Comparison of agar-based and checkerboard methods to assess phage-antimicrobial synergy in Pseudomonas aeruginosa.

BackgroundVibrio parahaemolyticus is a marine bacterium causing seafood-associated gastrointestinal illness in humans and acute hepatopancreatic necrosis disease (AHPND) in shrimp. Bacteriophages have emerged as promising biocontrol agents against V. parahaemolyticus. This study characterizes Vibrio phage VPK8, focusing on host specificity, efficiency of plating (EOP) variability across V. parahaemolyticus isolates from diverse sources and other Vibrio species, morphology, genomic features, and bacteriolytic potential.MethodsVibrio phage VPK8 was isolated from blood cockles in Thailand using a mixed-host approach and purified via the double-layer agar method. Host specificity was evaluated using spot assays and EOP measurements against 120 Vibrio strains, including AHPND-associated, clinical, and seafood isolates. Phage morphology was characterized by transmission electron microscopy (TEM), while genomic features were analyzed using next-generation sequencing. Lytic characteristics, including latent period and burst size, were determined through one-step growth curves, and bacterial growth reduction was evaluated over a 24-h.ResultsVibrio phage VPK8 is a lytic phage with a 42,866 bp linear double-stranded genome, G + C content of 49.4%, and 48 coding sequences. Phylogenetic analysis grouped it within the Autographiviridae family, showing 95.96% similarity to Vibrio phage vB_VpaP_MGD1. Viral proteomic analysis placed VPK8 within the Pseudomonadota host group. Spot assays indicated broad lytic activity, but EOP analysis revealed high infectivity in clinical and seafood V. parahaemolyticus isolates, as well as some V. cholerae and V. mimicus strains. TEM revealed an icosahedral head (~ 60 nm) and a short tail. At a multiplicity of infection of 0.01, VPK8 exhibited a latent period of 25 min, a burst size of 115, and effectively inhibited the reference host V. parahaemolyticus PSU5124 within 6 h, maintaining its lytic activity and stability for over 24 h.ConclusionsThis study provides a detailed characterization of Vibrio phage VPK8 which exhibits targeted infectivity with high EOP in clinical and seafood V. parahaemolyticus isolates, as well as selected Vibrio species. Its stable lytic performance, rapid replication, and genomic safety suggest its potential for phage-based applications. Further studies should explore its in vivo efficacy and the genetic features contributing to phage resistance mechanisms, enhancing its potential applicability in managing Vibrio-related diseases.

The prevalence and impact of bacteriophages in the ecology of bacterial communities coupled with their ability to control pathogens turn essential to understand and predict the dynamics between phage and bacteria populations. To achieve this knowledge it is essential to develop mathematical models able to explain and simulate the population dynamics of phage and bacteria. We have developed an unstructured mathematical model using delay-differential equations to predict the interactions between a broad-host-range Salmonella phage and its pathogenic host. The model takes into consideration the main biological parameters that rule phage-bacteria interactions likewise the adsorption rate, latent period, burst size, bacterial growth rate, and substrate uptake rate, among others. The experimental validation of the model was performed with data from phage-interaction studies in a 5 L bioreactor. The key and innovative aspect of the model was the introduction of variations in the latent period and adsorption rate values that are considered as constants in previous developed models. By modelling the latent period as a normal distribution of values and the adsorption rate as a function of the bacterial growth rate it was possible to accurately predict the behaviour of the phage-bacteria population. The model was shown to predict simulated data with a good agreement with the experimental observations and explains how a lytic phage and its host bacteria are able to coexist.

Using bacteriophages as a tool to the control of pathogens is a complementary to antibiotic therapy. We have isolated a lytic bacteriophage, designated vB_SauM_JS25, from sewage effluent on a dairy farm in Jiangsu, Mid-east of China for use as a biocontrol agent against Staphylococcus aureus infections. Phage vB_SauM_JS25 was morphologically classified as Myoviridae. The phage showed broad host ranges within S. aureus strains, lysing 51 of 56 strains (91.1 %). Its latent period and burst size were approximately 20 min and 21 PFU/cell, respectively. Phage vB_SauM_JS25 was able to survive in a pH range between 6 and 9. However, a treatment of 70 or 80 °C for 10 min completely inactivated the phage. Moreover, morphologic analysis of vB_SauM_JS25 revealed that it was closely related to other Myoviridae phages infecting Staphylococcus species. The bacteriolytic activity of phage vB_SauM_JS25 at a multiplicity infection (MOI) 1 indicted its efficiency for reducing bacterial growth. These findings suggest that phage vB_SauM_JS25 could be considered a potential therapeutic or prophylactic candidate against S. aureus infection.

