Isolation Of Bacteriophages Research Articles

Synergistic effects of zP-1 phage and ampicillin against methicillin-resistant Staphylococcus aureus isolated from hospital staff.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a major cause of hospital-acquiredinfections, exacerbated by increasing antibiotic resistance. This study aimed to isolate bacteriophages targeting MRSA and evaluate their antimicrobial activity incombination with antibiotics. Nasal samples from hospital staff (n = 50) were used to isolate MRSA strains, and sewage samples wereprocessed for phage isolation using the double agar overlay technique. The microtitration plate method evaluatedthe synergistic effects of isolated phages and antibiotics. Out of 50 samples, 11 MRSA strains were positive, showing high resistance to multiple antibiotics, includingoxacillin (95%), and vancomycin (85%). Phage zP-1, belonging to the Myoviridae family, exhibited > 90% lyticactivity, stable across temperatures (20-50 °C) and pH (6-8). A statistically significant synergistic effect wasobserved at a MIC of 0.137 μg/ml for gentamicin (p-value 0.02). These findings suggest that phage-antibiotic synergy may offer a promising strategy to combat MRSAinfections, warranting further in vivo studies to assess its clinical potential.

Isolation and characterization of a broad-spectrum bacteriophage against multi-drug resistant Escherichia coli from waterfowl field.

Escherichia coli (E. coli) is a significant pathogen responsible for intestinal infections and foodborne diseases. The rise of antibiotic resistance poses a significant challenge to global public health. Traditional antibiotic therapy is becoming increasingly ineffective, highlighting the urgent need for innovative control strategies. This study explores the potential of bacteriophages as a sustainable alternative to traditional antibiotics. From 2021 to 2022, a total of 183 non-repetitive duck source fecal samples were collected from Mianyang City, Sichuan Province, and 126 strains of E. coli were isolated. The minimum inhibitory concentration (MIC) test showed that these strains exhibited high resistance to piperacillin (96.8%), tetracycline (88.9%), and chloramphenicol (86.5%). It is concerning that 93.7% of the isolates are classified as multidrug-resistant (MDR), posing a significant threat to existing treatment options. 20 bacteriophages were isolated from fecal and soil samples, among which 5 bacteriophages were selected for further analysis. Bacteriophage YP6 showed excellent lytic effects on MDR strains, especially strain MY104, as well as representative serotypes O1 (E. coli MY51) and O18 (E. coli MY106). The identification of YP6 as a member of the Myoviridae family was conducted using transmission electron microscopy, and it was found to have an optimal infection factor of 0.1. Bacteriophages exhibit significant thermal and pH stability, maintaining survival at temperatures up to 60 °C and pH ranges of 4 to 10. Whole genome sequencing confirmed that YP6 has a double stranded DNA genome of 139,323 base pairs (bp), and no antibiotic resistance or virulence genes were found, indicating a low possibility of horizontal gene transfer. In addition, YP6 effectively inhibits the formation of E. coli biofilm, which is a key factor in chronic infections. The in vivo experiments using Galleria mellonella (G. mellonella) larvae have shown that it has a significant protective effect against MDR E. coli infection. In summary, bacteriophage YP6 is expected to become a therapeutic agent against MDR E. coli infection due to its broad host range, environmental stability, and biofilm inhibition properties. Future research should optimize bacteriophage preparations, evaluate the safety and efficacy of animal models, and establish clinical application plans in the field of food safety.

Isolation of Bacteriophage Against the Drug Resistant Clinical Pathogens and Host Range Analysis

