Universität zu Köln

Lucaßen, Kai (2022). Acinetobacter baumannii RND efflux pump regulators and their impact on antimicrobial susceptibility. PhD thesis, Universität zu Köln.

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Abstract

A. baumannii has been assigned as one of the most urgent threats among antimicrobial resistant bacterial organisms. The fundamental mechanism of action of antimicrobial agents is to bind to its bacterial target site and to interfere with cellular processes causing impaired bacterial cell growth or lead to bacterial cell death. A. baumannii is known for a broad spectrum of intrinsic and acquired resistance mechanisms, which are based on enzymatic inactivation of the drug, alterations in the structure of antimicrobial target sites preventing binding of antimicrobial agents. These mechanisms require specific adjustments of antimicrobial agents or targets. Thus, they are specific to single antimicrobials or antimicrobial classes. Contrary to this, mechanisms such as reduced permeability and increased efflux activity are able to diminish the intracellular concentration of antimicrobials and affect a large variety of agents. Among efflux systems, the RND family embodies the highest clinical relevance. These efflux pumps are associated with a broad spectrum of substrates, including antimicrobials as well as antiseptics and disinfectants commonly used in healthcare. Characterized RND efflux pumps in A. baumannii are AdeABC, AdeFGH, and AdeIJK, whose expression are controlled by the two-component systems AdeRS (AdeABC), the LysR-like transcriptional regulator AdeL (AdeFGH), or the TetR-like repressor AdeN (AdeIJK). Overexpression of AdeABC or AdeIJK has been shown to cause reduced antimicrobial susceptibility. During the present thesis the prevalence of regulatory mutations putatively causing RND overexpression in 113 carbapenem-resistant A. baumannii isolates obtained from a global collection was investigated. The most commonly and worldwide detected mechanism was the disruption of the AdeIJK repressor adeN, either by IS elements or nucleotide deletions causing premature stop codons. Furthermore, amino acid substitutions and disruption by IS elements within the two-component regulatory system adeRS, which regulates expression of the AdeABC efflux pump, were found. These mutations were detected less often but correlated with higher tigecycline MICs than mutations in adeN and adeL, which were assigned a minor role in reduced antimicrobial susceptibility. However, adeRS of A. baumannii ATCC 17978 was shown to have no impact on antimicrobial susceptibility in contrast to contemporary clinical isolates. Therefore, adeRS of A. baumannii ATCC 19606 and ATCC 17978 were compared and analyzed and an L172P amino acid substitution in AdeS was identified in A. baumannii ATCC 19606. Our studies revealed that the introduction of this amino acid substitution into AdeS of 17978 increased expression of adeB, increased efflux activity and reduced antimicrobial susceptibility. Residue 172 of AdeS is part of the DHp domain, which includes the phosphorylation residue H149 and might therefore affect the activity of the sensor kinase. This substantial difference between two reference strains commonly used in A. baumannii research demands to consider genetic plasticity and strain-dependent differences in studies analyzing fundamental bacterial mechanisms. Characterization of AdeRS was enhanced to investigate amino acid substitutions identified during the prevalence study. The AdeS amino acid substitution T156M was identified frequently, and the corresponding residue is positioned within the DHp domain in close proximity to the autophosphorylation site H149. An increased adeB expression, efflux activity, as well as reduced antimicrobial susceptibility was found to be induced by the AdeS T156M amino acid substitution. Additionally, the AdeR D21V and D26N double substitution, which was identified in the present and previous studies, was investigated. Although the amino acid substitution D21V has been detected in multiple isolates with high tigecycline MICs and is in close proximity to a magnesium binding pocket, which affects the regulators’ conformation, the contribution of this exchange to increased AdeABC efflux was disproven in the present study, since it did not cause increased efflux pump expression or reduced antimicrobial susceptibility. In contrast to these findings, the AdeR D26N mutation revealed a significant impact on AdeABC expression, efflux activity, and subsequently on antimicrobial susceptibility. Moreover, expression of RND efflux pumps and regulatory genes in different growth phenotypes and under the impact of various substrates was monitored. In this scope, motility was the only investigated growth condition in A. baumannii ATCC 17978 which inhibited expression of regulatory (adeRS), and RND efflux pump genes (adeABC, adeIJK) respectively. In addition, a correlation between the motile phenotype and increased antimicrobial susceptibility was observed. Summarizing, this thesis describes the worldwide distribution, the mechanism, and impact of RND regulatory mutations, as well as the effect of the mode of growth on gene expression and antimicrobial susceptibility in A. baumannii.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Lucaßen, Kai kai.lucassen@outlook.de UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-552813
Date:	2022
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Medicine > Medizinische Mikrobiologie, Immunologie und Hygiene > Institut für Medizinische Mikrobiologie, Immunologie und Hygiene
Subjects:	Life sciences Medical sciences Medicine
Uncontrolled Keywords:	Keywords Language Mikrobiologie UNSPECIFIED Antibiotika UNSPECIFIED Acinetobacter UNSPECIFIED
Date of oral exam:	28 January 2022
Referee:	Name Academic Title Seifert, Harald Prof. Schnetz, Karin Prof.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/55281