Activation of the SOS response increases the frequency of small colony variants

BACKGROUND: In Staphylococcus aureus sub-populations of slow-growing cells forming small colony variants (SCVs) are associated with persistent and recurrent infections that are difficult to eradicate with antibiotic therapies. In SCVs that are resistant towards aminoglycosides, mutations have been identified in genes encoding components of the respiratory chain. Given the high frequencies of SCVs isolated clinically it is vital to understand the conditions that promote or select for SCVs. RESULTS: In this study we have examined how exposure to sub-inhibitory concentrations of antibiotics with different mechanism of action influence the formation of SCVs that are resistant to otherwise lethal concentrations of the aminoglycoside, gentamicin. We found that exposure of S. aureus to fluoroquinolones and mitomycin C increased the frequency of gentamicin resistant SCVs, while other antibiotic classes failed to do so. The higher proportion of SCVs in cultures exposed to fluoroquinolones and mitomycin C compared to un-exposed cultures correlate with an increased mutation rate monitored by rifampicin resistance and followed induction of the SOS DNA damage response. CONCLUSION: Our observations suggest that environmental stimuli, including antimicrobials that reduce replication fidelity, increase the formation of SCVs through activation of the SOS response and thereby potentially promote persistent infections that are difficult to treat

Novel pathways for ameliorating the fitness cost of gentamicin resistant small colony variants

Small colony variants (SCVs) of the human pathogen Staphylococcus aureus are associated with persistent infections. Phenotypically, SCVs are characterized by slow growth and they can arise upon interruption of the electron transport chain that consequently reduce membrane potential and thereby limit uptake of aminoglycosides (e.g. gentamicin). In this study, we have examined the pathways by which the fitness cost of SCVs can be ameliorated. Five gentamicin resistant SCVs derived from S. aureus JE2 were independently selected on agar plates supplemented with gentamicin. The SCVs carried mutations in the menaquinone and hemin biosynthesis pathways, which caused a significant reduction in exponential growth rates relative to wild type (0.59-0.72) and reduced membrane potentials. Fifty independent lineages of the low-fitness, resistant mutants were serially passaged for up to 500 generations with or without sub-lethal concentrations of gentamicin. Amelioration of the fitness cost followed three evolutionary trajectories and was dependent on the initial mutation type (point mutation vs. deletion) and the passage condition (absence or presence gentamicin). For SCVs evolved in the absence of gentamicin, 12 out of 15 lineages derived from SCVs with point mutations acquired intra-codonic suppressor mutations restoring membrane potential, growth rate, gentamicin susceptibility and colony size to wild type levels. For the SCVs carrying deletions, all lineages enhanced fitness independent of membrane potential restoration without alterations in gentamicin resistance levels. By whole genome sequencing, we identified compensatory mutations in genes related to the σB stress response (7 out of 10 lineages). Inactivation of rpoF that encode for the alternative sigma factor SigB (σB) partially restored fitness of SCVs. For all lineages passaged in the presence of gentamicin, fitness compensation via membrane potential restoration was suppressed, however selected for secondary mutations in fusA and SAUSA300_0749. This study is the first to describe fitness compensatory events in SCVs with deletion mutations and adaptation of SCVs to continued exposure to gentamici

The evolution of antimicrobial peptide resistance in Pseudomonas aeruginosa is shaped by strong epistatic interactions

AbstractColistin is an antimicrobial peptide that has become the only remaining alternative for the treatment of multidrug-resistant Gram-negative bacterial infections, but little is known of how clinical levels of colistin resistance evolve. We use in vitro experimental evolution and whole-genome sequencing of colistin-resistant Pseudomonas aeruginosa isolates from cystic fibrosis patients to reconstruct the molecular evolutionary pathways open for high-level colistin resistance. We show that the evolution of resistance is a complex, multistep process that requires mutation in at least five independent loci that synergistically create the phenotype. Strong intergenic epistasis limits the number of possible evolutionary pathways to resistance. Mutations in transcriptional regulators are essential for resistance evolution and function as nodes that potentiate further evolution towards higher resistance by functionalizing and increasing the effect of the other mutations. These results add to our understanding of clinical antimicrobial peptide resistance and the prediction of resistance evolution.</jats:p

