Abstract:Pathogen evolution and subsequent phenotypic heterogeneity during chronic infection are proposed to enhance Staphylococcus aureus survival during human infection. We tested this theory by genetically and phenotypically characterizing strains with mutations constructed in the mismatch repair (MMR) and oxidized guanine (GO) system, termed mutators, which exhibit increased spontaneous-mutation frequencies. Analysis of these mutators revealed not only strain-dependent increases in the spontaneous-mutation frequenc… Show more
“…Their emergence is an eventual consequence of the genetic structure of asexuals, which allows mutator alleles to hitchhike with beneficial mutations occurring in the same genome [16,17]. Mutators pose a serious concern in clinical infections because they can readily evolve further adaptations, such as those promoting evasion of the immune system [18], increasing resistance to antibiotics [19] or alleviating the resistance fitness cost [20]. Different mutators, however, display a characteristic tendency to elevate only some types of transitions, transversions or frameshifts.…”
Genetic constraints can block many mutational pathways to optimal genotypes in real fitness landscapes, yet the extent to which this can limit evolution remains to be determined. Interestingly, mutator bacteria elevate only specific types of mutations, and therefore could be very sensitive to genetic constraints. Testing this possibility is not only clinically relevant, but can also inform about the general impact of genetic constraints in adaptation. Here, we evolved 576 populations of two mutator and one wild-type Escherichia coli to doubling concentrations of the antibiotic cefotaxime. All strains carried TEM-1, a b-lactamase enzyme well known by its low availability of mutational pathways. Crucially, one of the mutators does not elevate any of the relevant first-step mutations known to improve cefatoximase activity. Despite this, both mutators displayed a similar ability to evolve more than 1000-fold resistance. Initial adaptation proceeded in parallel through general multi-drug resistance mechanisms. High-level resistance, in contrast, was achieved through divergent paths; with the a priori inferior mutator exploiting alternative mutational pathways in PBP3, the target of the antibiotic. These results have implications for mutator management in clinical infections and, more generally, illustrate that limits to natural selection in real organisms are alleviated by the existence of multiple loci contributing to fitness.
“…Their emergence is an eventual consequence of the genetic structure of asexuals, which allows mutator alleles to hitchhike with beneficial mutations occurring in the same genome [16,17]. Mutators pose a serious concern in clinical infections because they can readily evolve further adaptations, such as those promoting evasion of the immune system [18], increasing resistance to antibiotics [19] or alleviating the resistance fitness cost [20]. Different mutators, however, display a characteristic tendency to elevate only some types of transitions, transversions or frameshifts.…”
Genetic constraints can block many mutational pathways to optimal genotypes in real fitness landscapes, yet the extent to which this can limit evolution remains to be determined. Interestingly, mutator bacteria elevate only specific types of mutations, and therefore could be very sensitive to genetic constraints. Testing this possibility is not only clinically relevant, but can also inform about the general impact of genetic constraints in adaptation. Here, we evolved 576 populations of two mutator and one wild-type Escherichia coli to doubling concentrations of the antibiotic cefotaxime. All strains carried TEM-1, a b-lactamase enzyme well known by its low availability of mutational pathways. Crucially, one of the mutators does not elevate any of the relevant first-step mutations known to improve cefatoximase activity. Despite this, both mutators displayed a similar ability to evolve more than 1000-fold resistance. Initial adaptation proceeded in parallel through general multi-drug resistance mechanisms. High-level resistance, in contrast, was achieved through divergent paths; with the a priori inferior mutator exploiting alternative mutational pathways in PBP3, the target of the antibiotic. These results have implications for mutator management in clinical infections and, more generally, illustrate that limits to natural selection in real organisms are alleviated by the existence of multiple loci contributing to fitness.
