Mutant Subpopulations Research Articles

A unified anti-mutant dosing strategy

Antimicrobial dosing is currently attracting attention as a way to minimize the emergence of resistance. Three dose-based strategies have been advocated, each with shortcomings. Focus on killing susceptible cells overlooks resistant mutant subpopulations that may be present before treatment or generated during therapy; keeping therapeutic drug concentrations above the mutant prevention concentration (MPC; resistant mutant MIC) may be overly stringent; and dosage escalation modelling uses indirect estimates of resistant mutant subpopulation susceptibility (multiples of bulk population susceptibility, MIC) rather than direct estimates from MPC. The latter is significant because MPC and MIC are discordant with multiple pathogen isolates. Combining the strategies leads to MPC-based PK/PD thresholds (e.g. AUC(24)/MPC and t > MPC) for restricting resistant subpopulation enrichment and amplification. Using MPC-based thresholds to model dosing regimens that will restrict emergence of resistance requires generation of databases in which MPC is determined for many isolates.

Efflux system overexpression and decreased OprD contribute to the carbapenem heterogeneity inPseudomonas aeruginosa

Pseudomonas aeruginosa strains exhibiting a heterogeneous mode of growth against carbapenems have been described recently. This study investigated the underlying molecular mechanisms in four genetically unrelated P. aeruginosa clinical isolates that were previously characterized by population analyses as heterogeneously resistant against carbapenems. Mutant subpopulations of all four isolates had at least fourfold higher minimum inhibitory concentrations than those of native cells for imipenem and meropenem. The heterogeneous subpopulations, when compared with the respective native ones, had significantly increased transcription levels of the mexB and mexY genes (P<0.05), whereas transcription levels of the mexE gene remained unchanged. They also exhibited significantly decreased expression of the oprD gene (P<0.05) and decreased intensity of the protein band of the porin OprD. Upregulation of efflux systems, in part, and the decrease of OprD contribute to the heterogeneous growth against carbapenems in our P. aeruginosa clinical isolates.

Dose-related selection of fluoroquinolone-resistant Escherichia coli

To investigate the effects of clinically used doses of norfloxacin, ciprofloxacin and moxifloxacin on survival and selection in Escherichia coli populations containing fluoroquinolone-resistant subpopulations and to measure the value of the pharmacodynamic index AUC/mutant prevention concentration (MPC) that prevents the growth of pre-existing resistant mutants. Mixed cultures of susceptible wild-type and isogenic single (gyrA S83L) or double (gyrA S83L, Delta marR) fluoroquinolone-resistant mutants were exposed to fluoroquinolones for 24 h in an in vitro kinetic model. Antibiotic concentrations modelled pharmacokinetics attained with clinical doses. All tested doses eradicated the susceptible wild-type strain. Norfloxacin 200 mg administered twice daily selected for both single and double mutants. Ciprofloxacin 250 mg administered twice daily eradicated the single mutant, but not the double mutant. For that, 750 mg administered twice daily was required. Moxifloxacin 400 mg once daily eliminated the single mutant, but did not completely remove the double mutant. The MPC of ciprofloxacin was determined and based on those dose simulations that eradicated mutant subpopulations, an AUC/MPC(wild-type) of 35 prevented selection of the single mutant, whereas an AUC/MPC(single mutant) of 14 (equivalent to an AUC/MPC(wild-type) of 105) prevented selection of the double mutant. All tested clinical dosing regimens were effective in eradicating susceptible bacteria, but ciprofloxacin 750 mg twice daily was the only dose that prevented the selection of single- and double-resistant E. coli mutants. Thus, among approved fluoroquinolone dosing regimens, some are significantly more effective than others in exceeding the mutant selection window and preventing the enrichment of resistant mutants.

