The phage was recovered from sewage water collected in Karabük, Türkiye, using the standard laboratory strain E. coli ATCC 25922 as the indicator and propagation host. After enrichment and four consecutive rounds of single-plaque purification, the researchers obtained a homogeneous phage preparation producing circular, clear plaques with well-defined edges. Transmission electron microscopy of negatively stained particles revealed a polyhedral head roughly 45 nanometers in diameter and a long, flexible, non-contractile tail of about 176 nanometers, giving a total particle length of approximately 220 nanometers. The absence of a contractile sheath places KBU_SV1 firmly in the siphovirus morphotype within the class Caudoviricetes, the broad group of tailed double-stranded DNA phages that dominates the bacterial virosphere.

Host-range testing against a panel of 106 bacterial strains revealed a pattern of selective rather than broad lytic activity. Spot assays produced visible clearance on 16 strains, corresponding to a host range index of 0.15. Notably, clearance was detected on 7 of 20 carbapenem-resistant E. coli clinical isolates and 4 of 20 carbapenem-susceptible E. coli isolates, meaning that lysis was actually more frequent among the resistant strains. Four of 40 carbapenem-resistant Klebsiella pneumoniae isolates also showed spot clearance, while none of the 20 Enterococcus faecalis clinical isolates or the other reference strains tested were affected. The researchers caution that spot clearance alone does not prove productive infection, since clearing can arise through mechanisms that do not generate progeny phages, such as lysis from without, and efficiency-of-plating measurements will be needed to confirm true susceptibility, particularly for K. pneumoniae.

In liquid culture, KBU_SV1 suppressed the growth of E. coli ATCC 25922 across the entire range of multiplicities of infection tested, from 100 down to 0.01. During the first 14 hours of incubation, optical density in all phage-treated cultures remained below 0.10, a reduction of at least 93 percent relative to the untreated control, which reached an optical density of 1.70 by 24 hours. Regrowth was eventually observed under all conditions, a common feature of phage-bacterium systems that is often attributed to the emergence of phage-resistant subpopulations. Interestingly, the late growth response showed an apparent non-monotonic relationship with the initial phage dose: cultures treated at multiplicities of 0.1 and 0.01 ended up with lower optical densities at 24 hours than those treated at 10 and 1. The authors emphasize that the mechanism behind this pattern remains unresolved and that it should not be taken as evidence that lower doses are generally superior.

Physicochemical stability is a key practical consideration for any phage envisaged as a therapeutic candidate, and here KBU_SV1 performed well. The phage retained infectivity after one hour of incubation across a pH range from 4 to 12, with only modest declines of roughly 0.25 log units at the extremes of that interval compared with the neutral pH reference. No infectivity was detected at pH 2, however, a point worth noting given the acidic environment of the stomach. Temperature testing showed stable titers at 4, 23 and 37 degrees Celsius, but exposure to 65 degrees Celsius eliminated detectable infectivity. Taken together, these results suggest that KBU_SV1 is robust under moderate conditions relevant to storage, formulation and physiological temperature, although long-term storage stability was not evaluated.

Biofilms, structured bacterial communities embedded in an extracellular polymeric matrix, are a major driver of treatment failure because they dramatically reduce susceptibility to both antibiotics and immune defenses. KBU_SV1 demonstrated meaningful antibiofilm activity in crystal violet assays. Treatment of established biofilms of E. coli ATCC 25922 reduced biomass by 84.5 to 86.0 percent across all tested multiplicities of infection, while inhibition of biofilm formation reached 78.0 to 83.7 percent. Against the carbapenem-resistant clinical isolate Ec59, the effects were smaller but still substantial, with biomass reductions of 22.3 to 54.2 percent and inhibition of formation ranging from 24.6 to 57.5 percent, with the strongest effects at the highest multiplicity. The authors note that crystal violet staining measures total attached biomass rather than viable cells, so these findings reflect reductions in biofilm mass rather than proof of bacterial killing or complete eradication.

