Phage-mediated control of melioidosis in Southern China: molecular insights and therapeutic potential of N4-like viruses from Hainan.

BACKGROUND: Melioidosis, caused by Burkholderia pseudomallei, is a tropical disease that poses a serious risk of fatal sepsis and imposes a substantial financial burden on healthcare systems, driven by its complicated antibiotic resistance profile. With the successful isolation of vB_BpP_HN01 in Hainan, a theoretical basis for phage therapy has been established. Whilst the observation of certain clinical isolates non-susceptible to its infection, along with concerns about the potential emergence of tolerance during monophage treatment, highlights the urgent need to develop a broader and more diverse phage arsenal. METHODS: Phage screening was performed on environmental samples collected at sites geographically distinct from the original isolation location via B. pseudomallei HNBP001, a Hainan representative strain previously employed as the isolation host. Candidates characterized by host range and plaque properties were subjected to assays assessing environmental stability, infection and proliferation traits. Genetic features were analyzed by sequencing and comparative bioinformatics, followed by efficacy evaluation in A549 cell infection assays and Galleria mellonella larvae. Statistical analyses included one-way ANOVA and Kaplan-Meier survival curves with log-rank tests. RESULTS: A total of four podoviruses (vB_BpP_HN02 - HN05), with highly efficient (≥ 92%) and complementary lytic activities, were recovered from natural sources in Wenchang and Qiongzhong. Each exhibited distinct plaque morphology and robust viability, although compromised by exposure to high temperatures (≥ 60 °C) or extremely acidic conditions (pH ≤ 3). Highest titer production was demonstrated in vB_BpP_HN02 and vB_BpP_HN05 (2.5 × 10¹⁰ and 1.0 × 10¹¹ PFU/mL at optimal multiplicities of infection of 0.1 and 1, respectively), whilst complete bacterial clearance was achieved by the former. Genomic analysis classified them into the Schitoviridae family, sharing conserved N4-like architecture. Phage treatments restored the viability of infected A549 cells from 29.29% to 60.07-75.81%, with vB_BpP_HN02 displaying superior bactericidal capacity, even against the ceftazidime-resistant strain, further supported in the G. mellonella model, albeit modestly under single-dose administration. CONCLUSIONS: As a continuation of our previous work, the identification of these phages addresses the natural occurrence of vB_BpP_HN01-insensitive strains and enhances understanding of B. pseudomallei phage diversity in Hainan. However, owing to close genetic similarity, cocktail formulation remains unfeasible, warranting further efforts to isolate more diverse novel phages.