Frequent Frameshift and Point Mutations in the SH Gene of Human Metapneumovirus Passaged In Vitro

During the preparation of recombinant derivatives of the CAN97-83 clinical isolate of human metapneumovirus (HMPV), consensus nucleotide sequencing of the recovered RNA genomes provided evidence of frequent sequence heterogeneity at a number of genome positions. This heterogeneity was suggestive of sizable subpopulations containing mutations. An analysis of molecularly cloned cDNAs confirmed the presence of mixed populations. The biologically derived virus on which the recombinant system is based also contained sizeable mutant subpopulations, whose presence was confirmed by biological cloning and nucleotide sequencing. Most of the mutations occurred in the SH gene. For example, partial consensus sequencing of 40 independent preparations of recombinant HMPV (wild-type and various derivatives) showed that 31 of these preparations contained a total of 41 instances of small insertions in the SH gene and a total of five small insertions elsewhere. In each of these 31 preparations, there was at least one insert in SH that changed the reading frame and would yield a truncated protein. Nearly all of these insertions involved adding one or more A residues to various tracks of four or more A residues, with the most frequent site being a tract of seven A residues. There were also two instances of nucleotide deletions and numerous instances of nucleotide substitution point mutations, mostly in the SH gene. The occurrence of mutant subpopulations was greatly reduced by the replacement of the SH gene with a synthetic version in which these oligonucleotide tracts were eliminated by silent nucleotide changes. We suggest that we frequently detected subpopulations in which the expression of full-length SH protein was ablated because it provided a modest selective advantage to this clinical isolate in vitro. Adaptation involving the functional loss of a gene is unusual for an RNA virus.

Pharmacokinetic–pharmacodynamic integration of danofloxacin after intravenous, intramuscular and subcutaneous administration to rabbits

The pharmacokinetics of danofloxacin was studied following intravenous (i.v.), intramuscular (i.m.) and subcutaneous (s.c.) administration of 6 mg/kg to healthy rabbits. Danofloxacin concentration were determined by high-performance liquid chromatography assay with fluorescence detection. Minimal inhibitory concentrations (MICs) assay of danofloxacin against 30 strains of Staphylococcus aureus from several European countries was performed in order to compute pharmacodynamic surrogate markers. The danofloxacin plasma concentration versus time data after i.v. administration could best be described by a two-compartment open model. The disposition of i.m. and subcutaneously administered danofloxacin was best described by a one-compartment model. The terminal half-life for i.v., i.m. and s.c. routes was 4.88, 6.70 and 8.20 h, respectively. Clearance value after i.v. dosing was 0.76 L/kg.h. After i.m. administration, the absolute bioavailability was mean (+/-SD) 102.34 +/- 5.17% and the Cmax was 1.87 mg/L. After s.c. administration, the absolute bioavailability was mean (+/-SD) 96.44 +/- 5.95% and the Cmax was 1.79 mg/L. Danofloxacin shows a favourable pharmacokinetics profile in rabbits reflected by parameters such as a long half-life and a high bioavailability. However, in consideration of the low AUC/MIC indices obtained, its use by i.m. and s.c. route against the S. aureus strains assayed in this study cannot be recommended given the risk for selection of first mutant subpopulations.

The Mutant Selection Window in Rabbits Infected withStaphylococcus aureus

The mutant selection window hypothesis, originally based on agar plate assays, may lead to new antimicrobial dosing strategies that severely restrict the acquisition of resistance. However, it has not been directly tested in an animal model of infection. Local infection with Staphylococcus aureus was established in rabbits, and the infected animals were treated orally with various doses of levofloxacin. Changes in levofloxacin concentration, levofloxacin susceptibility, and counts of total and resistant viable bacteria were monitored at the site of infection. S. aureus lost levofloxacin susceptibility when drug concentrations at the site of infection fluctuated between the lower and upper boundaries of the window, defined in vitro as the minimum inhibitory concentration (MIC)(99) and the mutant prevention concentration (MPC), respectively. The upper boundary of the selection window in vivo was estimated as an AUC(24)/MPC value of ~25 h, where AUC(24) is the area under the drug concentration time curve in a 24-h interval. The lower boundary was estimated as an AUC(24)/MIC value of ~20 h. The mutant selection window exists in vivo, and its boundaries fit well with those determined in vitro. Maintenance of antimicrobial concentrations above the window is expected to suppress the outgrowth of resistant mutant subpopulations.

