Determinants of extreme ss-lactam tolerance in the Burkholderia pseudomallei complex [PublishAheadOfPrint]

Slow-growing bacteria are insensitive to killing by antibiotics, a trait known as antibiotic tolerance. In this study, we characterized the genetic basis of an unusually robust ß-lactam (meropenem) tolerance seen in Burkholderia species. We identified tolerance genes under three different slow-growth conditions by extensive transposon mutant screening (Tn-seq) followed by single mutant validation. There were three principle findings. First, mutations in a small number of genes reduced tolerance under multiple conditions. Most of the functions appeared to be specific to peptidoglycan synthesis and the response to its disruption by meropenem action rather than associated with more general physiological processes. Top tolerance genes are involved in immunity toward a Type VI toxin targeting peptidoglycan (BTH_I0069), peptidoglycan recycling (ldcA), periplasmic regulation by proteolysis (prc), and an envelope stress response (rpoE, degS). Second, most of the tolerance functions did not contribute to growth in the presence of meropenem (intrinsic resistance), indicating that the two traits are largely distinct. Third, orthologues of many of the top B. thailandensis tolerance genes were also important in B. pseudomallei. Overall, these studies show that the determinants of meropenem tolerance differ considerably depending on cultivation conditions, but that there are a few shared functions that have strong mutant phenotypes and are important in multiple Burkholderia species.

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