First-step mupirocin-resistant mutants.
Selection of antibiotic-resistant mutants through chromosomal mutations occurs in a mutant selection window in which the drug concentration lies between the MIC and the MPC (33
). A mutant selection index of 31 (i.e., MPC divided by MIC) was obtained for S. aureus
8325-4 on the basis of an MPC of 0.5 μg/ml and MIC of 0.016 μg/ml. This indicates an intermediate vulnerability to the development of mupirocin resistance in S. aureus
, which is less than that exhibited by rifampin but greater than that displayed by the majority of fluoroquinolones (33
). We isolated and characterized mutants of strain 8325-4 that arose spontaneously after selection with 0.064 μg of mupirocin/ml (four times the MIC). The mutants displayed low-level resistance to mupirocin with MICs in the range 1 to 16 μg/ml (Table 1
). The mutants arose with an overall frequency of (7.2 ± 0.9) × 10−8
, which is consistent with single mutational events. Several mutants selected for sequencing of ileS
revealed the V588
F and V631
F IRS amino acid substitutions also found in clinical isolates (Table 1
). However, a third mutant class was identified involving a G593
V mutation (Table 1
). This genotype has not previously been identified in mupirocin-resistant S. aureus
, but mutation at the equivalent locus in Methanobacterium thermoautotrophicum
D) confers mupirocin resistance (17
) (Fig. 1
Second-step mutations in ileS conferring elevated mupirocin resistance.
Mutants of S. aureus
exhibiting elevated levels of resistance to mupirocin (MIC ≥ 16 μg/ml) have been isolated in vitro after stepwise exposure to increasing concentrations of the antibiotic (28
). Although these resistant derivatives probably arise by accumulation of separate mutations within ileS
, there are no DNA sequence data to support this hypothesis. Strains JGH-1, JGH-4, JGH-5, JGH-6, and JGH-8, which contain single mutations in ileS
), were therefore used as starting strains for the selection of higher-level resistant second-step mutants.
Second-step mutants were selected by plating onto ISA containing mupirocin at four times the respective MICs for the starting strain. This resulted in the recovery of mutants for which mupirocin MICs were in the range 16 to 128 μg/ml (Table 1
). Short regions of the ileS
were resequenced and resistance mutations detected. However, where no mutations were identified (JGH-13, JGH-15), the entire ileS
gene was sequenced to reveal second-step mutations. In many cases the second mutation occurred at one of the sites (V588
F or V631
F) already identified in low-level mutants, resulting in the generation of double V588
F or G593
F mutants (Table 1
). Antonio et al. (2
) have suggested that V631
F and V588
F mutations may be mutually exclusive since considerable distortion of the hydrophobic domain of IRS would occur. Nevertheless, we show here that these mutations can coexist in IRS. Similarly, G593
V and V631
F mutations can be simultaneously tolerated in IRS. Three second-step mutations occurred at loci not previously implicated in mupirocin resistance (Table 1
) involving the following changes: R816
C (strain JGH-13), F563
L (strain JGH-14) and H67
Q (strain JGH-15).
Second step mutants arose with an overall frequency in the range of 4 × 10−10
to 8 × 10−10
, which is less than the frequency for selection of first-step mutants. The reason that second-step mutations occur less frequently is unclear. However, this does not appear to be related to mutational target size since the numbers of sites at which new mutations arose during first- and second-step selections were similar (Fig. 1
In vitro fitness of first-step mupirocin-resistant mutants.
Resistance to antibiotics arising from mutations in target genes can result in fitness costs that affect bacterial growth and survival (1
). Fitness costs associated with development of mutational resistance to mupirocin could be a factor that influences the prevalence of certain staphylococcal resistance genotypes in the clinic. In preliminary studies, we recently reported that the V588
F mutation in clinical isolates of GISA is associated with low fitness costs (15
). The fitness of other genotypes has not been examined, but it is possible that fitness costs may account for the low frequency of detection of the V631
F resistance allele and, similarly, the apparent absence of G593
V in clinical specimens. We have now expanded our preliminary analysis by determining the in vitro fitness of a number of strains derived from 8325-4 that contain V588
F, or G593
V mutations in IRS. Fitness was assessed by determining the doubling times of mutants and also by comparing their performance with the parental strain 8325-4 in mixed culture competition assays (Table 1
and Fig. 2
The doubling times of six mutants containing the V588
F alteration, two mutants containing the V631
F alteration and two mutants containing the G593
V alteration were not considerably different from the parental starting strain 8325-4 (Table 1
). In mixed culture competition assays five of six strains containing the V588
F mutation displayed a small loss of fitness, but the relative competitive fitness (W
) never fell to <0.9. The G593
V mutation was also associated with a small loss (6 to 8%) of fitness detectable by the mixed culture competition assay.