Mutant prevention concentration for ciprofloxacin and levofloxacin with Pseudomonas aeruginosa

The mutant prevention concentration (MPC) was determined by population analysis using six fluoroquinolone-susceptible isolates of Pseudomonas aeruginosa applied to fluoroquinolone-containing agar plates. The MPCs were 3 mg/L and 9.5 mg/L for ciprofloxacin and levofloxacin, respectively. At high concentrations of either compound, single-step gyrA resistance mutants were recovered. Using a modified method for estimating the MPC, 151 clinical isolates were surveyed. Modal MPCs were 2 mg/L and 8 mg/L, respectively, for ciprofloxacin and levofloxacin. Thus, ciprofloxacin is three to four times more active than levofloxacin against resistant mutant subpopulations. For individual isolates, the MPC correlated poorly with the minimum inhibitory concentration ( r 2 = 0.41 and 0.39 for ciprofloxacin and levofloxacin, respectively).

Comparison of Two Nonradioactive, Single-Strand Conformation Polymorphism Electrophoretic Methods for Identification of rpoB Mutations in Rifampin- Resistant Isolates of Mycobacterium tuberculosis.

Background: Diverse mutations in an 81 bp region of the rpoB gene are found in approximately 95% of rifampin-resistant (RIFr) Mycobacterium tuberculosis isolates. Various methods to detect these mutations have been evaluated for their usefulness as rapid screens for rifampin resistance. Methods and Results: Two nonradioactive variations of single-strand conformation polymorphism (SSCP) electrophoresis were optimized and evaluated for their ability to distinguish nine rpoB mutations present in a collection of 51 RIFr M. tuberculosis isolates. One of the methods used polymerase chain reaction products (128 bp) encompassing the 81 bp region of the rpoB gene, which were denatured in the presence of methyl mercury hydroxide, subjected to polyacrylamide gel electrophoresis (PAGE) and detected by staining with ethidium bromide. For the second method, fluorogenically labeled primers were used to generate products that were electrophoresed in an ABI Model 310 Genetic Analyzer equipped with a 3% GeneScan Polymer Column (Applied Biosystems Inc; Foster City, CA). Mobility shifts for all nine mutations were clearly discernible from the wild-type pattern by methods when tested in blind analyses. When an additional 30 isolates were tested by both SSCP methods in a blinded fashion, correlations with RIF susceptibility testing were complete for susceptible and homogeneously resistant isolates. Among three isolates with heterogeneously resistant populations, however, two were correctly identified by fluorescent SSCP compared with one by the PAGE SSCP method. Subpopulations of the His 526-->Tyr rpoB mutant, which is frequently encountered among RIFr strains, could be detected using templates prepared from mixtures of broth cultures with a susceptible strain. Conclusions: SSCP electrophoresis is useful for rapid screening for RIF resistance in susceptible and fully resistant isolates of M. tuberculosis. However, conventional susceptibility testing is still necessary for two reasons: (1) <100% of RIF strains have mutations in the 81 bp hotspot rpoB genomic region, and (2) SSCP may not offer sufficient sensitivity to detect clinically important emergent mutant subpopulations, especially those present as <10% of the total population in a sample. Whereas PAGE SSCP is less costly than fluorescent SSCP, the latter method is somewhat easier to perform and generates quantitative data.

The Crisis of Resistance: Identifying Drug Exposures to Suppress Amplification of Resistant Mutant Subpopulations

Antibiotic resistance is seen in both the hospital and community settings. Approaches are required to minimize the increase in resistant strains, such as good antibiotic stewardship and the limiting of antibiotic use to appropriate circumstances. There are instances when drug dose and/or schedule can be used to minimize the probability that mutants will take over the bacterial population. Over the past several years, significant advances have been made in understanding the relationship between drug concentrations and amplification of resistant mutant subpopulations. In this review, we examine the use of preclinical models for facilitating this understanding. We also use mathematical techniques, including Monte Carlo simulation, to bridge between the identification of exposures to minimize resistance and the examination of candidate drug doses to achieve this end. Examples are provided for Pseudomonas aeruginosa, Streptococcus pneumoniae, Staphylococcus aureus, and Mycobacterium tuberculosis. In each instance, quinolone antimicrobials were examined. More investigations with other pathogens and drug classes are required.