The occurrence and prevalence of mcr gene in Gram-negative bacteria from animals was firstly reported in 2015, which greatly threat the values of the human last-resort antimicrobial-colistin. Here we have isolated a lytic bacteriophage, designated vB_EcoM_mcr1, from sewage effluent of a chicken farm in Jiangsu, Mid-east of China, which could infect mcr-positive Escherichia coli strains. Electron microscopy showed Phage vB_EcoM_mcr1 does not resemble to any known family of the phage, with an isometric head (110 nm) and a short contractile tail (35 nm). The phage showed a narrow host ranges against E. coli isolates of chicken origin, lysing 3 of 34 (8.8%) mcr-positive strains. Its latent period and burst size were approximately 5 min and 34 PFU/cell, respectively. The genome of phage vB_KpnM_mcr1 was double stranded DNA and estimated to be about 60 kb. The bacteriolytic activity of this phage at a multiplicity infection (MOI) of ≥0.001 indicted its efficiency for reducing bacterial growth. It is the first time to identify the phage, vB_EcoM_mcr1,which could infect mcr-positive E. coli. These findings suggest that phage vB_EcoM_mcr1 could be considered as a potential therapeutic or prophylactic candidate against mcr-positive E. coli infection in chicken.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen frequently causing healthcare-associated infections. The apocalyptic rise of antimicrobial resistance has rekindled interest in age-old phage therapy that uses phages (viruses that infect bacteria) to kill the targeted pathogenic bacteria. Because of its specificity, phages are often considered as potential personalized therapeutic candidate for treating bacterial infections. In this study, we isolated and purified lytic phages against multi-drug resistant P. aeruginosa using soft agar overlay technique. Phage characteristics like thermal and pH stability, latent period and burst size were determined using one-step growth assay while multiple host range spectrum was determined by spot assay. The phages were further characterized using protein profiling. Three Pseudomonas phages (øCDBT-PA31, øCDBT-PA56 and øCDBT-PA58) were isolated from the holy rivers of Kathmandu valley. Among 3 phages, øCDBT-PA31 demonstrated multiple host range and could lyse multi-drug resistant strain of P. aeruginosa. Further, øCDBT-PA31 showed latent period of 30 minutes with corresponding burst sizes of 423-525 PFU/cell. Interestingly, øCDBT-PA31 also tolerated a wide range of adverse conditions, such as high temperature (50°C) and pH 3-11. Further, protein profiling revealed that øCDBT-PA31 has 4 and øCDBT-PA11 had 3 distinct bands in the gradient gel ranging from approximately 3.5-29 kilodaltons (kDa) suggesting them to be morphologically distinct from each other. As multi-drug resistant bacteria are emerging as a global problem, lytic phages can be an alternative treatment strategy when all available antibiotics fail.