Antibiotic crisis is a major challenge being encountered by the global population as a result of development of antibiotic-resistant bacteria. Thus, shifting the focus to explore alternative methods to treat bacterial infection. Bacteriophage, the natural predator of the bacteria is one of those alternatives. This study is aimed to isolate bacteriophages from different types of water samples which are capable of infecting drug-resistant clinical isolates and understand its host range capacity. The isolation of bacteriophage was done by Double Layer Agar Assay (DLAA) from 3 different water samples i.e sewage sample from Bagmati and ALKA hospital and from Lele river water. For the isolation of phage, host organisms used were Multi Drug Resistant (MDR) E. coli and K. oxytoca resistant to Amoxyclav, Cefotaxime, Ciproflaxcin, Erythromycin, and Nalidixic Acid. Phage against E. coli II was isolated from both sewage samples while K. oxytoca phage was isolated from Bagamti sewage only. Phage could not be isolated against E. coli I and K. oxytoca phage could not be sub cultured as no plaques were formed on sub culturing and it showed temperate properties. The host range analysis of E. coli II phage showed that the phage has narrow host range as it did not formed plaques against bacteria of other genera. This indicates that sewage is the best source for discovery of phage and the isolated bacteriophage are capable of lytic activity on MDR bacterial isolates. Even though the isolated bacteriophage has potential to be used in phage therapy, still further research should be done for its application. Int. J. Appl. Sci. Biotechnol. Vol 12(4): 201-206.

Temperate phage-antibiotic synergy is widespread-extending to Pseudomonas-but varies by phage, host strain, and antibiotic pairing.

The recent discovery that otherwise therapeutically unusable temperate phages can potentiate the activity of antibiotics, resulting in a potent synergy, has only been tested in E. coli, and with a single model phage. Here, working with clinical isolates of Pseudomonas and phages from these isolates, we highlight the broad applicability of this synergy-across a variety of mechanisms but also highlight the limitations of predicting the phage, host, and antibiotic combinations that will synergize.

Isolation and characterization of a novel bacteriophage as a biological control agent against multidrug resistant Escherichia coli in compost and agricultural irrigation water

Background Escherichia coli is a critical priority pathogen due to its significant morbidity, mortality, and growing antimicrobial resistance, underscoring the urgent need for novel control strategies. This bacterium is frequently implicated in outbreaks associated with horticultural products, particularly those cultivated in organic farming systems. The aim of this study was to isolate and evaluate the potential of a bacteriophage as a biocontrol agent against E. coli in compost and agricultural irrigation water. Methods E. coli presence in compost samples (n=17) was determined through microbiological assays, and the bacterial identity was confirmed by PCR amplification of the phoA gene. Antimicrobial resistance profiles of the isolates were assessed using the disk diffusion method. Bacteriophage isolation was conducted from livestock fecal samples using a double-layer agar technique. The stability of the bacteriophage under varying pH levels and temperatures was evaluated, along with its replication dynamics. Additionally, the phage’s efficacy in reducing E. coli populations in compost and irrigation water was assessed. Genomic sequencing and bioinformatic analyses of the bacteriophage were conducted to characterize its genetic profile. Results E. coli strains isolated exhibiting multidrug resistance were isolated from compost samples. The isolated bacteriophage, named Alux-21, exhibited stability at neutral pH and retained viability at both 4°C and 40°C over a six-month period. Importantly, the phage achieved a significant reduction of E. coli counts, exceeding 3.8 logs in compost and 3 logs in irrigation water, demonstrating its superior efficacy compared to previously reported phages in similar substrates. Genomic analysis confirmed the absence of virulence-associated, lysogeny, and antibiotic resistance genes. Conclusion The findings highlight Alux-21 as a sustainable biocontrol agent for E. coli in compost and irrigation water. Field validation will be crucial to establish its scalability and efficacy under real-world agricultural conditions.

Isolation and characterization of bacteriophages targeting methicillin-resistant Staphylococcus aureus (MRSA) from burn patients and sewage water: a genomic and proteomic study.

