The impact of antibiotic resistance on the rampant spread of infectious diseases in Pakistan: Insights from a narrative review.

Background and Aims: Antibiotic resistance (ABR) is a global public health emergency which has seen an uptick in low- to middle-income countries in recent times due to a plethora of aggravating factors and has led to a whole host of setting-specific pathogens registering high rates of resistance, causing outbreaks with graver mortality and morbidity. This review analyzes available literature to determine the causes and effects of ABR and recommend solutions to the problem in a Pakistani setting. Methods: Sources for this narrative review were identified via electronic databases using keyword search methods. The information was retrieved using databases such as PubMed and Science Direct. Additionally, websites such as CDC and World Health Organization were used to attain pertinent information. All the sources were selected as per their relevance and appropriateness toward the purpose of this review. Results: This review details the causes by dividing them into three primary strata, namely (1) under-regulation, (2) over-prescription and self-medication, and (3) lack of medical stewardship. This is made much graver when the COVID-19 pandemic and the subsequent erratic treatment response is considered, with the pandemic augmenting already high levels of consumption. These factors have led a cascade of effects including, but not limited to, a considerable increase in ABR in pathogens to first-line drugs. Conclusion: ABR is a serious and growing issue which will result in undesirable personal, local, and national consequences if unchecked. Mitigation and reversal of this trend is necessary by developing existing programs and investing in novel therapies and pharmaceutical research and strengthening regulatory policies and mechanisms.

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.

Large-scale genomic survey with deep learning-based method reveals strain-level phage specificity determinants.

BACKGROUND: Phage therapy, reemerging as a promising approach to counter antimicrobial-resistant infections, relies on a comprehensive understanding of the specificity of individual phages. Yet the significant diversity within phage populations presents a considerable challenge. Currently, there is a notable lack of tools designed for large-scale characterization of phage receptor-binding proteins, which are crucial in determining the phage host range. RESULTS: In this study, we present SpikeHunter, a deep learning method based on the ESM-2 protein language model. With SpikeHunter, we identified 231,965 diverse phage-encoded tailspike proteins, a crucial determinant of phage specificity that targets bacterial polysaccharide receptors, across 787,566 bacterial genomes from 5 virulent, antibiotic-resistant pathogens. Notably, 86.60% (143,200) of these proteins exhibited strong associations with specific bacterial polysaccharides. We discovered that phages with identical tailspike proteins can infect different bacterial species with similar polysaccharide receptors, underscoring the pivotal role of tailspike proteins in determining host range. The specificity is mainly attributed to the protein's C-terminal domain, which strictly correlates with host specificity during domain swapping in tailspike proteins. Importantly, our dataset-driven predictions of phage-host specificity closely match the phage-host pairs observed in real-world phage therapy cases we studied. CONCLUSIONS: Our research provides a rich resource, including both the method and a database derived from a large-scale genomics survey. This substantially enhances understanding of phage specificity determinants at the strain level and offers a valuable framework for guiding phage selection in therapeutic applications.

In vitro and in vivo evaluation of the biofilm-degrading Pseudomonas phage Motto, as a candidate for phage therapy.

Infections caused by Pseudomonas aeruginosa are becoming increasingly difficult to treat due to the emergence of strains that have acquired multidrug resistance. Therefore, phage therapy has gained attention as an alternative to the treatment of pseudomonal infections. Phages are not only bactericidal but occasionally show activity against biofilm as well. In this study, we describe the Pseudomonas phage Motto, a T1-like phage that can clear P. aeruginosa infections in an animal model and also exhibits biofilm-degrading properties. The phage has a substantial anti-biofilm activity against strong biofilm-producing isolates (n = 10), with at least a twofold reduction within 24 h. To demonstrate the safety of using phage Motto, cytotoxicity studies were conducted with human cell lines (HEK 293 and RAW 264.7 macrophages). Using a previously established in vivo model, we demonstrated the efficacy of Motto in Caenorhabditis elegans, with a 90% survival rate when treated with the phage at a multiplicity of infection of 10.

Oral feed-based administration of phage cocktail protects rohu fish (Labeo rohita) against Aeromonas hydrophila infection.

