oklahomensis strains. To show that live bacteria are needed for killing of G. mellonella, B. thailandensis CDC272 or CDC301 were inactivated by heating to 80°C for 1 hour and then injected into G. mellonella larvae at the same concentration as live bacteria. After 24 hrs, all larvae infected with heat killed bacteria were still alive, whereas those infected with live bacteria had all died (data not shown). Figure 4 Virulence and intracellular survival of Burkholderia strains in Galleria mellonella larvae. Groups of 10 insect larvae were challenged with 100 cfu of different strains of Burkholderia as described in the method section. A) Percentage of surviving selleck kinase inhibitor larvae at 24 hrs post infection.
B) Number of bacteria present inside the haemocoel at 20 hrs post infection (calculated as cfu/ml). In both panels, results are shown as means and standard error of the mean of three independent experiments. B. pseudomallei = black bars; B. thailandensis = white bars and B. oklahomensis strains = grey bars. ND = not detected. At higher challenge doses
of 10,000 cfu bacteria, all of the strains caused 100% mortality of the cohort of larvae at 24 hrs post injection, except B. pseudomallei 708a, B. thailandensis DW503 and B. oklahomensis E0147. At lower inocula of 10 cfu bacteria, Etomoxir order all of the B. pseudomallei strains were able to kill G. mellonella by 72 hrs post challenge, but no dead larvae were recorded up to 5 days after challenge with B. thailandensis or B. oklahomensis. Discussion In this study, we set out to identify inexpensive alternative infection models that would reflect the virulence of B. pseudomallei, B. thailandensis or B. oklahomensis in mice and the association of these isolates with human disease. We have chosen B. pseudomallei isolates with different degree of virulence in mice, with strain 576 representing one of the most virulent isolates Amylase tested to date, and 708a one of the least . B. thailandensis and B. oklahomensis are not normally
considered to be human pathogens. However, occasional cases of disease do occur. We have included clinical isolates of B. thailandensis in our study alongside B. thailandensis isolates that have not been associated with disease (E264 and Phuket), as well as clinical isolates of B. oklahomensis. In general, our results confirm that cell culture or Galleria infection models can be used to discriminate B. pseudomallei, B. thailandensis and B. oklahomensis isolates and these results parallel those found in mice. With the exception of strain 708a and compared with B. thailandensis and B. oklahomensis isolates, the B. pseudomallei isolates we tested grew more rapidly in macrophages, caused a greater degree of cellular damage and caused greater mortality of G. mellonella larvae. The B. oklahomensis isolates we tested were the least virulent in all of these models. Our finding that we are able to distinguish between B. pseudomallei and B. thailandensis isolates on the basis of their virulence in G.