The spectrum of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) ranges from minor conditions to potentially life-threatening diseases. The rising antibiotic resistance in MRSA often leads to treatment failures, underscoring the urgent need for novel eradication strategies. This study focuses on isolating MRSA from burn patients, determining its antibiogram profile, and isolating and characterizing bacteriophages from sewage water that target MRSA, alongside conducting genomic analysis of the phages. A total of 70 samples were collected from burn patients, with MRSA identification and characterization performed using a combination of biochemical and molecular techniques, as well as antibiotic sensitivity testing. Based on host range analysis, a specific phage (phage-3) was selected for detailed characterization, including proteomic analysis, genetic mapping, phylogenetic studies, and analysis of open reading frames (ORFs) and motifs. The prevalence of MRSA in the samples was found to be 28.6%. Antibiotic susceptibility tests indicated that 94% of the MRSA isolates were sensitive to tobramycin and gentamicin, while vancomycin exhibited the lowest sensitivity, with only 2% effectiveness. Using the soft agar overlay method, three bacteriophages (phage-1, phage-2, and phage-3) were successfully isolated from sewage water. Among these, phage-3 exhibited the broadest host range. Further analysis showed that phage-3 demonstrated optimal activity at pH levels between 6 and 8, and within a temperature range of 20-40°C. Phage-3 also displayed a rapid adsorption phase within the first 0-5min, and its one-step growth curve revealed a latent period lasting up to 30min, followed by a significant increase in titer from 30 to 50min. Proteomic analysis of phage-3 identified the presence of 33kDa and 65kDa proteins. Phylogenetic analysis showed that phage-3 shares 96.6% similarity with Mammallicoccus phage vB_MscM-PMS3. The ORF analysis identified 80 potential ORFs within the phage's entire genome.

Isolation, partial characterization, therapeutic, and safety evaluation of carbapenem-resistant Acinetobacter baumannii lytic phage in a mouse model

BackgroundAntimicrobial resistance (AMR) is a major worldwide health concern, characterized by the ability of microorganisms to withstand the effects of medications that once effectively treated infections. Phage therapy has emerged as a promising alternative for management of multidrug-resistant (MDR) bacterial infections. Acinetobacter baumannii (A. baumannii) exemplifies the emergence of bacteria resistant to clinically relevant antimicrobials, leading to severe nosocomial infections and exhibiting extensive and pan drug-resistant (XDR and PDR) traits. In response, this study isolated A. baumannii virulent phage designated as vB_AbaP_PhE54 against carbapenem-resistant A. baumannii (CRAB) pathogen and examined its morphological characteristics using an electron micrograph. Phage stability at different temperatures, pH, chloroform, safety, therapeutic evaluation, and growth kinetics have been analyzed.ResultsThe A. baumannii phage vB_AbaP_PhE54 belongs morphologically to the Podoviridae family with very short, noncontractile tails, the phage demonstrated high thermal tolerance and infectivity across a pH range of 4–11, although it displayed a narrow host range. One-step growth kinetics indicated a burst size of 85 PFU (Plaque Forming Unit) per infected cell and a latent period of 20 min. Additionally, therapeutic efficiency in a mouse model showed total elimination of CRAB pathogen from lungs homogenates of mice and recovery from lung inflammation in all infected mice. On the other hand, safety evaluation of isolated phage revealed no adverse effects on structural or morphological tissue integrity.ConclusionsThese findings suggest that A. baumannii phage vB_AbaP_PhE54 could be a viable safe therapeutic option against A. baumannii infections, warranting further research into its clinical applications.

Isolation and Characterization of Lytic Phages Infecting Clinical Klebsiella pneumoniae from Tunisia.

Background: Klebsiella pneumoniae is an opportunistic pathogen that causes a wide range of infections worldwide. The emergence and spread of multidrug-resistant clones requires the implementation of novel therapeutics, and phages are a promising approach. Results: In this study, two Klebsiella phages, KpTDp1 and KpTDp2, were isolated from wastewater samples in Tunisia. These phages had a narrow host range and specifically targeted the hypervirulent K2 and K28 capsular types of K. pneumoniae. Both phages have double-stranded linear DNA genomes of 49,311 and 49,084 bp, respectively. Comparative genomic and phylogenetic analyses placed phage KpTDp2 in the genus Webervirus, while phage KpTDp1 showed some homology with members of the genus Jedunavirus, although its placement in a new undescribed genus may be reconsidered. The replication efficiency and lytic ability of these phages, combined with their high stability at temperatures up to 70 °C and pH values ranging from 3.5 to 8.2, highlight the potential of these phages as good candidates for the control of hypervirulent multidrug-resistant K. pneumoniae. Methods: Phage isolation, titration and multiplicity of infection were performed. The stability of KpTDp1 and KpTDp2 was tested at different pH and temperatures. Genomic characterization was done by genome sequencing, annotation and phylogenetic analysis. Conclusions: The ability of KpTDp1 and KpTDp2 to lyse one of the most virulent serotypes of K. pneumoniae, as well as the stability of their lytic activities to pH and temperature variations, make these phages promising candidates for antibacterial control.

