The maintenance and evolution of antibiotic resistance genes in the absence of antibiotic selection : a thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology & Genetics at Massey University, Albany Campus, New Zealand
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2023
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Massey University
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Abstract
The rise of new antibiotic resistance in pathogenic bacteria combined with the stagnation of
drug development has led to a crisis in treating bacterial infection. An understanding of factors
that can influence the development and prevalence of antibiotic resistance in bacteria can help
combat resistance. Bacteria can acquire antibiotic resistance via two types of genetic changes—
antibiotic resistance mutations (ARMs) and acquisition of antibiotic resistance genes (ARGs).
In the presence of antibiotics, these genetic changes are beneficial to bacteria but in the absence
of antibiotic any fitness cost of the resistance genotype imposed on the bacteria is uncovered.
The fitness cost of a resistance genotype creates a fitness difference between resistant and
susceptible bacteria leading to purifying selection against resistant bacteria. As a result, the
fitness cost of a resistance genotype can play an important role in the maintenance of resistant
bacteria in a population, especially when the antibiotic selection is absent or weak. Previous
studies have focused on the degree and mechanistic basis of the fitness cost of ARMs and of
ARGs embedded in mobile genetic elements (MGEs), such as plasmids. Little is known about
the fitness cost of individual ARGs, let alone its mechanistic basis. Moreover, ARGs are often
associated with MGEs, which subject ARGs to frequent gene flow between bacteria. Because
of this movement between host strains, any variation in the fitness cost of an ARG between
different strains can influence its prevalence at the population level. Despite the potential
importance of this effect in determining the success of ARGs, direct measurements of host specific
fitness costs have been made for only a few distinct ARGs. Finally, compensatory
evolution can alleviate the fitness cost of resistance genotypes so that both immediate and longterm
costs of ARGs must be considered. In this thesis, I aim to investigate the fitness cost of
individual ARGs and test its evolutionary significance. In Chapter 2, I quantify the fitness
costs of six ARGs prevalent in published Escherichia coli genomes and determine the variation
in costs across twelve Escherichia strains. While on average the fitness cost of the six ARGs
is small, consistent with their high prevalence, the costs of most ARGs vary between hosts. I
show that this variation can be consequential, resulting in host-dependent evolutionary
dynamics of an ARG plasmid. In Chapter 3, I use whole genome sequencing and reverse
genetics to dissect the genetic basis of the compensatory evolution observed in Chapter 2. I
identify a mutation on a phage gene that can alleviate the fitness cost of a b-lactamase, and
moreover, I demonstrate that the host-dependent cost of the b-lactamase is due to the negative
interaction between the b-lactamase and the phage gene. Chapter 4 extends work on
measuring ARG costs and determining their effect on ARG maintenance to investigate the
influence of costs on the molecular evolution of an ARG. In Chapter 4, I examine if the host dependent
fitness cost of the b-lactamase can influence the accumulation of genetic variation
in that gene. Together, these chapters characterize the influence of the fitness cost of ARGs on
their maintenance and evolution and demonstrate that, even without antibiotic selection, other
selective forces continue to influence the persistence of antibiotic resistance genes in bacterial
populations.
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Figures 1.1 and 1.2 are reproduced under a Creative Commons Attribution 4.0 International (CC BY 4.0) license and Creative Commons Attribution-NonCommercial-NoDerivs 4.0 (CC BY-NC-ND) International licence respectively.
Keywords
Drug resistance in microorganisms, Pathogenic bacteria, Antibiotics