Stem and crown gall disease caused by the plant pathogen Rhizobium radiobacter has a significant impact on highbush blueberry (Vaccinium corymbosum) production. Current methods for controlling the bacterium are limited. Lytic phages, which can specifically target host bacteria, have been widely gained interest in agriculture. In this study, 76 bacteriophages were recovered from sewage influent and screened for their inhibitory effect against Rhizobium spp. The phages were genetically characterized through whole-genome sequencing, and their lytic cycle was confirmed. Five potential candidate phages (isolates IC12, IG49, AN01, LG08, and LG11) with the ability to lyse a broad range of hosts were chosen and assessed for their morphology, environmental stability, latent period, and burst size. The morphology of these selected phages revealed a long contractile tail under transmission electron microscopy. Single-step growth curves displayed that these phages had a latent period of 80-110 min and a burst size ranging from 8 to 33 phages per infected cell. None of these phages contained any antimicrobial resistance or virulence genes in their genomes. Subsequently, a combination of two-, three- and four-phage cocktails were formulated and tested for their efficacy in a broth system. A three-phage cocktail composed of the isolates IC12, IG49 and LG08 showed promising results in controlling a large number of R. radiobacter strains in vitro. In a soil/peat-based model, the three-phage cocktail was tested against R. radiobacter PL17, resulting in a significant reduction (p < 0.05) of 2.9 and 1.3 log10 CFU/g after 24 and 48 h of incubation, respectively. These findings suggest that the three-phage cocktail (IC12, IG49 and LG08) has the potential to serve as a proactive antimicrobial solution for controlling R. radiobacter on blueberry.

Alfalfa sprouts have been linked to numerous North American outbreaks of Salmonella in recent years. Conventionally, treatments involving chlorine, heat, and irradiation are used for alfalfa seed sanitation. However, such treatments may be highly variable in their efficacy for pathogen control and/or detrimental to sprout quality, therefore negatively perceived by consumers advocating for natural alternatives. The usage of bacteriophages for pathogen control in sprouts has been previously explored, although with conflicting and inconsistent results. Lytic phages, viral predators of bacteria, represent an attractive approach as they provide several advantages compared to conventional treatments, such as their high specificity for bacterial targets and their ubiquity in nature. In this study, four Salmonella phages were isolated from British Columbia, Canada and characterized with respect to host range, burst size, latent period, and environmental stability to assess their potential to control Salmonella. Phage isolate SI1 showed the greatest host range, highest burst size and shortest latent period, greatest stability across all pH and temperatures and was the most effective in control of S. Enteritidis in vitro. Therefore, SI1 was chosen for treatment of sprouting alfalfa seeds artificially contaminated with S. Enteritidis with a multiplicity of infection (MOI) of ∼110 PFU/CFU. A significant (p < 0.05) reduction of 38.3 ± 3.0% of viable Salmonella cells was observed following two h of phage treatment. On days two to six of the sprouting process, reductions of Salmonella were also observed, but were not significant compared to the control (p > 0.05). It was further demonstrated that the sprout yield was not significantly (p > 0.05) affected by phage treatment. These results highlight the potential of phages recovered from the British Columbia environment for use as biocontrol agents against Salmonella, although differing efficacies in vitro was observed. Moreover, the effectiveness of SI1 to significantly (p < 0.05) control Salmonella on sprouting alfalfa seeds on day 1 of treatment was demonstrated. Although promising, future work should aim to optimize this treatment to achieve more effective, and longer lasting, biocontrol of Salmonella in sprouting alfalfa seeds.

The multidrug-resistant (MDR) Klebsiella pneumoniae is an important pathogen that threatens public health directly with life-threatening infections. The need for the development of new effective and well tolerated alternative treatments for these infections is crucial. Therefore, the interest in phage therapy as a promising alternative is increasing. Here, a novel phage named ΦAYH was isolated from the Tigris River water, Baghdad, IRAQ near sewage of Baghdad Medical City with its specific host from the same site. Phage ΦAYH belongs to Podoviridae family in the order Caudovirales. The ΦAYH maintained stability at different temperatures (−10 to 60°C) and pH values (5–11). The phage was able to lyse 8 from 32 clinical K. pneumoniae isolates in vitro with no lytic activity against other tested species. For one-step growth, latent period was 10 min with burst size ∼64 virions/cell at multiplicity of infection (MOI) 10. The SDS-PAGE test revealed one major structural protein and different structural proteins ranging from 28 to 89 kDa in size. The phage host and 32 clinical K. pneumoniae isolates were tested for phenotypic identification and antibiotics profile by VITEK-2 system and genotypically using rpob gene. All clinical K. pneumoniae isolates showed resistance to the most antibiotics tested while phage host was resistant only to amoxicillin. Biofilm production by all clinical isolates including the host isolate was tested. These isolates showed different ability as following: 72.72% as weak, 6.06% as moderate, and 21.21% as strong biofilm producer. Together these results demonstrate that ΦAYH is a promising alternative against MDR K. pneumoniae.