Mechanisms of adaptation to the fitness cost of antibiotic resistance

The dissemination and persistence of antibiotic resistance, is not only depending on the volume of drugs used but also on the resistance mechanisms effect of bacterial fitness (reproductive ability). Fitness is a multifactorial parameter that is comprised of the relative growth rate of the resistant pathogen in the host and in the environment, as well as the clearance and transmission rates compared to susceptible strains. The focus of this thesis has been to determine (i) the connection between the mechanisms of resistance and their effect on growth in vitro and in a host-model, and (ii) how different adaptive mechanisms can partly or fully reverse the deleterious effects of resistance mutations. More specifically the fitness effect of mutational resistance towards the translational inhibitors mupirocin and streptomycin, targeting the isoleucyl–tRNA synthetase (IleRS) and the ribosomal 30S subunit, respectively were investigated using Salmonella typhimurium as a model organism. These studies showed that a fitness cost was associated with the resistance mutations and that suppression of their deleterious effect could be achieved by both intragenic and extragenic compensatory events. Three compensatory mechanisms that could restore fitness were identified, (i) intragenic mutations in the target protein (IleRS), (ii) extragenic (i.e. outside the target protein) mutations in the ribosomal proteins of the 30S and 50S subunits and (iii) extragenic compensatory events increasing the expression of the target protein (IleRS). The mechanism behind the resistant and compensatory mutations effect on respective target protein could be determined by in vivo and in vitro kinetical measurements of ribosomal translation (rate and accuracy) and the IleRS aminoacylation activity. The impact on activity for the resistant and compensatory mutations was shown to correlate with their effect on growth rate. However, since the fitness impact of the resistance mutations has been seen to vary between different in vitro conditions and between in vitro and in vivo conditions, we investigated and validated the nematode Caenorhabditis elegans (C. elegans) as an in vivo model for determining the fitness effects of resistance against several classes of antibiotics (including mupirocin and streptomycin). The fitness impact of the resistance mutations measured in C. elegans, correlated well with what had been detected in the mouse model of typhoid fever. It is worth noting that for all resistant strains, relative fitness in the two hostmodels was lower compared to fitness measured in the Luria Bertani broth laboratory medium. In conclusion, we have shown how resistance and compensatory mechanisms can at the protein and cellular level affect the stability of resistance in different in vitro and in vivo models

Caenorhabditis elegans as a Model To Determine Fitness of Antibiotic-Resistant Salmonella enterica Serovar Typhimurium

We used the ability of Salmonella enterica serovar Typhimurium to colonize the gut of Caenorhabditis elegans to measure the fitness costs imposed by antibiotic resistance mutations. The fitness costs determined in the nematode were similar to those measured in mice, validating its use as a simple host model to evaluate bacterial fitness

The agr quorum sensing system in Staphylococcus aureus cells mediates death of sub-population

Abstract Objective In the human pathogen, Staphylococcus aureus, the agr quorum sensing system controls expression of a multitude of virulence factors and yet, agr negative cells frequently arise both in the laboratory and in some infections. The aim of this study was to examine the possible reasons behind this phenomenon. Results We examined viability of wild type and agr mutant cell cultures using a live-dead stain and observed that in stationary phase, 3% of the wild type population became non-viable whereas for agr mutant cells non-viable cells were barely detectable. The effect appears to be mediated by RNAIII, the effector molecule of agr, as ectopic overexpression of RNAIII resulted in 60% of the population becoming non-viable. This effect was not due to toxicity from delta toxin that is encoded by the hld gene located within RNAIII as hld overexpression did not cause cell death. Importantly, lysed S. aureus cells promoted bacterial growth. Our data suggest that RNAIII mediated cell death of agr positive but not agr negative cells provides a selective advantage to the agr negative cell population and may contribute to the common appearance of agr negative cells in S. aureus populations