (p < 0.005) at all the doses administered, particularly at optimal MOI of 1 PFU/CFU, compared to the control. Morphological analysis using high resolution transmission electron microscopy (HR-TEM) revealed an icosahedral capsid of ~ 55 nm in diameter on average with a short, non-contractile tail. The genome of vB_PmeS_STP12 is a linear, dsDNA containing 36,212 bp in size with a GC content of 58.87% harbouring 46 open reading frames (ORFs). The 46 predicted ORFs encode proteins with functional information categorized as lysis, replication, packaging, regulation, assembly, infection, immune, and hypothetical. However, the genome of vB_PmeS_STP12 appeared to be devoid of tRNAs, integrase gene, toxins genes, virulence factors, antimicrobial resistance genes (ARGs) and CRISPR arrays. The blast analysis with phylogeny revealed that vB_PmeS_STP12 is genetically similar to Pseudomonas phage PMBT14, Pseudomonas phage Almagne and Serratia phage Serbin with a highest identity of 74.00%, 74.93% and 59.48% respectively. CONCLUSIONS: Taken together, characterization, morphological analysis and genome-informatics indicated that vB_PmeS_STP12 is podovirus morphotype belonging to the class Caudoviticetes, family Zobellviridae which appeared to be devoid of integrase gene, ARGs, CRISPR arrays, virulence factors and toxins genes, exhibiting stability and infectivity at wide range of pH (4 to10) and temperature (-20 to 70 °C), thereby making vB_PmeS_STP12 suitable for phage therapy or biocontrol. Based on the bibliometric analysis and data availability with respect to sequences deposited in GenBank, this is the first report of a phage infecting Pseudomonas mendocina.

Predictive phage therapy for Escherichia coli urinary tract infections: Cocktail selection for therapy based on machine learning models.

This study supports the development of predictive bacteriophage (phage) therapy: the concept of phage cocktail selection to treat a bacterial infection based on machine learning (ML) models. For this purpose, ML models were trained on thousands of measured interactions between a panel of phage and sequenced bacterial isolates. The concept was applied to Escherichia coli associated with urinary tract infections. This is an important common infection in humans and companion animals from which multidrug-resistant (MDR) bloodstream infections can originate. The global threat of MDR infection has reinvigorated international efforts into alternatives to antibiotics including phage therapy. E. coli exhibit extensive genome-level variation due to horizontal gene transfer via phage and plasmids. Associated with this, phage selection for E. coli is difficult as individual isolates can exhibit considerable variation in phage susceptibility due to differences in factors important to phage infection including phage receptor profiles and resistance mechanisms. The activity of 31 phage was measured on 314 isolates with growth curves in artificial urine. Random Forest models were built for each phage from bacterial genome features, and the more generalist phage, acting on over 20% of the bacterial population, exhibited F1 scores of >0.6 and could be used to predict phage cocktails effective against previously untested strains. The study demonstrates the potential of predictive ML models which integrate bacterial genomics with phage activity datasets allowing their use on data derived from direct sequencing of clinical samples to inform rapid and effective phage therapy.

PqsA mutation-mediated enhancement of phage-mediated combat against Pseudomonas aeruginosa.

Phage therapy is a potential approach in the biocontrol of foodborne pathogens. However, the emergence of phage resistance and the narrow host range of most phage isolates continue to limit the antimicrobial efficacy of phages. Here, we investigated the potential of the pqsA gene, encoding the anthranilate-CoA ligase enzyme, as an adjuvant for phage therapy. The knockout of the pqsA gene significantly enhanced the bactericidal effect of phages vB_Pae_QDWS and vB_Pae_S1 against Pseudomonas aeruginosa. Under phage infection pressure, the growth of the PaΔpqsA was significantly inhibited within 8 h compared to the wild-type PAO1. Furthermore, we found that altering phage adsorption is not how PaΔpqsA responds to phage infection. Although pqsA represents a promising target for enhancing phage killing, it may not be applicable to all phages, such as types vB_Pae_W3 and vB_Pae_TR. Our findings provide new material reserves for the future design of novel phage-based therapeutic strategies.

Advances in phage-host interaction prediction: in silico method enhances the development of phage therapies.

Phages can specifically recognize and kill bacteria, which lead to important application value of bacteriophage in bacterial identification and typing, livestock aquaculture and treatment of human bacterial infection. Considering the variety of human-infected bacteria and the continuous discovery of numerous pathogenic bacteria, screening suitable therapeutic phages that are capable of infecting pathogens from massive phage databases has been a principal step in phage therapy design. Experimental methods to identify phage-host interaction (PHI) are time-consuming and expensive; high-throughput computational method to predict PHI is therefore a potential substitute. Here, we systemically review bioinformatic methods for predicting PHI, introduce reference databases and in silico models applied in these methods and highlight the strengths and challenges of current tools. Finally, we discuss the application scope and future research direction of computational prediction methods, which contribute to the performance improvement of prediction models and the development of personalized phage therapy.

Bacteriophages in nature: recent advances in research tools and diverse environmental and biotechnological applications.

Bacteriophages infect and replicate within bacteria and play a key role in the environment, particularly in microbial ecosystems and bacterial population dynamics. The increasing recognition of their significance stems from their wide array of environmental and biotechnological uses, which encompass the mounting issue of antimicrobial resistance (AMR). Beyond their therapeutic potential in combating antibiotic-resistant infections, bacteriophages also find vast applications such as water quality monitoring, bioremediation, and nutrient cycling within environmental sciences. Researchers are actively involved in isolating and characterizing bacteriophages from different natural sources to explore their applications. Gaining insights into key aspects such as the life cycle of bacteriophages, their host range, immune interactions, and physical stability is vital to enhance their application potential. The establishment of diverse phage libraries has become indispensable to facilitate their wide-ranging uses. Consequently, numerous protocols, ranging from traditional to cutting-edge techniques, have been developed for the isolation, detection, purification, and characterization of bacteriophages from diverse environmental sources. This review offers an exploration of tools, delves into the methods of isolation, characterization, and the extensive environmental applications of bacteriophages, particularly in areas like water quality assessment, the food sector, therapeutic interventions, and the phage therapy in various infections and diseases.