The in vivo experiments used the greater wax moth Galleria mellonella, a widely used invertebrate infection model. Larvae infected with a lethal dose of E. coli ATCC 25922 died rapidly, with 40 percent mortality at 12 hours and complete mortality by 18 hours. Larvae that received phage treatment one hour after infection survived significantly longer: mortality was distributed across the 48-hour observation period, and 40 percent of treated larvae were still alive at 24 hours. Kaplan-Meier survival analysis confirmed a statistically significant delay in mortality compared with the infection control, with a log-rank test yielding a p-value of 1.2 × 10⁻⁷. Importantly, no deaths occurred in larvae receiving phage alone or mock injections, indicating no detectable toxicity of the preparation under the tested conditions. The authors stress, however, that the phage delayed rather than prevented death, and that G. mellonella does not fully reproduce mammalian physiology, so these results should be viewed as preliminary evidence supporting further investigation rather than demonstration of therapeutic efficacy.

Genomic sequencing provided the most reassuring safety-relevant findings. The KBU_SV1 genome is a 45,190-base-pair double-stranded DNA molecule with a G plus C content of 53.56 percent, confirmed as complete by CheckV with 100 percent estimated completeness, zero contamination and a high-confidence 272-base-pair direct terminal repeat at both ends. Sequencing depth averaged nearly 24,000-fold with complete positional coverage. The genome encodes 71 putative open reading frames organized into five functional modules covering DNA and RNA replication, DNA metabolism, structural proteins, assembly and lysis, and contains no tRNA genes. Critically, screening with ResFinder, VirulenceFinder, the Comprehensive Antibiotic Resistance Database and the Virulence Factor Database detected no known antimicrobial resistance genes, no virulence determinants and no lysogeny-associated genes such as integrases, excisionases or phage repressors. Machine-learning-based lifestyle prediction tools PhageAI and PhageLeads both classified the phage as virulent, meaning strictly lytic, a property generally considered desirable for therapeutic candidates because it rules out the temperate lifestyle that could mediate horizontal gene transfer.

Comparative genomics clarified where KBU_SV1 fits in the phage taxonomy. BLASTn searches revealed 99.12 percent nucleotide identity with Escherichia phage EPIMAM03, and VIRIDIC intergenomic similarity calculations showed values above the 95 percent species-level threshold with several related genomes, including Dhillonvirus LM40P1 at 98.7 percent. Independent taxonomic analysis with taxMyPhage assigned KBU_SV1 to the genus Dhillonvirus within the class Caudoviricetes, and phylogenetic trees based on the large terminase subunit and whole-proteome comparisons were congruent with this placement. The complete genome has been deposited in GenBank under accession number PZ050807.1. Rather than representing an entirely new species, KBU_SV1 belongs to an existing species-level genomic cluster, which the authors note is consistent with contemporary genome-based approaches to phage classification.

The study has limitations that the authors themselves enumerate candidly. Efficiency-of-plating measurements were not performed on the spot-test-positive isolates, one-step growth experiments to determine adsorption kinetics, latent period and burst size were not carried out, the mechanism of bacterial regrowth was not investigated, and the DNA packaging mechanism implied by the direct terminal repeat was not experimentally resolved. The growth-reduction and in vivo experiments used the reference strain rather than carbapenem-resistant clinical isolates, and endotoxin levels in the phage preparation were not quantified. Nevertheless, the overall profile of KBU_SV1, combining lytic activity against carbapenem-resistant E. coli, broad pH and temperature stability, antibiofilm effects, delayed mortality in an infection model and a genome apparently free of resistance, virulence and lysogeny genes, marks it as a promising candidate for further development, whether deployed alone or as a component of phage cocktails. Before any clinical application, the authors conclude, future work must address productive host range, replication parameters, resistance development, optimized dosing and efficacy in mammalian infection models.

