Acinetobacter baumannii is one of the most challenging nosocomial pathogens, whose infections are often associated with epidemic spread of multi- and extensively-drug resistant (M/XDR) strains. A. baumannii resistant to carbapenems and colistin are an emergent threat as they limit therapeutic alternatives threatening patient care and public health. We investigated the role of efflux pumps (EPs) and porins in A. baumannii Portuguese clinical isolates from hospital environment.
Drug susceptibility was assessed by disc diffusion. Isolates were screened for the presence of β-lactamases, metallo-β-lactamases, carbapenemases, ISAba1 and mutations in genes associated colistin resistance by PCR and DNA sequencing. The existence of active efflux was studied by checkerboard assays with efflux inhibitors (EIs), ethidium bromide real-time assays and analysis of mRNA transcriptional levels of selected efflux pump genes and porins in response to imipenem or colistin.
Of the 74 strains studied, 72 were MDR with additional resistance to carbapenems (XDR). Of these, eight evolved to TDR, presenting high-level resistance to colistin. The β-lactamases OXA-23 were detected in few strains and OXA-24 in the majority. OXA-51 were detected in all but ISAba1 was not detected upstream this oxacillinase excluding its contribution to carbapenem resistance. The results showed the existence of synergistic interactions between EIs, carbapenems, colistin and ethidium bromide extrusion. Efflux assays demonstrated a significantly increased efflux activity that can be inhibited in the presence of EIs, mainly thioridazine. The EPs adeB, adeJ, adeG, craA, amvA, abeS and abeM were overexpressed in response to carbapenems and colistin. An association between carbapenem resistance and expression of the porins ompA, carO or oprD was not found.
This study demonstrated the contribution of EPs to carbapenems and colistin resistance in M/XDR A. baumannii clinical strains where resistance is a combination between increased efflux activity, mutations in antibiotic target genes and β-lactamases production. Overexpression of EPs impacts the clinical outcome of A. baumannii infections and treatment should urgently consider alternative therapeutic combinations such as the use of EIs.