Bacteriophages (phages) are viruses that infect bacteria. The emergence of resistant bacteria has been reported worldwide. The use of phages to treat bacterial infection or to reduce bacteria load has become a new area of interest. The viral colonization of rodents represents an important source for a specific biotope. The objective of this study is to characterize the effect of novel phages isolated from rodents (Mastomys) using clinical Enterobacteria strains. Three novel phages were isolated from the intestinal tract of rodents. The phages belong to Myoviridae family. By using three Random Polymerase Chain Reactions (PCR), genomic characterization of the phages shows a high diversity that reveals several DNA bands. 3 virulent phages exhibited different patterns, indicating that the rodents’ phages are genetically unique and could be distinguished by typing-PCR. The virulence spectrum of the phages shows phages Mα3 and Mα7 have 50% (7/15) lytic activity on enteropathogenic and enterohemorrhagic Escherichia coli strains while phage Mα11 has 26.6% (4/15) lytic activity. These phages have no lytic activity on Salmonella strains. This study is the first to isolate novel phages from rodents with lytic activity. The candidate rodents’ phages (Mα3, Mα7, Mα11) can be used to control virulent E. coli in West Africa. Keys words: Lytic phages, rodents, Escherichia coli strains, West Africa, enteropathogenic, enterohemorrhagic.

Klebsiella pneumoniae is a common pathogen responsible for various infections, with multidrug-resistant (MDR) strains increasingly complicating treatment. Phage therapy has shown significant potential in treating difficult bacterial infections; however, research specifically focused on phages targeting this bacterium remains limited. This study aimed to isolate and characterize lytic bacteriophages that target multidrug-resistant K. pneumoniae. Phages were isolated from 66 environmental samples through spot assays conducted on 10 multidrug-resistant strains. Phages were isolated via spot assays and purified via streak plating. Characterization included PCR-based identification and classification, determination of the latent period, efficiency of plating, burst size, stability testing, and evaluation of in vitro bactericidal activity. From a total of 660 spots tested against 10 multidrug-resistant (MDR) isolates, 102 phages were successfully isolated. These phages demonstrated individual lytic activity ranging from 8% (4/46) to 63% (29/46). PCR-based classification of the 60 bacteriophages identified six distinct virulent phage genera, with Taipeivirus being the most prevalent at 18.3% (11/60) and Webervirus the least common at 10.0% (6 out of 60). Stability assessments of pH and temperature demonstrated optimal activity between pH 5 and 9 and at temperatures up to 50 °C. These results endorse phage therapy as a viable alternative for treating MDR and hypervirulent K. pneumoniae infections. The data offer critical insights into local bacteriophage diversity and underscore its potential for developing targeted therapeutic agents. Genome sequencing, in vivo studies, and clinical trials are required to validate the efficacy and safety of these phages.

Bovine mastitis causes severe economic losses to dairy farmers. Staphylococcus aureus, is one of the most important pathogen implicated in etiology of clinical and subclinical mastitis in bovines. In view of increasing antimicrobial resistance alternatives to antibiotic therapy are much needed. The present decade has witnessed a renewed interest in phage based therapeutics and diagnostics. The present study, describes isolation and characterization of two lytic phages SAJK-IND and MSP against Staphylococcus aureus having a potential to be used in therapy against mastitis. SAJK-IND and MSP phages belonged to Myoviridae and Podoviridae families, respectively. TEM imaging of the two phages revealed an iscosahedral head. MSP phage has a short non contractile tail. SAJK-IND and MSP have a burst size of 44 ± 3 and 25 ± 5 PFU/ infected cell, respectively. SAJK-IND and MSP phages revealed ̴ 12 and ̴16 proteins, respectively on SDS-PAGE analysis. The lytic activity of the phages was specific for Staphylococcus aureus. SAJK-IND revealed 100% lytic activity against several strains of Staphylococcus aureus isolated from mastitis milk samples whereas, MSP had only 40% lytic activity. SAJK-IND phage genome was sequenced, assembled and deposited in Genbank under accession no MG010123.

