Prevalence of resistance-nodulation-cell division-type efflux pumps and their contribution to antimicrobial resistance in <em>Acinetobacter baumannii</em>

Abstract

Over the past three decades <em>A. baumannii</em> has evolved into a clinically important nosocomial pathogen causing epidemic outbreaks worldwide. Due to its intrinsic antimicrobial resistance and its ability to easily acquire new resistance determinants, <em>A. baumannii</em> can display resistance to all of today’s currently available antimicrobials, leaving limited treatment options. Of all the described antimicrobial resistance mechanisms, chromosomally encoded efflux pumps contribute most to multidrug resistance owing to their broad substrate specificity. In <em>A. baumannii</em> particularly, the overexpression of RND-type efflux pumps, extruding antimicrobials from the periplasm to the extracellular environment, plays a significant role in the appearance of MDR clones worldwide. <br /> The aim of this PhD project was to investigate and characterize RND-type efflux transporters in <em>A. baumannii</em>. <br /> Using BLAST analysis five putative RND pumps in addition to the three characterized systems AdeABC, AdeIJK and AdeFGH were identified. Testing the prevalence of these eight exporter-encoding genes within a genotypically and geographically diverse pool of clinical <em>A. baumannii</em> isolates using a PCR-based approach, five of eight RND-type efflux genes were present in all isolates. Two of them were present in ≥87% of isolates, highlighting the great distribution and relevance of these efflux pumps for this nosocomial pathogen.<br /> Further investigation of the putative exporter A1S_2660 (locus tag in <em>A. baumannii</em> ATCC 17978), low-level expression of this pump was induced by antimicrobials (carbapenems, chloramphenicol), disinfectants (ethanol) and NaCl, substances which are frequently used in the healthcare environment. Carbapenems are used as a last resort antibiotic against <em>A. baumannii</em> infections. Overexpression of this exporter was toxic in the <em>A. baumannii</em> reference strain ATCC 17978, but did not have a fitness cost in the AdeABC-deficient isolate NIPH 60. Testing a large collection of antimicrobials, bile salts, disinfectants and organic dyes, substrates of A1S_2660 could not be identified. <br /> The characterisation of amino acid substitutions in the two-component regulatory system AdeRS, which regulates the expression of the clinically most significant RND-type efflux pump AdeABC, are rarely investigated and remain merely associated to increased <em>adeB</em> expression and decreased antimicrobial susceptibility. Within this thesis, we report that the Asp→Asn20 substitution in the response regulator AdeR induced increased <em>adeB</em> expression levels, which was accompanied by decreased susceptibility to a multitude of structurally diverse antimicrobials. With the application of the ethidium accumulation assay enhanced efflux was determined to be the cause of the changed susceptibility phenotype. <br /> Being aware of the significant contribution of RND-type efflux pumps to antimicrobial resistance and understanding their structure, regulation and transport mechanism will help to find new treatment options against infections caused by MDR pathogens.