A novel lytic phage potentially effective for phage therapy against Burkholderia pseudomallei in the tropics

Abstract

Background: Burkholderia pseudomallei is a tropical pathogen that causes melioidosis. Its intrinsic drug-resistance is a leading cause of treatment failure, and the few available antibiotics require prolonged use to be effective. This study aimed to assess the clinical potential of B. pseudomallei phages isolated from Hainan, China.

Methods: Burkholderia pseudomallei strain (HNBP001) was used as the isolation host, and phages were recovered from domestic environmental sources, which were submitted to the host range determination, lytic property assays, and stability tests. The best candidate was examined via the transmission electron microscope for classification. With its genome sequenced and analyzed, its protective efficacy against B. pseudomallei infection in A549 cells and Caenorhabditis elegans was evaluated, in which cell viability and survival rates were compared using the one-way ANOVA method and the log-rank test.

Results: A phage able to lyse 24/25 clinical isolates was recovered. It was classified in the Podoviridae family and was found to be amenable to propagation. Under the optimal multiplicity of infection (MOI) of 0.1, an eclipse period of around 20 min and a high titer (10¹² PFU/ml) produced within 1 h were demonstrated. This phage was found stabile at a wide range of temperatures (24, 37, 40, 50, and 60 °C) and pH values (3-12). After being designated as vB_BpP_HN01, it was fully sequenced, and the 71,398 bp linear genome, containing 93 open reading frames and a tRNA-Asn, displayed a low sequence similarity with known viruses. Additionally, protective effects of applications of vB_BpP_HN01 (MOI = 0.1 and MOI = 1) alone or in combination with antibiotics were found to improve viability of infected cells (70.6 ± 6.8%, 85.8 ± 5.7%, 91.9 ± 1.8%, and 96.8 ± 1.8%, respectively). A significantly reduced mortality (10%) and a decreased pathogen load were demonstrated in infected C. elegans following the addition of this phage.

Conclusions: As the first B. pseudomallei phage was isolated in Hainan, China, phage vB_BpP_HN01 was characterized by promising lytic property, stability, and efficiency of bacterial elimination during the in vitro/vivo experiments. Therefore, we can conclude that it is a potential alternative agent for combating melioidosis.

Keywords: Burkholderia pseudomallei; Melioidosis; Phage; Podovirus.

Fig. 1

Morphologies of the plaque and the virus particle. Transparent plaques were formed after B. pseudomallei strain HNBP001 was infected by phi9 (A). The corresponding transmission electron microscope image of a viral particle confirmed that phi9 belongs to the Podoviridae family (B)

Fig. 2

Determination of optimal MOI for phi9. When the MOI was adjusted to 0.1, the titer was highest at approximately 10¹² PFU/ml. (***P < 0.001, one-way ANOVA). MOI: Multiplicity of infection

Fig. 3

One-step growth curve of phi9 and the growth curves of phi9 infected and uninfected B. pseudomallei HNBP001. Through one-step growth curve of phi9, the burst was initiated 20 min post-infection, and the highest titer was 10¹² PFU/ml (A). In the presence of phi9, the viable B. pseudomallei HNBP001 were eliminated within 150 min (B). MOI: Multiplicity of infection; LB: Luria–Bertani

Fig. 4

Temperature and pH stability of phi9. After a 30 min incubation, there were no significant changes in phage survival within the temperature range of 24–60 °C (A) or at pH 3–12 (B). (*P < 0.05, ***P < 0.001, where no asterisks are shown no difference was detected, one-way ANOVA)

Fig. 5

The linear genome of vB_BpP_HN01. Parts of functional elements and ORFs were exhibited and labeled, with the redundant fragment for DNA packaging and tRNA-Asn indicated as blue and red, respectively. ORFs: Open reading frames

Fig. 6

Bioinformatic analyses of the genome of vB_BpP_HN01. According to the results generated by VipTree, the genome-wide similar viruses are those members that infect the Achromobacter species (A). Alignments based on the similarities of the terminases (B), the portal proteins (C), or the major capsid proteins (D) indicated that the homologs should be the Rhizobium phage, the Erwinia phage, or a Pseudomonas phage

Fig. 7

Protection efficacy of phage vB_BpP_HN01 on A549 cells. Asterisks indicate statistically significant differences between treated groups and blank control (*P < 0.05, **P < 0.01, ***P < 0.001, where no asterisks are shown no difference was detected, one-way ANOVA)

Fig. 8

Therapeutic efficacy of phage vB_BpP_HN01 against B. pseudomallei infection in C. elegans. Survival curves of the blank control (open circle), C. elegans infected by B. pseudomallei HNBP001 (open triangle), infected C. elegans treated with phage vB_BpP_HN01 (open diamond), and the C. elegans treated with phage alone (open square) were plotted using the Kaplan–Meier method, in which results represent the average of triplicates. Through the log-rank test, a statistically significant difference (P < 0.05) was demonstrated in the treatment groups (A). Through the fluorescent probe, the presence of B. pseudomallei in the infected C. elegans (B) is seen, particularly in the intestine, whilst this is considerably diminished following phage treatment (C). Bp: B. pseudomallei