“…Furthermore, growth of S. aureus in CF lungs is associated with hypermutability (22,266), in vitro exposure of hypermutators to gentamicin resulted in an increased emergence of SCVs (268), and antibacterials other than gentamicin have not been studied. S. aureus hypermutators were found to be more virulent (27). Thus, SCV colonial morphotype, heteroresistance, and hypermutability serve as catalysts for persistent S. aureus infections in CF patients.…”
Section: Emergence Of Resistance In Pk/pd Studies and In Cf Patientsmentioning
confidence: 98%
“…Such data are considered to provide important information about the risk of resistance emerging in a treated patient. However, pathoadaptive processes favor the emergence of bacteria displaying a hypermutator phenotype (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27), exhibiting significantly increased rates of mutation, or a small-colony morphotype (30)(31)(32)(33)(34), facilitating recurrent and persistent infections. Both adaptive responses confer antibiotic resistance, which, however, is not analyzed by routine procedures.…”
Section: Emergence Of Resistance In Pk/pd Studies and In Cf Patientsmentioning
confidence: 99%
“…In addition to the poor penetration of antibacterials into the focus of infection, antibacterial therapy of CF patients is aggravated by several factors, such as biofilm formation, which impairs drug penetration and protects the pathogens from immune defense (7)(8)(9)(10)(11)(12)(13)(14); the development of hypermutator strains, characterized by high mutation rates, which result in pronounced resistance against antimicrobials and pathoadaptation ( [15][16][17][18][19][20][21][22][23][24][25][26][27]; drug adsorption to sputum components (28,29), which limits antibiotic activities; and slow growth as small-colony morphotypes (30)(31)(32)(33)(34), which facilitates recurrent and persistent infections and reduces the antibacterial activities of several antimicrobials (35,36). The result of the multiplicity of factors impairing the activities of antibacterials is that the pathogen cannot be eliminated, and infection persists in a chronic state.…”
SUMMARY
Bacteria adapt to growth in lungs of patients with cystic fibrosis (CF) by selection of heterogeneously resistant variants that are not detected by conventional susceptibility testing but are selected for rapidly during antibacterial treatment. Therefore, total bacterial counts and antibiotic susceptibilities are misleading indicators of infection and are not helpful as guides for therapy decisions or efficacy endpoints. High drug concentrations delivered by aerosol may maximize efficacy, as decreased drug susceptibilities of the pathogens are compensated for by high target site concentrations. However, reductions of the bacterial load in sputum and improvements in lung function were within the same ranges following aerosolized and conventional therapies. Furthermore, the use of conventional pharmacokinetic/pharmacodynamic (PK/PD) surrogates correlating pharmacokinetics in serum with clinical cure and presumed or proven eradication of the pathogen as a basis for PK/PD investigations in CF patients is irrelevant, as minimization of systemic exposure is one of the main objectives of aerosolized therapy; in addition, bacterial pathogens cannot be eradicated, and chronic infection cannot be cured. Consequently, conventional PK/PD surrogates are not applicable to CF patients. It is nonetheless obvious that systemic exposure of patients, with all its sequelae, is minimized and that the burden of oral treatment for CF patients suffering from chronic infections is reduced.
“…The mutation frequency was determined by plating an overnight bacterial culture on LB agar containing 100 mg of rifampin/liter (36,37). The mutation frequency was defined as the proportion of bacterial cells that grew on drug containing agar divided by the total population.…”
e Development of spontaneous mutations in Pseudomonas aeruginosa has been associated with antibiotic failure, leading to high rates of morbidity and mortality. Our objective was to evaluate the pharmacodynamics of polymyxin B combinations against rapidly evolving P. aeruginosa mutator strains and to characterize the time course of bacterial killing and resistance via mechanism-based mathematical models. Polymyxin B or doripenem alone and in combination were evaluated against six P. aeruginosa strains: wild-type PAO1, mismatch repair (MMR)-deficient (mutS and mutL) strains, and 7,8-dihydro-8-oxo-deoxyguanosine system (GO) base excision repair (BER)-deficient (mutM, mutT, and mutY) strains over 48 h. Pharmacodynamic modeling was performed using S-ADAPT and facilitated by SADAPT-TRAN. Mutator strains displayed higher mutation frequencies than the wild type (>600-fold). Exposure to monotherapy was followed by regrowth, even at high polymyxin B concentrations of up to 16 mg/liter. Polymyxin B and doripenem combinations displayed enhanced killing activity against all strains where complete eradication was achieved for polymyxin B concentrations of >4 mg/liter and doripenem concentrations of 8 mg/liter. Modeling suggested that the proportion of preexisting polymyxin B-resistant subpopulations influenced the pharmacodynamic profiles for each strain uniquely (fraction of resistance values are ؊8.81 log 10 for the wild type, ؊4.71 for the mutS mutant, and ؊7.40 log 10 for the mutM mutant). Our findings provide insight into the optimization of polymyxin B and doripenem combinations against P. aeruginosa mutator strains.
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