O.170 Characterization and functional analyses of hepatitis B virus (HBV) isolated from liver tissue of patients with occult HBV infection

The present study was aimed to obtain baseline information of basal core promoter A1762T/G1764A and precore G1896A mutations of hepatitis B virus (HBV) in 192 HBeAg-positive chronically-infected Chinese patients, who were potential candidates for antiviral treatment. The detection of these mutations (including minor mutant subpopulations) was achieved by direct sequencing, whose sensitivity for minor mutant subpopulations identification was confirmed by clone sequencing. Patients enrolled were infected with either genotype B (46.35%) or C (53.65%) HBV identified by routine tests in our laboratory. The A1762T/G1764A or G1896A mutations were detected in 125 specimens (125/192, 65.10%), in which 77 (77/125, 61.60%) existed as subpopulations. The A1762T/G1764A mutations were found to be more prevalent in genotype C than that in genotype B HBV [62.14% (64/103) vs. 20.22% (18/89), P < 0.0001]. There is no statistically significant link between G1896A and genotypes. The emergence of A1762T/G1764A mutations was also found to be associated with an older age, an elevated ALT/AST level, and a lower HBsAg level in serum [wild-type vs. mutant: 4.57 (3.46–5.42) vs. 3.93 (2.51–5.36), P < 0.0001]. In conclusion, HBV basal core promoter mutations A1762T/G1764A are associated with genotype C and a low serum HBsAg level in chronically-infected HBeAg-positive Chinese patients.

Low Correlation between MIC and Mutant Prevention Concentration

Soon after the term mutant prevention concentration (MPC) was coined to define the MIC of the least susceptible mutant subpopulation of a microbial culture (2), we noticed that MPCs and MICs correlated poorly (r2 = 0.39) for a set of closely related fluoroquinolones when determined with Mycobacterium smegmatis (9). Subsequently, isolated examples were described in which correlation was low for a variety of fluoroquinolones with strains of Escherichia coli, Salmonella enterica, and Staphylococcus aureus (5, 8, 10), and a set of 20 clinical isolates of E. coli showed a low correlation (r2 = 0.58) for ciprofloxacin (6). To determine whether a low correlation between MICs and MPCs is likely to be a general phenomenon, we calculated the correlation coefficients for several quinolones with five bacterial species and for three macrolides with Streptococcus pneumoniae using data from published and unpublished studies of clinical isolates. As shown in Table ​Table1,1, r2,determined by linear regression, was below 0.5 for fluoroquinolones with E. coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, S. aureus, and S. pneumoniae (an exception was levofloxacin with K. pneumoniae [r2 = 0.7]). Values of r2 were slightly above 0.5 for three macrolides with S. pneumoniae (Table ​(Table11). TABLE 1. Relationship between MICs and MPCs Low correlations between MICs and MPCs with clinical isolates are likely to require a complex explanation. These isolates probably contain mutant subpopulations that vary considerably in relative abundance and drug susceptibility, which will contribute to a wide variation in MICs when the mutants are abundant enough to be scored. The isolates may also contain many different multistep mutants (1) which may or may not represent the least susceptible subpopulations that determine MPCs. Added complexity derives from some resistance mutations having a much larger effect on MPCs than on MICs (4). Indeed, isolates with the same MIC were found to have values of MPC that ranged over 5 twofold dilutions. A consequence of a low correlation between MICs and MPCs is that MPCs cannot be estimated accurately from MICs on an individual patient basis. Thus, using antimutant strategies for individual patients will require measurement of the MPC. Likewise, empirical estimates of antimutant activity that are keyed to MIC-based pharmacokinetic-pharmacodynamic indices, such as area under the concentration-time curve at 24 h/MIC, will tend to exhibit more patient-to-patient variability than indices using MPCs.

Evolutionary trajectories in rugged fitness landscapes

We consider the evolutionary trajectories traced out by an infinite population undergoingmutation–selection dynamics in static, uncorrelated random fitness landscapes. Startingfrom the population that consists of a single genotype, the most populated genotype jumpsfrom one local fitness maximum to another and eventually reaches the global maximum.We use a strong selection limit, which reduces the dynamics beyond the first time stepto the competition between independent mutant subpopulations, to study thedynamics of this model and of a simpler one-dimensional model which ignores thegeometry of the sequence space. We find that the fit genotypes that appear along atrajectory are a subset of suitably defined fitness records, and exploit severalresults from the record theory for non-identically distributed random variables.The genotypes that contribute to the trajectory are those records that are notbypassed by superior records arising further away from the initial population. Severalconjectures concerning the statistics of bypassing are extracted from numericalsimulations. In particular, for the one-dimensional model, we propose a simple relationbetween the bypassing probability and the dynamic exponent which describesthe scaling of the typical evolution time with genome size. The latter can bedetermined exactly in terms of the extremal properties of the fitness distribution.