Isolation, characterization, and potential application of Acinetobacter baumannii phages against extensively drug-resistant strains.

One of the significant issues in treating bacterial infections is the increasing prevalence of extensively drug-resistant (XDR) strains of Acinetobacter baumannii. In the face of limited or no viable treatment options for extensively drug-resistant (XDR) bacteria, there is a renewed interest in utilizing bacteriophages as a treatment option. Three Acinetobacter phages (vB_AbaS_Ftm, vB_AbaS_Eva, and vB_AbaS_Gln) were identified from hospital sewage and analyzed for their morphology, host ranges, and their genome sequences were determined and annotated. These phages and vB_AbaS_SA1 were combined to form a phage cocktail. The antibacterial effects of this cocktail and its combinations with selected antimicrobial agents were evaluated against the XDR A. baumannii strains. The phages exhibited siphovirus morphology. Out of a total of 30 XDR A. baumannii isolates, 33% were sensitive to vB_AbaS_Ftm, 30% to vB_AbaS_Gln, and 16.66% to vB_AbaS_Eva. When these phages were combined with antibiotics, they demonstrated a synergistic effect. The genome sizes of vB_AbaS_Ftm, vB_AbaS_Eva, and vB_AbaS_Gln were 48487, 50174, and 50043 base pairs (bp), respectively, and showed high similarity. Phage cocktail, when combined with antibiotics, showed synergistic effects on extensively drug-resistant (XDR) strains of A. baumannii. However, the need for further study to fully understand the mechanisms of action and potential limitations of using these phages is highlighted.

Debunking the dogma that RecBCD nuclease destroys phage.

For decades, it has been repeatedly claimed that the potent bacterial helicase-nuclease RecBCD (exonuclease V) destroys foreign (non-self) DNA, such as that of phages, but repairs and recombines cellular (self) DNA. While this would constitute a strong host-survival mechanism, no phage destroyed by RecBCD is ever specified in those claims. To determine which phages are destroyed by RecBCD, we searched for phage isolates that grow on Escherichia coli ΔrecBCD but not on recBCD+. In contrast to the prevailing claim, we found none among >80 new isolates from nature and >80 from previous collections. Based on these and previous observations, we conclude that RecBCD repairs broken DNA that can recombine but destroys DNA that cannot recombine and recycles the nucleotides.

Isolation, optimization, and redesigning of phages of methicillin-resistant Staphylococcus aureus from clinical hospital isolates in Baghdad

Background: A global health concern is methicillin-resistant Staphylococcus aureus (MRSA). The use of bacteriophages is one of the many novel control strategies against MRSA that are frequently sought. However, it is quite challenging to isolate enough lytic anti-MRSA phages. In order to extract, optimize, and remodel anti-MRSA phages, this study sought novel approaches. Methods: Two ATCC MRSA strains and nine clinical MRSA isolates were used to isolate wild anti-MRSA phages from hospital settings, dirt, and sewage. The wild phages were optimized using plaque-based biokinetic techniques. Using chemicals that weakened bacterial cell walls, the resulting highly lytic and specific anti-MRSA phages were subjected to unique physicochemical phage redesign processes. This allowed the phages to enter host bacteria and acquire the specificity of the new host. Three different protocols were tested using combinations of Tween 20, lysozyme, and nisin A. Results: Nisin A and lysozyme protocols at different rates were found to be successful in producing newly redesigned, transiently stable, anti-MRSA phages. Conclusion: Unlike self-depleting antibiotic-based applications, phage redesign is self-fortifying. In order to address the increasing number of epidemic MRSA strains, this model could prove to be a perfect platform for developing trustworthy control and treatment strategies. Additionally, it is believed to be an infinite supply of anti-MRSA lytic phages from which several permanent phage lysins can be isolated and refined.

