Layering vaccination with antibiotic therapy results in protection and clearance of Burkholderia pseudomallei in Balb/c mice.

Melioidosis is a disease that is difficult to treat due to the causative organism, Burkholderia pseudomallei being inherently antibiotic resistant and it having the ability to invade, survive, and replicate in an intracellular environment. Combination therapy approaches are routinely being evaluated in animal models with the aim of improving the level of protection and clearance of colonizing bacteria detected. In this study, a subunit vaccine layered with the antibiotic finafloxacin was evaluated in vivo against an inhalational infection with B. pseudomallei in Balb/c mice. Groups of mice vaccinated, infected, and euthanized at antibiotic initiation had a reduced bacterial load compared to those that had not been immunized. In addition, the subunit vaccine provided a synergistic effect when it was delivered with a CpG ODN and finafloxacin was initiated at 48 h post-challenge. Vaccination was also shown to improve the outcome, in a composite measure of survival and clearance. In summary, layering a subunit vaccine with the antibiotic finafloxacin is a promising therapeutic alternative for use in the treatment of B. pseudomallei infections.

Considerations in the design of animal infection pilot studies.

Ethical research with experimental systems (animals or humans) requires a rationale for the number of subjects to be included in a study. Standard methods for estimating sample size are not fit-for-purpose when the experimenter cannot predict the effect size/outcome with any certainty. These types of studies are often designated "pilot study"; however, there are few guidelines for sample size needed for a pilot study. Here we seek to address this issue. Concerning survival analysis it is noted that the experimenter can adjust the parameters of the experiment to improve the power. We propose that the experimenter needs to consider the "limit of interest" needed to represent an effect that the experimenter would be prepared to defend in terms of scientific or medical interest. Conventional power analysis is then used to estimate the n to deliver an alpha (false positive rate) of p < 0.2. This approach provides a balance that can inform a future study, demonstrate a strong effect or dismiss if no effect was observed. Where weight change or infection burden is considered, parametric analysis can be used. Here the main requirement for the pilot study is to establish a meaningful estimate of variability for subsequent power analysis. When considering the confidence intervals for standard deviations, it can be noted that a turning point is reached for n of four to six, beyond which we observe diminishing returns, suggesting that sample sizes should be greater than four. Finally, we discuss both the importance in statistical blocking and repeated measures in maximising the usefulness of the pilot study; and the importance of considering and outlining analysis techniques prior to performing the experiment. These findings are intended to be useful in the design of experiments in further prospective research.

Investigation of a combination therapy approach for the treatment of melioidosis.

The efficacy of finafloxacin as a component of a layered defense treatment regimen was determined in vitro and in vivo against an infection with Burkholderia pseudomallei. Doxycycline was down-selected from a panel of antibiotics evaluated in vitro and used in combination with finafloxacin in a Balb/c mouse model of inhalational melioidosis. When treatment was initiated at 24 h post-infection with B. pseudomallei, there were no differences in the level of protection offered by finafloxacin or doxycycline (as monotherapies) when compared to the combination therapy. There was evidence for improved bacterial control in the groups treated with finafloxacin (as monotherapies or in combination with doxycycline) when compared to mice treated with doxycycline. Survival comparisons of finafloxacin and doxycycline (as monotherapies) or in combination initiated at 36 h post-infection indicated that finafloxacin was superior to doxycycline. Doxycycline was also unable to control the levels of bacteria within tissues to the extent that doxycycline and finafloxacin used in combination or finafloxacin (as a sole therapy) could. In summary, finafloxacin is a promising therapy for use in the event of exposure to B. pseudomallei.