Subject of Research: Isolation and genomic characterization of the bacteriophage KBU_SV1 as a candidate therapeutic against carbapenem-resistant Escherichia coli

Article Title: Biological and genomic characterization of Escherichia phage KBU_SV1 active against carbapenem-resistant Escherichia coli

Article References: Öztaş, S., Karaynir, A., Ozkan, R. G., Bozdogan, B., & Dündar, D. (2026). Biological and genomic characterization of Escherichia phage KBU_SV1 active against carbapenem-resistant Escherichia coli. Frontiers in Microbiology, 17, Article 1927801. https://doi.org/10.3389/fmicb.2026.1927801

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.1927801

Keywords: bacteriophage, phage therapy, carbapenem-resistant E. coli, antimicrobial resistance, Dhillonvirus, biofilm, Galleria mellonella, Caudoviricetes, siphovirus, phage genome, antibiofilm activity, wastewater

The research, led by Tizazu Zelelie, Biniam Mekonnen, and Demiss Nigussie and published in Public Health in Practice, was designed around the One Health framework, the internationally endorsed recognition that human, animal, and environmental health are inseparable. First formalized in 2015 by the tripartite coalition of the Food and Agriculture Organization, the World Health Organization, and the World Organization for Animal Health, and later expanded to include the United Nations Environment Programme, the approach acknowledges that resistant bacteria and their genes do not respect the boundaries between hospital wards, farms, and waterways. Resistant organisms shed into the environment can transfer their resistance genes horizontally to other bacteria infecting humans and animals, creating reservoirs of resistance that no single-sector intervention can drain. East Africa presents particularly fertile ground for this dynamic, combining agricultural and pastoral livelihoods with patterns of antimicrobial misuse, limited access to clean water, poor sanitation, and inadequate infection prevention infrastructure.

To capture the full scope of the problem, the authors searched PubMed, ScienceDirect, and Google Scholar for studies reporting carbapenem-resistant E. coli isolated from humans, animals, food, or the environment in thirteen East African countries. Following the PRISMA reporting guidelines and registered prospectively on PROSPERO, the review screened more than 4,300 records, ultimately retaining twenty-nine studies that met rigorous eligibility criteria. Each study was assessed for bias by two independent reviewers using the Newcastle Ottawa Scale, and all were judged to carry low risk of bias, scoring at or above the six-star threshold. The researchers then performed a random-effects meta-analysis using restricted maximum likelihood estimation in the R statistical environment, quantifying heterogeneity with the I-squared statistic and probing publication bias with funnel plots and Egger’s test.

The numbers underlying the headline finding are substantial. Across the included studies, the combined sample size reached 11,581 participants and specimens, from which 3,908 E. coli isolates were recovered. Of those, 442 strains proved resistant to carbapenem antibiotics. The studies were heavily weighted toward clinical settings, with seventy-six percent hospital-based and human samples accounting for nearly eighty percent of all sources, but the remainder drew from environmental reservoirs, food, and animals, giving the analysis its distinctive cross-sector character. Geographically, Ethiopia dominated the evidence base with fourteen studies, followed by Uganda with seven, Sudan with four, Tanzania with two, and single studies from Djibouti and Kenya. No eligible studies emerged from the other seven countries in the region, itself a telling indicator of surveillance gaps.

Beneath the pooled estimate of 12.7 percent lies a sobering spread. The lowest reported prevalence was 0.5 percent in an Ethiopian study, while the highest, drawn from sewage at a Ugandan referral hospital, reached a remarkable 50 percent. In subgroup analyses, Sudan recorded the highest country-level pooled prevalence at 24.7 percent, followed by Uganda at 17.4 percent and Ethiopia at 9.9 percent. More striking still was the finding that the environment emerged as the most contaminated interface of all, with a pooled prevalence of 24.2 percent among environmental isolates, compared with 11.9 percent in humans, 7.7 percent in food, and 4.2 percent in animals. Clinical samples of multiple types yielded a pooled prevalence of 15.8 percent. These figures suggest that hospitals, farms, and communities are continuously seeding the environment with resistant organisms, which in turn serve as a reservoir capable of reinfecting the human population.