Novel isolation and optimization of anti-MRSA bacteriophages using plaque-based biokinetic methods

Background: MRSA (methicillin-resistant Staphylococcus aureus) is a global health problem. Many people are looking for new ways to combat MRSA, for example by using bacteriophages (phages). It has been extremely challenging to isolate a sufficient quantity of lytic anti-MRSA phages. Therefore, new techniques for separating, refining, and reworking anti-MRSA phages were sought in this study. Methods: Of 437 S. aureus isolates, nine clinical MRSA isolates were obtained from three hospitals in Baghdad, Iraq and two ATCC MRSA strains were used to separate wild anti-MRSA phages from sewage, filth, and hospital settings. The wild phage was optimized using plaque-based biokinetic methods. The resulting highly lytic and specific anti-MRSA phages were subjected to novel physico-chemical phage redesign protocols using agents to weaken the bacterial cell wall to allow phages to enter into host bacteria and acquire the specificity of the new host. Three protocols were tested using different combinations of benzethonium chloride (BZT-CH), alkaline ethanol, and chlorhexidine gluconate. Results: Five newly redesigned, transiently stable, anti-MRSA phages were produced using the BZT-CH and ethanol–alkali methods, albeit at varying rates. Conclusion: Although the resulting designed anti-MRSA phages are transiently stable, they represent a rare opportunity and an excellent endless source of lytic anti-MRSA phages from which a large number of permanent phage lysins can be separated and purified.

Isolation and Characterization of Highly Lytic Bacteriophages against Staphylococcus aureus in Malaysian Sewage Water

Staphylococcus aureus is a pathogen that can cause both minor and life-threatening infection to human. Recently, the emergence of antibiotic-resistant Staphylococcus aureus (MRSA) has become a global public health concern. As an alternative to antibiotics, bacteriophage therapy is receiving increasing attention. Isolation and characterization of more Staphylococcus aureus phages is an important pre-requisite for building a large repository of phages that can be used in the future for phage therapy. Here we report the isolation of bacteriophages against S. aureus ATCC 6538, the first of its kind in Malaysia. Twenty phages were isolated and two were examined in detail. These two phages, TJSb3 and TJSb6, were found to be highly lytic and belong to the order Caudovirales and the family Siphoviridae. TJSb3 and TJSb6 have high efficiency of plating (EOP value) of 0.907 ± 0.085 and 0.665 ± 0.114, respectively. These two phages exhibited a broad lytic effect against the 4 different S. aureus strains tested (one of which, S. aureus ATCC 43300, is a MRSA strain). TJSb3 and TJSb6 also have small genome size of 20-30k base pairs, making them smaller than 90% of the S. aureus phages recorded in the NCBI viral genome database. These traits make TJSb3 and TJSb6 very attractive as potential candidates for phage therapy.

The role of bacteriophages in facilitating the horizontal transfer of antibiotic resistance genes in municipal wastewater treatment plants

Bacteriophages play integral roles in the ecosystem; however, their precise involvement in horizontal gene transfer and the spread of antibiotic resistance genes (ARGs) are not fully understood. In this study, a coculture system involving consortia of bacteriophages and multidrug-resistant bacteria from an aerobic tank in a municipal wastewater treatment plant (WWTP) was established to investigate the functions of bacteriophages in ARG transfer and spread. The results of the cocultivation of the MRB and bacteriophage consortia indicated that the bacterial community remained stable throughout the whole process, but the addition of bacteriophages significantly increased ARG abundance, especially in bacteriophage DNA. Nine out of the 11 identified ARGs significantly increased, indicating that more bacteriophage particles carried ARGs in the system after cocultivation. In addition, 686 plasmids were detected during cocultivation, of which only 3.36 % were identified as conjugative plasmids, which is significantly lower than the proportion found among previously published plasmids (25.2 %, totaling 14,029 plasmids). Our findings revealed that bacteriophages may play important roles in the horizontal transfer of ARGs through both bacteriophage-mediated conduction and an increase in extracellular ARGs; however, conjugative transfer may not be the main mechanism by which multidrug-resistant bacteria acquire and spread ARGs. Unlike in most previous reports, a coculture system of diverse bacteria and bacteriophages was established in this study to assess bacteriophage functions in ARG transfer and dissemination in the environment, overcoming the limitations associated with the isolation of bacteria and bacteriophages, as well as the specificity of bacteriophage hosts.

