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Gram-negative bacteria belonging to the genus Burkholderia are remarkably resistant to broad-spectrum, cationic, antimicrobial peptides (AMPs). It has been proposed that this innate resistance is related to changes in the outer membrane lipopolysaccharide (OM LPS), including the constitutive, essential modification of outer membrane Lipid A phosphate groups with cationic 4-amino-4-deoxy-arabinose. This modification reduces the overall negative charge on the OM LPS which may change the OM structure and reduce the binding, accumulation, and permeation of cationic AMPs. Similarly, the Gram-negative pathogen Pseudomonas aeruginosa can quickly become resistant to many AMPs by multiple mechanisms, frequently, including activation of the arn operon, which leads, transiently, to the same modification of Lipid A. We recently discovered a set of synthetically evolved AMPs that do not invoke any resistance in P. aeruginosa over multiple passages and thus are apparently not inhibited by aminorabinosylation of Lipid A in P. aeruginosa. Here we test these resistance-avoiding peptides, within a set of 18 potent AMPs, against Burkholderia thailandensis. We find that none of the AMPs tested have measurable activity against B. thailandensis. Some were inactive at concentrations as high as 150 μM, despite all having sterilizing activity at ≤ 10 μM against a panel of common, human bacterial pathogens, including P. aeruginosa. We speculate that the constitutive modification of Lipid A in members of the Burkholderia genus is only part of a broader set of modifications that change the architecture of the OM to provide such remarkable levels of resistance to cationic AMPs., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Gram-negative bacteria secrete outer membrane vesicles (OMVs) that play critical roles in intraspecies, interspecies, and bacteria-environment interactions. Some OMVs, such as those produced by Pseudomonas aeruginosa, have previously been shown to possess antimicrobial activity against competitor species. In the current study, we demonstrate that OMVs from Burkholderia thailandensis inhibit the growth of drug-sensitive and drug-resistant bacteria and fungi. We show that a number of antimicrobial compounds, including peptidoglycan hydrolases, 4-hydroxy-3-methyl-2-(2-non-enyl)-quinoline (HMNQ) and long-chain rhamnolipid are present in or tightly associate with B. thailandensis OMVs. Furthermore, we demonstrate that HMNQ and rhamnolipid possess antimicrobial and antibiofilm properties against methicillin-resistant Staphylococcus aureus (MRSA). These findings indicate that B. thailandensis secretes antimicrobial OMVs that may impart a survival advantage by eliminating competition. In addition, bacterial OMVs may represent an untapped resource of novel therapeutics effective against bio-film-forming and multidrug-resistant organisms.

Burkholderia pseudomallei is the causative agent of melioidosis, an emerging bacterial disease that accounts for high rates of septicaemia and death in parts of Southeast Asia and Northern Australia. The closely related species Burkholderia thailandensis is considered avirulent in humans and has been used as a surrogate for B. pseudomallei in several studies. The pathogenesis of B. pseudomallei and the role of Toll-like receptors (TLRs) in host immunity to infection are not well-defined. In this study, we exposed four strains of inbred mice (BALB/c, C57BL/6, TLR4-deficient C3H/HeJ, and TLR4-competent C3H/HeN) to increasing doses of aerosolized B. thailandensis to determine strain susceptibility and the role of TLR4 during pulmonary infection. Our results indicate an increased susceptibility in the C57BL/6 and BALB/c strains, who displayed lethality, bacterial burden in organs, and pulmonary and systemic inflammation. C3H/HeJ were as resistant as C3H/HeN mice to B. thailandensis at the highest challenge dose examined, but TLR4-deficient animals exhibited a modest increase in chronic pulmonary inflammation. These results demonstrate that B. thailandensis can be used as a surrogate for experimental laboratory investigation of melioidosis in small animal models and that TLR4 may not play a prominent role during acute pneumonic melioidosis.

Several candidates for a vaccine against Burkholderia pseudomallei, the causal bacterium of melioidosis, have been developed, and a rational approach is now needed to select and advance candidates for testing in relevant nonhuman primate models and in human clinical trials. Development of such a vaccine was the topic of a meeting in the United Kingdom in March 2014 attended by international candidate vaccine developers, researchers, and government health officials. The focus of the meeting was advancement of vaccines for prevention of natural infection, rather than for protection from the organism's known potential for use as a biological weapon. A direct comparison of candidate vaccines in well-characterized mouse models was proposed. Knowledge gaps requiring further research were identified. Recommendations were made to accelerate the development of an effective vaccine against melioidosis. [ABSTRACT FROM AUTHOR]