Genetic analysis revealed the molecular arsenal behind this resistance. Half of the included studies, 51.7 percent, reported the detection of carbapenemase-encoding genes, including KPC and GES from the class A serine beta-lactamases, NDM-1 and NDM-5, VIM, IMP, and SPM among the metallo-beta-lactamases, and OXA-48 and OXA-181 oxacillinases. These genes encode enzymes that destroy carbapenems and are readily transferred between bacterial species on mobile genetic elements, meaning a resistance trait that emerges in one organism can rapidly colonize others. The World Health Organization ranks carbapenem-resistant pathogens first on its global priority list of antibiotic-resistant bacteria precisely because treatment options for them are so desperately limited: essentially reduced to polymyxins, fosfomycin, and tigecycline, older or more toxic drugs that represent imperfect substitutes for an entire antibiotic class.

The stakes of this trend are measured in lives. Global modeling published in the Lancet estimates that bacterial antimicrobial resistance was associated with nearly 5 million deaths in 2021, contributing to roughly one in eight deaths worldwide, and projects that as many as 8.22 million deaths annually could be attributable to AMR by 2050 if current trajectories continue. In the WHO African region alone, more than one million deaths were associated with resistant bacteria in 2019. Among Gram-negative bacteria, resistance to carbapenems has climbed faster than to any other antibiotic class, with associated deaths rising from 619,000 in 1990 to 1.03 million in 2021. Carbapenem-resistant Enterobacteriaceae infections are linked to longer hospital stays, higher healthcare costs, and elevated mortality, burdens that fall hardest on health systems with the least capacity to absorb them.

The review also captured a subtle temporal signal. Prevalence in studies published between 2021 and 2025 was marginally lower, at 12.6 percent, than in the 2015 to 2020 window, which averaged 13.5 percent, though the authors note this difference was not statistically significant and may simply reflect variation in study designs, settings, and target populations. Regional trend data from Ethiopia’s Amhara Public Health Institute, which showed resistance climbing to a peak in 2019 before declining, offers tentative grounds for the hope that sustained interventions can bend the curve. At the same time, the extraordinarily high statistical heterogeneity among the studies, with an I-squared value of 93.3 percent, underscores how differently the epidemic of resistance is unfolding across countries, populations, and sample types, and the authors caution that significant publication bias was detected and statistically adjusted in their estimates.

Perhaps the most consequential implication of the study is what it says about surveillance and regional coordination. The authors attribute the region’s elevated burden to weak laboratory capacity, poor health system governance, limited diagnostic infrastructure, and the destabilizing effects of conflict in countries such as Sudan and Ethiopia, all compounded by prolonged and often inappropriate antimicrobial use in both human and animal populations. Because routine testing for carbapenemase production is rarely performed in African clinical laboratories, the true prevalence may well exceed what published studies capture. The authors call for effective implementation of the One Health approach across sectors, strengthened routine diagnostic capacity, national and regional surveillance programs, rapid detection methods, and integration that extends beyond health policy alone. Without such coordinated action, the environmental reservoirs documented at prevalence rates approaching one in four isolates will continue to replenish the human burden, ensuring that one of medicine’s final antibiotics loses its power across one of the world’s most vulnerable regions.

Subject of Research: Prevalence and molecular epidemiology of carbapenem-resistant E. coli across human, animal, and environmental interfaces in East Africa

Article Title: Carbapenem-resistant E. coli with one-health approach in East Africa: Systematic review and meta-analysis

Article References: Zelelie, T., Mekonnen, B., & Nigussie, D. (2026). Carbapenem-resistant E. coli with one-health approach in East Africa: Systematic review and meta-analysis. Public Health in Practice, 12, Article 100843. https://doi.org/10.1016/j.puhip.2026.100843

Image Credits: AI Generated

DOI: 10.1016/j.puhip.2026.100843

Keywords: carbapenem-resistant E. coli, antimicrobial resistance, One Health, East Africa, systematic review, meta-analysis, carbapenemase genes, NDM, public health surveillance, Ethiopia, Uganda, Sudan