Experimental Phage Therapy for Burkholderia pseudomallei Infection

Abstract

Burkholderia pseudomallei is an intracellular Gram-negative bacterial pathogen intrinsically resistant to a variety of antibiotics. Phages have been developed for use as an alternative treatment therapy, particularly for bacterial infections that do not respond to conventional antibiotics. In this study, we investigated the use of phages to treat cells infected with B. pseudomallei. Phage C34 isolated from seawater was purified and characterised on the basis of its host range and morphology using transmission electron microscopy (TEM). Phage C34 was able to lyse 39.5% of B. pseudomallei clinical strains. Due to the presence of contractile tail, phage C34 is classified as a member of the family Myoviridae, a tailed double-stranded DNA virus. When 2 × 105 A549 cells were exposed to 2 × 107 PFU of phage C34, 24 hours prior to infection with 2 × 106 CFU of B. pseudomallei, it was found that the survivability of the cells increased to 41.6 ± 6.8% as compared to 22.8 ± 6.0% in untreated control. Additionally, application of phage successfully rescued 33.3% of mice infected with B. pseudomallei and significantly reduced the bacterial load in the spleen of the phage-treated mice. These findings indicate that phage can be a potential antimicrobial agent for B. pseudomallei infections.

Fig 1. Transmission electron microscopy image of phage C34.

The phages show an icosahedral head with contractile tail, which is a typical morphology of the family Myoviridae. Magnification: x100000.

Fig 2. One step growth curve of phage C34.

The infection cycle of phage C34 propagating on B. pseudomallei strain CMS. E, eclipse period (30 minutes); L, latent period(40 minutes); B, burst size of the phage (234).

Fig 3. Bacterial count of B. pseudomallei over the course of 6 hours.

Control (●), B. pseudomallei infected by different MOIs (■) and the corresponding phage titre of C34 (▲).The graph shows averages for three independent assays.

Fig 4. Growth curve of B. pseudomallei strain CMS (wild type) and the phage-resistant isolates.

Six of the isolates appeared to grow slower than the parental strain. The graph shows averages for three independent assays. Error bars indicate the standard error of the averages.

Fig 5. Survivability of infected A549 cells.

The survivability of infected A549 cells which received pre-infection and pre + post-infection treatment was significantly higher than the control. Survivability of non-infected A549 cells was calculated as 100% and thus not shown in the figure. The results are the averages of three independent assays. Pre: Pre-infection treatment; HT: Pre-infection treatment using heat-killed phage; Post: Post-infection treatment; Pre+post: Pre-infection and Post-infection treatment.

Fig 6. Mortality of B. pseudomallei strain CMS-infected mice.

Mice which received treatment 24 hours before the infection (■) or 2 hours post-infection (▲). Control mice without any treatment (○) were 100% moribund at day 11 while 33% of the mice (n = 5) in both of the treated groups survived at the end of 14 days.

Fig 7. Bacterial burdens in lung, spleen, and liver of mice.

Control (○), mice treated with i.p. phage treatment (2 × 10⁸ PFU of phage C34), administered 24 hours before the infection (■) or 2 hours post-infection (▲). The numbers of viable bacteria (CFU) were enumerated from the organs of mice on day 1, 2 and 3 post-infection (A-C). On day 3, the bacterial burden of mice which received post-infection treatment was significantly lower than that of the control in spleen tissues only.

Fig 8. Recovery of phages from the mice tissues following i.p. administration of 2 × 10⁸ PFU phage C34.

Presence of phages were examined at 24 hours post-injection in the mock-infected mice (○), at 24 hours after the infection in pre-infection treated mice (■) and 24 after the infection in post- infection treated mice (▲). Phages were recovered from the lung, liver and spleen of the mice which received post-infection treatment, and from the spleen of the mock-infected mice.