Genomic analysis and characterization of lytic bacteriophages that target antimicrobial resistant Escherichia coli in Addis Ababa, Ethiopia

The emergence of antibiotic resistance in E. coli strains has sparked a fervent investigation of alternative therapies, such as the use of lytic bacteriophages. Phage genome sequence analysis is a novel method for learning more about proteins and other biomolecules encoded by phages, particularly phage-lytic enzymes that are crucial to the lysis of bacterial cells. Seven potential lytic E. coli phages—EH-B-A (A1), EP-M-A, EP-B-K (E2), EI-SP-GF, ET-SD-TH, and ST-TK—isolated from activated dairy farm sludges, rivers, and hospital liquid waste genomes were described in this study. The Illumina NextSeq 550 sequencer was used for sequencing phage isolates. The virus nucleotide collection (nr/nt) (taxid:10239) was used to evaluate whole-genome sequences. Phylogenetic analysis was performed using the MEGA11 software. Genome sequencing revealed that each bacteriophage contained a linear double-stranded DNA genome. Phage isolates were taxonomically identified as the Caudoviricetes class with four genera of phages, including Kagunavirus, Vequintavirus, Dhillonvirus, and Jilinvirus. Phage genome lengths varied from 24,264 to 136,204 bp, and GC contents ranged from 44 % to 55 %. In total, 33–218 CDSs (coding sequences) were predicted, with 19–77 % of CDSs encoding functional proteins. All phages lacked tRNA genes in their genomes, except for EI-SP-GF, which possessed five tRNA genes. Based on the phylogenetic tree analysis, the phage isolates were related to Enterobacteria and E. coli phage sequences in the database. Screening did not show any genes encoding for a CRISPR-like system, virulence, antibiotic resistance, or lysogeny. Because of their stringent lytic nature, these phage isolates might be used in the future to treat E. coli infections. This study might also provide some primary data for developing phage control techniques and advance our understanding of the genetic composition of E. coli phages.

Novel Bacteriophage KG853 Exhibits Potent Lytic Activity and Biofilm Inhibition Against Pseudomonas aeruginosa

This study reports the isolation and characterization of a novel bacteriophage, KG853, specifically targeting Pseudomonas aeruginosa ATCC 27853. Morphological analysis using transmission electron microscopy revealed that bacteriophage KG853 belongs to the Bruynoghevirus genus. The phage demonstrated favorable characteristics for potential therapeutic applications, including a short latent period of 30 minutes and a large burst size of 136 plaque-forming units (PFU) per cell. KG853 exhibited stability across various temperatures and pH values, indicating its robustness under various environmental conditions. Genomic analysis showed that KG853 possesses a circular DNA genome of 45,390 base pairs with a GC content of 52.2%. No lysogenic or virulence genes were detected among the 84 open reading frames annotated in the genome, suggesting its safety for potential therapeutic use. Phylogenetic analysis revealed that phage KG853 is closely related to phage PaP3. Notably, KG853 demonstrated the ability to inhibit the formation of 4-hour biofilms by P. aeruginosa, a critical virulence factor in many infections. Host range analysis showed that KG853 is specific to P. aeruginosa, an important characteristic for targeted therapy. These findings suggest that bacteriophage KG853 represents a promising candidate for combating drug-resistant P. aeruginosa infections. Its specific host range, robust physical characteristics, lack of harmful genes, and anti-biofilm activity make it a potential alternative to conventional antibiotics. Further research is warranted to explore its efficacy in in vivo models and potential clinical applications.

Isolation and Antibiofilm Activity of Bacteriophages against Cutibacterium acnes from Patients with Periprosthetic Joint Infection.

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

A Vibrio cholerae anti-phage system depletes nicotinamide adenine dinucleotide to restrict virulent bacteriophages.