Evaluation of the therapeutic effect of a novel bacteriophage in the healing process of infected wounds with Klebsiella pneumoniae in mice.

OBJECTIVE: Bacterial wound infections have recently become a threat to public health. The emergence of multidrug-resistant (MDR) strains of Klebsiella pneumoniae highlights the need for a new treatment method. The effectiveness of bacteriophages has been observed for several infections in animal models and human trials. In this study, we assessed the effectiveness of bacteriophages in the treatment of wound infections associated with MDR and biofilm-producing K. pneumoniae and compared its effectiveness with that of gentamicin. METHODS: A lytic phage against MDR K. pneumoniae was isolated and identified. The effectiveness of phages in the treatment of wound infection in mice was investigated and its effectiveness was compared with gentamicin. RESULTS: The results showed that the isolated phage belonged to the Drexlerviridae family. This phage acts like gentamicin and effectively eliminates bacteria from wounds. In addition, mice in the phage therapy group were in better physical condition. CONCLUSION: Our results demonstrated the success of phage therapy in the treatment of mice wounds infected with K. pneumoniae. These results indicate the feasibility of topical phage therapy for the safe treatment of wound infections.

Collateral sensitivity increases the efficacy of a rationally designed bacteriophage combination to control Salmonella enterica.

The ability of virulent bacteriophages to lyse bacteria influences bacterial evolution, fitness, and population structure. Knowledge of both host susceptibility and resistance factors is crucial for the successful application of bacteriophages as biological control agents in clinical therapy, food processing, and agriculture. In this study, we isolated 12 bacteriophages termed SPLA phage which infect the foodborne pathogen Salmonella enterica. To determine phage host range, a diverse collection of Enterobacteriaceae and Salmonella enterica was used and genes involved in infection by six SPLA phages were identified using Salmonella Typhimurium strain ST4/74. Candidate host receptors included lipopolysaccharide (LPS), cellulose, and BtuB. Lipopolysaccharide was identified as a susceptibility factor for phage SPLA1a and mutations in LPS biosynthesis genes spontaneously emerged during culture with S. Typhimurium. Conversely, LPS was a resistance factor for phage SPLA5b which suggested that emergence of LPS mutations in culture with SPLA1a represented collateral sensitivity to SPLA5b. We show that bacteria-phage co-culture with SPLA1a and SPLA5b was more successful in limiting the emergence of phage resistance compared to single phage co-culture. Identification of host susceptibility and resistance genes and understanding infection dynamics are critical steps in the rationale design of phage cocktails against specific bacterial pathogens.IMPORTANCEAs antibiotic resistance continues to emerge in bacterial pathogens, bacterial viruses (phage) represent a potential alternative or adjunct to antibiotics. One challenge for their implementation is the predisposition of bacteria to rapidly acquire resistance to phages. We describe a functional genomics approach to identify mechanisms of susceptibility and resistance for newly isolated phages that infect and lyse Salmonella enterica and use this information to identify phage combinations that exploit collateral sensitivity, thus increasing efficacy. Collateral sensitivity is a phenomenon where resistance to one class of antibiotics increases sensitivity to a second class of antibiotics. We report a functional genomics approach to rationally design a phage combination with a collateral sensitivity dynamic which resulted in increased efficacy. Considering such evolutionary trade-offs has the potential to manipulate the outcome of phage therapy in favor of resolving infection without selecting for escape mutants and is applicable to other virus-host interactions.

Phage therapy: A renewed approach against oral diseases caused by Enterococcus faecalis infections.

Antibiotics play an important role in the treatment of infectious diseases. Long-term overuse or misuse of antibiotics, however, has triggered the global crisis of antibiotic resistance, bringing challenges to treating clinical infection. Bacteriophages (phages) are the viruses infecting bacterial cells. Due to high host specificity, high bactericidal activity, and good biosafety, phages have been used as natural alternative antibacterial agents to fight against multiple drug-resistant bacteria. Enterococcus faecalis is the main species detected in secondary persistent infection caused by failure of root canal therapy. Due to strong tolerance and the formation of biofilm, E. faecalis can survive the changes in pH, temperature, and osmotic pressure in the mouth and thus is one of the main causes of periapical lesions. This paper summarizes the advantages of phage therapy, its applications in treating oral diseases caused by E. faecalis infections, and the challenges it faces. It offers a new perspective on phage therapy in oral diseases.

Phage susceptibility determinants of the opportunistic pathogen Staphylococcus epidermidis.

Staphylococcus epidermidis is a common member of the human skin and nose microbiomes and a frequent cause of invasive infections. Transducing phages accomplish the horizontal transfer of resistance and virulence genes by mispackaging of mobile-genetic elements, contributing to severe, therapy-refractory S. epidermidis infections. Lytic phages on the other hand can be interesting candidates for new anti-S. epidermidis phage therapies. Despite the importance of phages, we are only beginning to unravel S. epidermidis phage interactions. Recent studies shed new light on S. epidermidis phage diversity, host range, and receptor specificities. Modulation of cell wall teichoic acids, the major phage receptor structures, along with other phage defense mechanisms, are crucial determinants for S. epidermidis susceptibility to different phage groups.