Aeromonas hydrophila is one of the major freshwater fish pathogens. In the current study, a cocktail of D6 and CF7 phages was given orally to Labeo rohita to assess phage survival in fish organs as well as to determine the therapeutic efficacy of phage treatment against fish mortality caused by A. hydrophila. In the phage-coated feed, prepared by simple spraying method, phage counts were quite stable for up to 2 months with a decline of ≤ 0.23 log10 and ≤ 1.66 log10 PFU/g feed during 4 oC and room temperature storage. Throughout the experimental period of 7 days, both phages could be detected in the gut of fish fed with phage-coated feed. Besides, both CF7 and D6 phages were also detected in fish kidneys indicating the ability of both the phage to cross the intestinal barrier. During challenge studies with LD50 dose of A. hydrophila, phage cocktail doses of 1 × 106 - 1 × 108 PFU/g feed prevented the mortality in L. rohita with relative percentage survival (RPS) of 8.7-65.2. When challenged with LD90 dose of A. hydrophila, an RPS value of 28.6 was obtained at a phage cocktail dose of 1 × 108 PFU/g feed. The RPS data showed that orally-fed phage cocktail protected the fish against the mortality caused by A. hydrophila in a dose-dependent manner. Simple practical approaches for phage cocktail development, medicated feed preparation and oral administration along with phage survival and protection data make the current study useful for farmer-level application.

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.

Controlling Recombination to Evolve Bacteriophages.

Recombination among different phages sometimes facilitates their ability to grow on new hosts. Protocols to direct the evolution of phage host range, as might be used in the application of phage therapy, would then benefit from including steps to enable recombination. Applying mathematical and computational models, in addition to experiments using phages T3 and T7, we consider ways that a protocol may influence recombination levels. We first address coinfection, which is the first step to enabling recombination. The multiplicity of infection (MOI, the ratio of phage to cell concentration) is insufficient for predicting (co)infection levels. The force of infection (the rate at which cells are infected) is also critical but is more challenging to measure. Using both a high force of infection and high MOI (>1) for the different phages ensures high levels of coinfection. We also apply a four-genetic-locus model to study protocol effects on recombinant levels. Recombinants accumulate over multiple generations of phage growth, less so if one phage outgrows the other. Supplementing the phage pool with the low-fitness phage recovers some of this 'lost' recombination. Overall, fine tuning of phage recombination rates will not be practical with wild phages, but qualitative enhancement can be attained with some basic procedures.

Phage-antibiotic combinations to control Pseudomonas aeruginosa-Candida two-species biofilms.

Phage-antibiotic combinations to treat bacterial infections are gaining increased attention due to the synergistic effects often observed when applying both components together. Most studies however focus on a single pathogen, although in many clinical cases multiple species are present at the site of infection. The aim of this study was to investigate the anti-biofilm activity of phage-antibiotic/antifungal combinations on single- and dual-species biofilms formed by P. aeruginosa and the fungal pathogen Candida albicans. The Pseudomonas phage Motto in combination with ciprofloxacin had significant anti-biofilm activity. We then compared biofilms formed by P. aeruginosa alone with the dual-species biofilms formed by bacteria and C. albicans. Here, we found that the phage together with the antifungal fluconazole was active against 6-h-old dual-species biofilms but showed only negligible activity against 24-h-old biofilms. This study lays the first foundation for potential therapeutic approaches to treat co-infections caused by bacteria and fungi using phage-antibiotic combinations.

Opportunities and challenges in phage therapy for cardiometabolic diseases.

The worldwide prevalence of cardiometabolic diseases (CMD) is increasing, and emerging evidence implicates the gut microbiota in this multifactorial disease development. Bacteriophages (phages) are viruses that selectively target a bacterial host; thus, phage therapy offers a precise means of modulating the gut microbiota, limiting collateral damage on the ecosystem. Several studies demonstrate the potential of phages in human disease, including alcoholic and steatotic liver disease. In this opinion article we discuss the potential of phage therapy as a predefined medicinal product for CMD and discuss its current challenges, including the generation of effective phage combinations, product formulation, and strict manufacturing requirements.

Critically evaluating the relative importance of phage in shaping microbial community composition.

The ubiquity of bacteriophages (phages) and the major evolutionary and ecological impacts they can have on their microbial hosts has resulted in phages often cited as key drivers shaping microbial community composition (the relative abundances of species). However, the evidence for the importance of phages is mixed. Here, we critically review the theory and data exploring the role of phages in communities, identifying the conditions when phages are likely to be important drivers of community composition. At ecological scales, we conclude that phages are often followers rather than drivers of microbial population and community dynamics. While phages can affect strain diversity within species, there is yet to be strong evidence suggesting that fluctuations in species' strains affects community composition.

Defense and anti-defense mechanisms of bacteria and bacteriophages. / 细菌和噬菌体间的防御与反防御机制.

