Drug efflux and lipid A modification by 4-L-aminoarabinose are key mechanisms of polymyxin B resistance in the sepsis pathogen Enterobacter bugandensis.

OBJECTIVES:A concern with the ESKAPE pathogen Enterobacter bugandensis and other species of the E. cloacae complex is the frequent appearance of multidrug resistance against last-resort antibiotics, such as polymyxins. METHODS:Here, we investigated the responses to polymyxin B (PMB) in two PMB-resistant E. bugandensis clinical isolates by global transcriptomics and deletion mutagenesis. RESULTS:In both isolates, the genes of the CrrAB-regulated operon, including crrC and kexD, displayed the highest levels of upregulation in response to PMB. ∆crrC and ∆kexD mutants became highly susceptible to PMB and lost the heteroresistant phenotype. Conversely, heterologous expression of CrrC and KexD proteins increased PMB resistance in a sensitive Enterobacter ludwigii clinical isolate and in the Escherichia coli K12 strain W3110. The efflux pump AcrABTolC, and the two component regulators PhoPQ and CrrAB, also contributed to PMB resistance and heteroresistance. Additionally, the lipid A modification with 4-L-aminoarabinose (L-Ara4N), mediated by the arnBCADTEF operon, was critical to determine PMB resistance. Biochemical experiments, supported by mass spectrometry and structural modeling, indicated that CrrC is an inner membrane protein that interacts with the membrane domain of the KexD pump. Similar interactions were modeled for AcrB and AcrD efflux pumps. CONCLUSION:Our results support a model whereby drug efflux potentiated by CrrC interaction with membrane domains of major efflux pumps combined with resistance to PMB entry by the L-Ara4N lipid A modification, under the control of PhoPQ and CrrAB, confers the bacterium high-level resistance and heteroresistance to PMB.