Strains carrying the V631
F mutation (JGH-3 and JGH-4) were as fit as the parental strain (Table 1
). However, although there were no significant differences in doubling times, there was an apparent reduction in W
of ∼0.2 for three other strains containing V631
F mutations (data not shown). In other systems, it is well known that compensatory mutations can arise in unfit antibiotic-resistant mutants that restore fitness and yet retain resistance (1
). Therefore, the V631
F mutation might be initially associated with a small fitness cost that is ameliorated during subculture through intragenic or extragenic suppression. Nevertheless, no evidence for intragenic compensatory adaptation was found in fit strains possessing the V631
The relatively modest fitness costs associated with the V588
F and V631
F mutations are in agreement with our earlier studies on GISA and consistent with the occurrence of these resistance genotypes in clinical isolates (2
). Since the G593
V resistance mutation does not appear to incur significant fitness costs, at least in vitro, it might arise in clinical isolates. Currently, this mutation has not been identified in clinical strains (2
). However, more extensive surveys may reveal this genotype in the future.
In vivo fitness of first- and second-step mupirocin-resistant mutants.
Significant costs of resistance observed in vitro usually indicate situations where resistance would impose a fitness burden on the organism in natural populations (1
). Nevertheless, the survival of S. aureus
8325-4 and selected first- and second-step mupirocin-resistant mutants, which carry mupirocin resistance genotypes previously identified in clinical strains, was also examined in a murine wound abscess infection model (Fig. 3
). The number of viable organisms in the infectious dose was established at the start of the experiment, and the number of survivors after 7 days of infection was determined by plating samples of homogenized skin lesions onto ISA. The surviving fraction of strain JGH-12 (double IRS mutant, V631
F) was significantly lower than the wild-type 8325-4 (Fig. 3
). In contrast, survival of the first-step mupirocin-resistant mutants JGH-4 (V631
F) and JGH-6 (V588
F) was not significantly different from 8325-4. The mupirocin MICs and doubling times of independent colonies recovered from the mice were similar to the values for the strains used to initiate the infections (data not shown). This implies genetic stability of mupirocin resistance within ileS
during the murine infections.
Although the experiments reported here did not reveal a statistically significant difference in the in vivo fitness of strains 8325-4 and JGH-6, there is, nevertheless, an indication that the V588
F mutation in strain JGH-6 might be associated with increased fitness compared to the parental starting strain 8325-4 (Fig. 3
). However, further experiments involving infection of five mice with mixed cultures of strains 8325-4 and JGH-6 provided no evidence that JGH-6 was fitter than 8325-4 (data not shown).
Overall, the murine data provide a correlation between in vitro and in vivo fitness and further indicate that the clinically occurring V588F and V631F mutations are not associated with significant fitness costs, whereas second-step mutants, such as JGH-12 (V631F/V588F), which have not been reported clinically, possess significant fitness burdens.
Stability of second-step mupirocin-resistant mutants.
In the absence of continued antibiotic selection pressure, the fitness of unfit antibiotic-resistant mutants can be restored by several mechanisms, one of which involves reversion of resistant alleles by back mutation (1
). Consequently, if mupirocin-resistant mutants with more than one mutation in ileS
can arise in the clinic, the failure to detect them in clinical isolates could reflect reversion to produce fitter strains containing only one resistance allele. We therefore examined whether resistance was lost and fitness restored when unfit second-step mupirocin-resistant mutants (JGH-11 to JGH-15) were serially subcultured in the absence of mupirocin for up to 9 days by daily transfer into fresh medium.
At the end of each subculture, samples were taken for determination of mupirocin MICs. Serial subculture of mupirocin-resistant strains JGH-11, JGH-13, JGH-14, and JGH-15 led to progressive reduction of organisms expressing higher levels of resistance to mupirocin within the cultures and replacement by derivatives for which mupirocin MICs were lower (Fig. 4
). For instance, the MIC of mupirocin against JGH-15 was initially 128 μg/ml. However, after serial subculture in the absence of mupirocin for 4 days, the culture contained daughter cells with only low-level resistance (mupirocin MIC = 8 μg/ml), which remained stable for the remainder of the serial subculture. With the exception of JGH-12 (see below), similar results were obtained with the other higher level mupirocin-resistant mutants, which all gave rise to stable, lower-level mupirocin-resistant populations within 4 to 7 days of serial subculture (Fig. 4
In contrast to the other higher level mupirocin-resistant strains, resistance was stable in strain JGH-12 during the course of 9 days serial subculture since starting and final MICs were 32 μg/ml. However, organisms recovered at the end of this experiment were fitness compensated (see below).