Wastewater treatment plants (WWTPs) are large repositories of enteropathogenic bacteria as they serve as the foundation for environmental enteropathogenic pollution and disease transmission in the environment. This threatens public health, thus undermining wastewater effluent and sludge reuse. Convetional biological methods used in Kenya WWTPs have limitations in controlling multidrug resistant (MDR) pathogens.Therefore, better biocontrol strategies to reduce the enteropathogenic load and control MDR pathogens in sewage effluent and sludge for reuse are required. Bacteriophages are viruses that infect and lyse bacteria. They have emerged as promising biocontrol agents in reducing bacterial loads including MDR strains following ONE HEALTH strategies including wastewater treatment. However their application in wastewater biocontrol remains under explored in Kenya.This study aimed in isolating and characterizing enteropathogens and bacteriophages in Ruai and Kariobangi WWTPs. Wastewater and sludge samples were aseptically collected, transported under cold chain conditions, and processed as soon as possible. Bacterial isolates were obtained through standard culture methods and characterized using morphological, biochemical following API20E system, and molecular techniques, including 16S rRNA sequencing. The 18 isolates molecularly confirmed were tested against 8 antibiotics are carriers of two resistant genes following PCR techniques. Bacteriophages were isolated following direct plate method and tested for pH, temparute stability, host range activity and growth curve determined. Data from this work show that the CFU counts of treated wastewater (5962028 per 100 mL) and treated sludge (6019053.667 per 100 mL) were above the World Health Organization (WHO) and National Environmental Management (NEMA), which stipulate that a CFU count of 1000.0/100 mL and below is allowed for discharge. The dominant genera included Escherichia, Enterobacter, Klebsiella, Citrobacter, and Serratia. Antibiotic susceptibility testing of 18 molecularly confirmed isolates revealed that14 were multidrug-resistant, with high resistance rates to ampicillin, co-trimoxazole, and sulfamethoxazole. PCR analysis identified 12 isolates carrying the sul2 gene and 11 carrying the Amp gene, with seven harboring both. The five lytic bacteriophages Psvu, Encl, Klpn, Sepr, and Enfa targeted P. vulneris, E. cloacae, K. pneumoniae, S. proteamaculans, and E. faecalis, respectively.The Phage stability studies showed that lytic activity peaked at pH 7.5 and 37 °C, with substantial variation in burst sizes and plaque morphologies among the isolates.The phages were used to prepare a cocktail for a laboratory-based CFU reduction experiment. The CFU count in the effluent was reduced by 20.24-fold and in sludge by 22.25-fold. These findings highlight the prevalence of multidrug resistance and associated resistance genes in WWTP enteropathogen isolates and demonstrate the potential of locally isolated bacteriophages as effective biocontrol agents for reducing enteropathogenic loads. This study provides evidence of the application of bacteriophage therapy in environmental biotechnology, paving the way for scalable, sustainable interventions in enteropathogen management in treated wastewater.

Antimicrobial peptides are promising therapeutic agents in view of increasing resistance to conventional antibiotics. Antimicrobial peptides usually fold in α-helical, β-sheet, and extended/random-coil structures. The α-helical antimicrobial peptides are often unstructured in aqueous solution but become structured on bacterial membrane. The α-helical structure allows the partitioning into bacterial membrane. Therefore it is important to understand the mechanism of unfolding and refolding of α-helical structure in antimicrobial peptides. It is not very easy to obverse and study the process of unfolding and refolding of α-helical antimicrobial peptides because of their rapidity. Therefore, molecular simulation provides a way to observe and explain this phenomenon. Plantaricin A is a 26 amino-acid antimicrobial pheromone peptide and can spontaneously unfold and refold under physiological condition. This study demonstrated the unfolding and refolding of plantaricin A by means of molecular simulation, and its mechanism was discussed with its implication to the Levinthal paradox.