In the post-antibiotic era, the overuse of antimicrobials has led to a massive increase in antimicrobial resistance, leaving medical doctors few or no treatment options to fight infections caused by superbugs. The use of bacteriophages is a promising alternative to treat infections, supplementing or possibly even replacing antibiotics. Using phages for therapy is possible, since these bacterial viruses can kill bacteria specifically, causing no harm to the normal flora. However, bacteria have developed a multitude of sophisticated and complex ways to resist infection by phages, including abortive infection and the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. Phages also can evolve and acquire new anti-defense strategies to continue predation. An in-depth exploration of both defense and anti-defense mechanisms would contribute to optimizing phage therapy, while we would also gain novel insights into the microbial world. In this paper, we summarize recent research on bacterial phage resistance and phage anti-defense mechanisms, as well as collaborative win-win systems involving both virus and host.

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.

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.

Phage Delivery Strategies for Biocontrolling Human, Animal, and Plant Bacterial Infections: State of the Art.

This review aims at presenting the main strategies that are currently available for the delivery of bacteriophages to combat bacterial infections in humans, animals, and plants. It can be seen that the main routes for phage delivery are topical, oral, systemic, and airways for humans. In animals, the topical and oral routes are the most used. To combat infections in plant species, spraying the plant's phyllosphere or drenching the soil are the most commonly used methods. In both phage therapy and biocontrol using phages, very promising results have been obtained so far. However, more experiments are needed to establish forms of treatment and phage doses, among other parameters. Furthermore, in general, there is a lack of specific standards for the use of phages to combat bacterial infections.

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.

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.

Genomic analysis of seven mycobacteriophages identifies three novel species with differing phenotypic stabilities.

Recently, case studies have been published regarding the application of mycobacteriophage (MP) therapy (MPT) in patients with multi-antibiotic-resistant infections. A major limitation in the development of MPT is the paucity of therapeutically useful MP. As there are approximately 10,000 MP that have yet to be sequenced, it is possible that characterization of this cohort would increase the repertoire of useful MP. This study aims to contribute to such a strategy, by characterizing a cohort of 7 mycobacteriophages. Sequencing analyses revealed that the MP have unique sequences, and subsequent gene annotation revealed differences in gene organization. Notably, MP LOCARD has the largest genome and operons encoding for glycosyltransferases. Taxonomic analysis executed with VIRIDIC, Gegenees and VICTOR revealed that LOCARD belongs to a different genus than the other phages and is the foundational member of one of three novel species identified in this study. LOCARD, LOCV2, and LOCV5 were selected as representative members of their species and subjected to phenotypic analyses to compare their stability under biologically and industrially relevant conditions. Again LOCARD stood out, as it was unaffected by the typical temperatures (37 °C) and salinity (0.9%) experienced in mammals, while the viability of LOCV2 and LOCV5 was significantly reduced. LOCARD was also tolerant to pH 10, low levels of antiviral detergent and was the least impacted by a single freeze-thaw cycle. When all these results are considered, it indicates that LOCARD in particular, has potential therapeutic and/or diagnostics applications, given its resilience towards physiological and storage conditions.

Preclinical characterization and in silico safety assessment of three virulent bacteriophages targeting carbapenem-resistant uropathogenic Escherichia coli.

Phage therapy has recently been revitalized in the West with many successful applications against multi-drug-resistant bacterial infections. However, the lack of geographically diverse bacteriophage (phage) genomes has constrained our understanding of phage diversity and its genetics underpinning host specificity, lytic capability, and phage-bacteria co-evolution. This study aims to locally isolate virulent phages against uropathogenic Escherichia coli (E. coli) and study its phenotypic and genomic features. Three obligately virulent Escherichia phages (øEc_Makalu_001, øEc_Makalu_002, and øEc_Makalu_003) that could infect uropathogenic E. coli were isolated and characterized. All three phages belonged to Krischvirus genus. One-step growth curve showed that the latent period of the phages ranged from 15 to 20 min, the outbreak period ~ 50 min, and the burst size ranged between 74 and 127 PFU/bacterium. Moreover, the phages could tolerate a pH range of 6 to 9 and a temperature range of 25-37 °C for up to 180 min without significant loss of phage viability. All phages showed a broad host spectrum and could lyse up to 30% of the 35 tested E. coli isolates. Genomes of all phages were approximately ~ 163 kb with a gene density of 1.73 gene/kbp and an average gene length of ~ 951 bp. The coding density in all phages was approximately 95%. Putative lysin, holin, endolysin, and spanin genes were found in the genomes of all three phages. All phages were strictly virulent with functional lysis modules and lacked any known virulence or toxin genes and antimicrobial resistance genes. Pre-clinical experimental and genomic analysis suggest these phages may be suitable candidates for therapeutic applications.