Analysis of resistance nodulation division efflux pumps in Acinetobacter baumannii

Strains from Canadian hospitals were classified as A. baumannii, A. pitti, or A. osocomialis and the most commonly expressed resistance nodulation division (RND) efflux pump was adeFGH. A collection of A. baumannii isolates from the environment showed decreased susceptibility to at least three antibiotics and expression of three RND efflux pumps (adeFGH, adeABC, adeIJK). From the clinical collection two blood isolates, AB030 and AB031, were chosen to evaluate the outcomes of three virulence models. Using Caenorhabditis elegans and Galleria mellonella a statistically significant difference was shown in AB030 the extremely drug resistant strain. Investigation of the physiological role of adeFGH pump revealed its expression may be more important to cell metabolism and independent of factors controlling expression of the other pumps. This was further demonstrated through examination of local regulator knockout (adeL) in ATCC17978 showing expression levels of adeFGH pump increased only 2-fold. Expression in sub-lethal concentrations of chloramphenicol or florfenicol showed >5-fold induction of adeFGH expression as well as adeABC, independently of AdeL. These data show that the AdeFGH pump is likely regulated by AdeL, but more so by another unknown regulatory mechanism that responds to aberrant peptide formation and not DNA synthesis error or nitrosative stress. In addition strain specific differences likely also account for differences in phenotypic observations since nitrosative stress has improved tolerance and induced adeFGH expression in ATCC19606 and AB031. Finally analysis of efflux pumps in two triclosan resistant mutants, AB042 and AB043, revealed overexpression of AdeIJK and mutation in the adeIJK regulator, AdeN. Complementation repressed adeIJK expression levels and increased susceptibility to moxifloxacin and ciprofloxacin. The AB043 strain did not have any mutations in adeN, promoter region or RND operon. Osmotic stress tolerance revealed more C16 fatty acids and less C18 compared to the parent strain. The more triclosan resistant strain AB043, showed biofilm formation exclusively at 30??C. Twitching was observed and caused by mutation in the histone-like nucleoid structuring (HNS) protein but did not affect biofilm formation. Using RNA-seq and proteomic analysis putative targets of AdeIJK regulation were identified