Abstract. Narulita E, Utami DAW, Izzaturrohmah L, Arrachmi HI, Febrianti RA, Ludfillah RZ, Widianto AH, Addy HS, Utami BR, Kuswati, Fikri K. 2025. Genomic and evolutionary perspectives on bacteriophage ?Afa-NA1 targeting MDR Gram-negative bacteria in diabetic ulcer. Biodiversitas 26: 6014-6024. The rise of Multidrug-Resistant (MDR) bacterial infections poses a significant challenge in clinical settings, particularly for diabetic ulcer patients who are highly susceptible to persistent infections. This study reports the isolation, characterization, and genomic analysis of a novel lytic bacteriophage, ?Afa-NA1, specifically targeting MDR Gram-negative bacteria associated with diabetic ulcers in Indonesia. The bacteriophage was isolated from wound dressings and tested against four clinical isolates: Alcaligenes faecalis T17, Pseudomonas sp. FP1911, and the Gram-positive control Streptomyces violaceoruber S21. ?Afa-NA1 exhibited strong lytic activity against three Gram-negative isolates but none against the Gram-positive control, confirming host specificity. ?Afa-NA1 produced clear plaques with an average diameter of 3.0±0.5 mm on Alcaligenes faecalis T17, indicative of its potent lytic activity. Stability tests showed the phage retained &gt;90% infective titer after 24 hours at -20°C and 4°C, and exhibited robust stability across a wide pH range (pH 6 to 11). One-step growth analysis revealed a short latent period of 21.7±8.1 minutes and a burst size of 27.7±1.5 PFU per infected cell. Genomic analysis showed ?Afa-NA1 possesses a 78,105 bp double-stranded DNA genome with 45.8% G+C content encoding 83 predicted open reading frames. Comparative genomics and phylogenetic reconstruction confirmed its placement among T7-like Podoviruses, consistent with its obligate lytic lifecycle and genomic architecture. However, the potential for therapeutic application remains preliminary, as host range testing was conducted on a limited panel of single clinical isolates, and all assays were performed in vitro.

Background: Multidrug-resistant (MDR) enterococci have become a major problem in recent times and have been reported increasingly around the world. Lytic phages infect bacteria leading to rapid host death with limited risk of phage transduction, underlining the increasing interest in potential phage therapy in the future. Objective (s): The aim of this study is to use phage therapy as alternative approach for treatment of Enterococcus faecalis infections that recorded as MDR in Iraq to tackle this problem. Materials and Methods: Thirty E. faecalis isolates were collected from patients with different infectious diseases such as urinary tract infection (UTI), diabetic foot, septicemia, and wound infections. The isolation of specific lytic phages was from different environmental sources such as (sewage, and wastewater). The biokinetic assays were carried out to measure the characteristics of the isolated phage. The study of the bacteriophage and the formed phage cocktail infectivity against isolates E.faecalis was tested by the top layer assay. The phage endolysin was extracted from the best bacteriophage that gave best results. Results: All the isolated E.faecalis was reported as MDR in this study. About 75 E.faecalis specific phages were isolated and purified. All the isolated bacteria were 100% sensitive to the lytic phages. The formed phage cocktail was capable to create inhibition zones on the most bacterial isolates' lawns. The molecular weight and the concentration of the extracted endolysin was evaluated in this study and found to be as (48 kDa) and (0.5mg/ml), respectively. The antibacterial activity of the extracted endolysin was evaluated by the turbidity reduction assay. A clear decline in the bacterial growth was manifested (5x107 CFU/ml) to (1x104 CFU/ml), in which the bacterial growth was reduced by (3.63 log). The endolysin found to be effective against 90% of E.faeclais isolates. Conclusion: The activity of the isolated specific phage together with the activity of the formed phage cocktail, were efficient as successful and inexpensive method of therapy against MDR E.faecalis. The potential of the extracted endolysin over the phage therapy was verified in this study. The coverage rate and the absence of resistant E.faecalis to the phage and its endolysin had emphasized on the importance of this alternative therapy to commonly used antibiotics. List of abbreviation: MDR = multiple drug-resistant, E. faecalis = Enterococcus faecalis, BT = burst time, BS = burst size, IP = infective percentage, Phage =bacteriophage, CFU = Colony forming unit, kDa = Kilodalton, OD = Optical Densities.