An important mechanism for generation of new genes is by duplication-divergence of existing genes. Duplicationdivergence includes several different submodels, such as subfunctionalization where after accumulation of neutral mutations the original function is distributed between two partially functional and complementary genes, and neofunctionalization where a new function evolves in one of the duplicated copies while the old function is maintained in another copy. The likelihood of these mechanisms depends on the longevity of the duplicated state, which in turn depends on the fitness cost and genetic stability of the duplications. Here, we determined the fitness cost and stability of defined gene duplications/amplifications on a low copy number plasmid. Our experimental results show that the costs of carrying extra gene copies are substantial and that each additional kilo base pairs of DNA reduces fitness by approximately 0.15%. Furthermore, gene amplifications are highly unstable and rapidly segregate to lower copy numbers in absence of selection. Mathematical modeling shows that the fitness costs and instability strongly reduces the likelihood of both sub-and neofunctionalization, but that these effects can be offset by positive selection for novel beneficial functions.

Plasmid-encoded β-lactamases, including non-ESBL enzymes, have a strong influence on the frequency and resistance level of spontaneous carbapenem-resistant mutants. The fitness cost associated with the loss of OmpC/OmpF in E. coli most likely reduces the survivability of porin mutants and could explain why they have not emerged as a clinical problem in this species.

Many pathogenic intracellular bacteria manipulate the host phago-endosomal system to establish and maintain a permissive niche. The fate and identity of these intracellular compartments is controlled by phosphoinositide lipids. By mechanisms that have remained undefined, a Francisella pathogenicity island-encoded secretion system allows phagosomal escape and replication of bacteria within host cell cytoplasm. Here we report the discovery that a substrate of this system, outside pathogenicity island A (OpiA), represents a family of wortmannin-resistant bacterial phosphatidylinositol (PI) 3-kinase enzymes with members found in a wide range of intracellular pathogens, including Rickettsia and Legionella spp. We show that OpiA acts on the Francisella-containing phagosome and promotes bacterial escape into the cytoplasm. Furthermore, we demonstrate that the phenotypic consequences of OpiA inactivation are mitigated by endosomal maturation arrest. Our findings suggest that Francisella, and likely other intracellular bacteria, override the finely tuned dynamics of phagosomal PI(3)P in order to promote intracellular survival and pathogenesis.

The combination of ESBL production and porin loss in E. coli can result in reduced susceptibility to ertapenem. Porin-deficient subpopulations frequently emerged in ESBL-producing E. coli during exposure to ertapenem at concentrations simulating human pharmacokinetics. Inappropriate use of ertapenem should be avoided to minimize the risk of selection of ESBL-producing bacteria with reduced susceptibility to carbapenems.

Our results show that antibiotic resistance evolution can occur via several parallel pathways and that new mechanisms may appear after the most common pathways (i.e. β-lactamases and loss of porins) have been eliminated. These findings suggest that strategies to target the most commonly observed resistance mechanisms might be hampered by the appearance of previously unknown parallel pathways to resistance.