Oculodentodigital Dysplasia-causing Connexin43 Mutants Are Non-functional and Exhibit Dominant Effects on Wild-type Connexin43

Oculodentodigital dysplasia, a rare condition displaying congenital craniofacial deformities and limb abnormalities, has been associated with over 20 known human connexin43 (Cx43) mutations. The localization of two of these mutants, G21R and G138R, was examined in Cx43-positive normal rat kidney cells (NRK) and Cx43-negative gap junctional intercellular communication-deficient HeLa cells. Green fluorescent protein-tagged and untagged Cx43 G21R and G138R mutants were transported to the plasma membrane and formed punctate structures reminiscent of gap junction plaques in both NRK and HeLa cells. Further localization studies revealed no significant trafficking defects as subpopulations of Cx43 mutants were found in both the Golgi apparatus and lysosomes, not unlike wild-type Cx43. Dual patch clamp functional analysis of the mutants expressed in gap junctional intercellular communication-deficient N2A cells revealed that neither G21R nor G138R formed functional gap junction channels, although they successfully reached cell-cell interfaces between cell pairs. Importantly, when either mutant was expressed in NRK cells, dye coupling experiments revealed that both mutants inhibited endogenous Cx43 function. These studies suggest that, although patients suffering from oculodentodigital dysplasia possess one wild-type Cx43 allele, it is likely that Cx43-mediated gap junctional intercellular communication is reduced below 50% because of a dominant-negative effect of mutant Cx43 on wild-type Cx43.

Superoxide is a mediator of an altruistic aging program in Saccharomyces cerevisiae.

Aging is believed to be a nonadaptive process that escapes the force of natural selection. Here, we challenge this dogma by showing that yeast laboratory strains and strains isolated from grapes undergo an age- and pH-dependent death with features of mammalian programmed cell death (apoptosis). After 90–99% of the population dies, a small mutant subpopulation uses the nutrients released by dead cells to grow. This adaptive regrowth is inversely correlated with protection against superoxide toxicity and life span and is associated with elevated age-dependent release of nutrients and increased mutation frequency. Computational simulations confirm that premature aging together with a relatively high mutation frequency can result in a major advantage in adaptation to changing environments. These results suggest that under conditions that model natural environments, yeast organisms undergo an altruistic and premature aging and death program, mediated in part by superoxide. The role of similar pathways in the regulation of longevity in organisms ranging from yeast to mice raises the possibility that mammals may also undergo programmed aging.

Is ‘dosing-to-cure’ appropriate in the face of antimicrobial resistance?

In vitro studies have defined antimicrobial concentration ranges within which resistant bacterial mutants are enriched. Each pathogen–antimicrobial combination has a characteristic range called the mutant selection window. Traditional antimicrobial dosing strategies, which are designed to cure disease, tend to place drug concentrations inside selection windows. Removal of resistant mutant subpopulations then relies on the action of host defences rather than antimicrobial treatment. Such strategies will lead increasingly to antimicrobial resistance because immunosuppression is becoming more widespread. Alternative approaches for closing the selection window and for keeping antimicrobial concentrations outside the window are discussed.

Normally functioning murF is essential for the optimal expression of methicillin resistance in Staphylococcus aureus.

A carboxy-terminal fragment of murF was used to construct and insert a suicide plasmid into the chromosomal copy of the gene in the highly and homogeneously methicillin-resistant Staphylococcus aureus (MRSA) strain COL by Campbell type integration. The plasmid insertion generated a mutant in which the MIC value for oxacillin was reduced from 400 microg/ml of the parental strain to 0.75 microg/ml in 90% of the cells of the mutant cultures that were heterogeneous: they contained subpopulations of bacteria with a frequency of 10(-3) that were capable of expressing resistance at nearly the parental level. The impact of the murF mutation on antibiotic resistance was selective for beta-lactam antibiotics: there was no change in the susceptibility of the mutant to D-cycloserine, fosfomycin, beta-D-chloro-alanine, moenomycin, bacitracin, or vancomycin. Analysis of the mutant peptidoglycan showed decrease in the percentage of oligomeric components in rough proportion to the accumulation of several abnormal muropeptide components, which were identified as structural variants of the disaccharide tripeptide monomer. An abnormal cell wall precursor identified as UDP MurNac tripeptide was also detected in the cytoplasmic pool of the mutant strain. A normal proportion of oligomers and a greatly reduced representation of the disaccharide tripeptide were demonstrated in the cell wall of the murF mutant's subpopulation that has retained the parental level of resistance. Northern analysis demonstrated a drastic reduction in the transcription rate of mecA in mutant F9 whereas mecA transcription increased in the subpopulation of bacteria that retained high-level resistance.