Bacteria and their predatory viruses (bacteriophages or phages) are in a perpetual molecular arms race. This has led to the evolution of numerous phage defensive systems in bacteria that are still being discovered, as well as numerous ways of interference or circumvention on the part of phages. Here, we identify a unique molecular battle between the classical biotype of Vibrio cholerae and virulent phages ICP1, ICP2, and ICP3. We show that classical biotype strains resist almost all isolates of these phages due to a 25-kb genomic island harboring several putative anti-phage systems. We observed that one of these systems, Nezha, encoding SIR2-like and helicase proteins, inhibited the replication of all three phages. Bacterial SIR2-like enzymes degrade the essential metabolic coenzyme nicotinamide adenine dinucleotide (NAD+), thereby preventing replication of the invading phage. In support of this mechanism, we identified one phage isolate, ICP1_2001, which circumvents Nezha by encoding two putative NAD+ regeneration enzymes. By restoring the NAD+ pool, we hypothesize that this system antagonizes Nezha without directly interacting with its proteins and should be able to antagonize other anti-phage systems that deplete NAD+.IMPORTANCEBacteria and phages are in a perpetual molecular arms race, with bacteria evolving an extensive arsenal of anti-phage systems and phages evolving mechanisms to overcome these systems. This study identifies a previously uncharacterized facet of the arms race between Vibrio cholerae and its phages. We identify an NAD+-depleting anti-phage defensive system called Nezha, potent against three virulent phages. Remarkably, one phage encodes proteins that regenerate NAD+ to counter the effects of Nezha. Without Nezha, the NAD+ regeneration genes are detrimental to the phage. Our study provides new insight into the co-evolutionary dynamics between bacteria and phages and informs the microbial ecology and phage therapy fields.

Exploration of Phage-Agrochemical Interaction Based on a Novel Potent Phage LPRS20-Targeting Ralstonia solanacearum.

Phage therapy has the potential to alleviate plant bacterial wilt. However, the knowledge gap concerning the phage-agrochemical interaction impedes the broader application of phages in agriculture. This study characterized a phage isolate and investigated its interactions with agrochemicals. A novel species within the Ampunavirus genus was proposed, serving phage LPRS20 as a type phage with a broad lytic range and significant antibacterial activity against Ralstonia solanacearum strains infecting tobacco, chili, or tomato. Sensory evaluation of the morphology of tobacco leaves suggested that phage application resulted in negligible harm to plants. Investigations into phage-agrochemical interactions revealed synergisms when LPRS20 was delivered 4 h before thiodiazole-copper as well as LPRS20 in combination with low-concentration berberine. Overall, our findings reveal that phage LPRS20 represents a novel, effective, and eco-friendly biocontrol agent against tobacco bacterial wilt in vivo and in vitro and contributes to the potential integration of phages and agrochemicals for controlling soil-borne pathogens.

Mycobacteriophages and Their Applications.

Mycobacterial infections caused by tuberculous and non-tuberculous strains pose significant treatment challenges, especially among immunocompromised patients. Conventional antibiotic therapies often fail due to bacterial resistance, highlighting the need for alternative therapeutic strategies. Mycobacteriophages are emerging as promising candidates for the treatment of mycobacteria. This review comprehensively explores phage isolation, characterization, and clinical applications. Despite the need for more extensive in vitro and in vivo studies, existing evidence shows their efficacy against both sensitive and antibiotic-resistant mycobacterial strains, even under disease-mimicking conditions, particularly when used in cocktails to minimize resistance development. Mycobacteriophages can be engineered and evolved to overcome limitations associated with lysogeny and narrow host range. Furthermore, they exhibit activity in ex vivo and in vivo infection models, successfully targeting mycobacteria residing within macrophages. Delivery methods such as bacterial and liposomal vectors facilitate their entry into human cells. Considering the potential for phage-treatment-induced bacterial resistance, as described in this review, the combination of mycobacteriophages with antibiotics shows efficacy in countering mycobacterial growth, both in the laboratory setting and in animal models. Interestingly, phage-encoded products can potentiate the activity of relevant antibiotics. Finally, the application of phages in different compassionate cases is reported. The positive outcomes indicate that phage therapy represents a promising solution for the treatment of antibiotic-resistant mycobacteria.