Sheida Heidarian: Heteroresistance in Bacteria –Prevalence, and Mechanisms

Abstract

Antibiotic heteroresistance (HR) is a phenotype characterized by small subpopulations of cells with elevated resistance present in a main susceptible bacterial population. HR often evades detection by routine antimicrobial susceptibility testing, potentially leading to treatment failure, and occurs across multiple bacterial species and antibiotic classes. Known mechanisms include (i) genetic alterations such as point mutations, insertions, and deletions, and (ii) increased resistance gene copy numbers through tandem gene amplifications, increased plasmid copy number and transposition of antibiotic resistance genes into high copy number plasmids or highly expressed loci. This thesis investigates HR prevalence in Gram-positive bacteria and the genetic mechanisms causing HR.

In Paper I, HR prevalence and mechanisms were studied in 40 clinical Staphylococcus aureus isolates from four European countries against six clinically relevant antibiotics. HR isolates were frequent against gentamicin (69.2%), oxacillin (27%), daptomycin (25.6%), and teicoplanin (15.4%), and absent for linezolid and vancomycin. Genomic analyses revealed that HR primarily arise from chromosomal mutations affecting various cellular pathways, contrasting with the dominant mechanisms of increased gene copies in Gram-negative bacteria.

In Paper II, 40 clinical Enterococcus faecalis isolates from the same four European countries as in paper I were analyzed and HR was observed for daptomycin (20%), gentamicin (13.2%), and tigecycline (35.9%), but absent for linezolid and vancomycin. Several different genetic mechanisms, including chromosomal mutations, deletions in regulatory region of an antibiotic resistance gene leading to overexpression, and transposon-mediated increased gene copy number were identified in HR isolates, highlighting parallels to HR in S. aureus.

In Paper III, we assessed the role of 19 recombination-associated genes in tandem amplification-driven HR in Escherichia coli. A clinical plasmid conferring tobramycin HR via tandem amplification of aac(3)-IId was introduced into 19 recombination-gene knockout strains. All constructed isolates exhibited HR, but deletions in recA and recB substantially reduced tandem amplifications, shifting the mechanism of the HR isolates towards increased plasmid copy number or mutations. Partial reductions in HR were observed in recC, recJ, ruvA, and ruvC mutants, while other deletions had minimal impact.

In conclusion, this thesis demonstrates the high prevalence of HR and advances the understanding of the genetic mechanisms of HR in Gram-positive and Gram-negative pathogens. The results underscore the need for improved HR detection methods and reveal the multitude of mechanisms that can generate HR, highlighting challenges in targeting HR-mediated antibiotic resistance.