Effect of chloramphenicol, erythromycin, moxifloxacin, penicillin and tetracycline concentration on the recovery of resistant mutants of Mycobacterium smegmatis and Staphylococcus aureus.

The effect of antimicrobial concentration on colony-forming ability of resistant mutant subpopulations of Mycobacterium smegmatis and Staphylococcus aureus was measured for chloramphenicol, erythromycin, moxifloxacin, penicillin and tetracycline. The relationship between drug concentration and the recovery of mutant colonies was distinct for each bacterium-antimicrobial combination; however, in each case application of large numbers of cells to drug-containing agar plates revealed a progressive reduction in mutant recovery as antimicrobial concentration increased. The minimal concentration that allowed no mutant recovery from more than 10(10) input cells was measured to estimate the minimum inhibitory concentration (MIC) of the least susceptible, single-step mutant subpopulation, a parameter also called the mutant prevention concentration (MPC). These data expand the number of antimicrobial-bacterial combinations for which a mutant selection window can be measured.

The mutant selection window and antimicrobial resistance.

The mutant selection window is an antimicrobial concentration range extending from the minimal concentration required to block the growth of wild-type bacteria up to that required to inhibit the growth of the least susceptible, single-step mutant. The upper boundary is also called the mutant prevention concentration (MPC). Placing antimicrobial concentrations inside the window is expected to enrich resistant mutant subpopulations selectively, whereas placing concentrations above the window is expected to restrict selective enrichment. Since window dimensions are characteristic of each pathogen-antimicrobial combination, they can be linked with antimicrobial pharmacokinetics to rank compounds and dosing regimens in terms of their propensity to enrich mutant fractions of bacterial populations. For situations in which antimicrobial concentrations cannot be kept above the window, restricting the enrichment of mutants requires combination therapy.

Fluoroquinolones: action and resistance.

Fluoroquinolones trap gyrase and topoisomerase IV on DNA as ternary complexes that block the movement of replication forks and transcription complexes. Studies with resistant mutants indicate that during complex formation quinolones bind to a surface alpha-helix of the GyrA and ParC proteins. Lethal action is a distinct event that is proposed to arise from release of DNA breaks from the ternary complexes. Many bacterial pathogens are exhibiting resistance due to alterations in drug permeability, drug efflux, gyrase-protecting proteins, and target topoisomerases. When selection of resistant mutants is described in terms of fluoroquinolone concentration, a threshold (mutant prevention concentration, MPC) can be defined for restricting the development of resistance. MPC varies among fluoroquinolones and pathogens; when combined with pharmacokinetics, MPC can be used to identify compounds least likely to enrich mutant subpopulations. Use of suboptimal doses and compounds erodes the efficacy of the class as a whole because resistance to one quinolone reduces susceptibility to others and/or increases the frequency at which resistance develops. When using fluoroquinolones in combination therapy, the development of resistance may be minimized by optimizing regimens for pharmacokinetic overlap.

Bursts of sectors in expanding bacterial colonies as a possible model for tumor growth and metastases

All kinds of hypotheses have been proposed to explain the mechanism of tumorigenesis. Up until now, we have not had any generally acknowledged model that helps us understand the process. However, it is well accepted that cancer development and progression is dictated by a series of alterations in genes such as oncogenes, tumor suppressor genes, DNA replication genes and others. Segregation of cell populations is a key question in evolution theory. One important aspect when observing cell proliferation in general or bacterial colonies in particular is the relation between spatial organization and the composition of the populations. Here we study a specific example—sectors in expanding bacterial colonies. Such sectors are spatially segregated sub-populations of mutants. Bursts of sectors are observed during compact growth and during branching growth. For theoretical studies of these bursts we employ two mathematical models. Using these models we investigate the amount of segregation achieved by a neutral mutation, as well as by mutations having some advantage over the wild type.