Fitness of strains recovered from serial subculture and further genetic analysis.
During serial subculture the emergence of lower-level mupirocin-resistant subpopulations in cultures of JGH-11, JGH-13, JGH-14, and JGH-15 coincided with progressive increases in growth rates of the mixed populations (Fig. 4
). This is consistent with reversion of one of the mutated alleles in the ileS
gene of the parent strains to produce derivatives with restored fitness and reduced resistance or accumulation of further mutations that act to suppress both the resistance phenotype and fitness cost of the existing allele (pseudoreversion). Elevated growth rates were also recorded in cultures of JGH-12 after 3 days of subculture even though resistance of JGH-12 to mupirocin was unaltered through 9 days of serial subculture. This probably reflects the appearance of an intragenic or extragenic compensatory mutation that restores fitness but allows resistance to be retained (1
The more rapidly growing colonies obtained from the mupirocin-resistant strains were therefore presumptive fitness-restored mutants. A number of these colonies were retained for further analysis. They were designated strains JGH-16 to JGH-27 (Table 1
). Strains JGH-16 to JGH-27 exhibited doubling times (range, 38 to 52 min) and W
values (range, 0.79 to 1) that indeed indicated partial or complete restoration of fitness compared to their original unfit parental starting strains (Table 1
To determine whether compensatory or back mutations were responsible for fitness restoration, the entire ileS
gene of the fitness-restored mutants was sequenced and mutations were identified. Back mutation of the second-step mutation (Q67
H) in strain JGH-15 to create JGH-25 restored fitness by reinstating the HMGH sequence (Fig. 1
and Table 1
). However, back mutation of resistance alleles was not detected in other fitness-restored strains. Intragenic and extragenic compensatory mutations that suppressed the effects of the resistance genotypes were inferred from sequencing data. For example, in strains JGH-22 and JGH-23 (both derived from JGH-13) either E195
K (JGH-22) or A196
V (JGH-23) mutations were found, in addition to the previously identified resistance mutations V588
F and R816
C. One strain, JGH-21 (derived from strain JGH-13) seemed to have acquired an extragenic mutation in addition to the compensatory mutation E195
K. Acquisition of this uncharacterized extragenic mutation may explain why strain JGH-21 showed greater susceptibility to mupirocin (MIC = 0.5 μg/ml) compared to JGH-22 (MIC = 4 μg/ml). Compensatory mutations (E190
K or A196
V) were detected in strains JGH-16 to JGH-19 (each derived from JGH-11) in regions adjacent to compensatory mutations found in JGH-22 and JGH-23 (each derived from JGH-13). Intragenic mutations were not found in JGH-20 (derived from JGH-12) or JGH-24 (derived from JGH-14), a finding consistent with a role for extragenic compensatory mutations in restoring the fitness of these strains.
Restoration of fitness by intragenic suppression usually involves compensation by second site mutations that restore fitness and retain resistance (1
). Therefore, the situation described here is somewhat unusual since intragenic (and indeed some extragenic) mutations produced derivatives that exhibited lower-level resistance to mupirocin, despite retention of both original mutations that conferred high-level resistance. Fitness costs may be alleviated in these pseudorevertants by introduction of compensatory mutations that alter IRS conformation, restoring catalytic activity and susceptibility to mupirocin, despite the presence of mutations previously conferring high-level mupirocin resistance whose expression is now partially suppressed.
Comparison of computational predictions for mupirocin binding with in vitro IRS binding data.
It has been reported (3
) that an ethyl ester variant of mupirocin (therefore containing a much-shortened alkyl chain) shows a 10-fold reduction in activity against S. aureus
IRS compared to mupirocin (ki
= 0.19 versus 0.017 nM, respectively). Using the equation ΔG
(wherein ΔG is the change in free energy, R
is the gas constant, T
is the temperature, and ki
is the dissociation constant of the enzyme-inhibitor complex), free-energy changes upon dissociation (ΔG
) of 13.25 and 14.68 kcal/mol, respectively, are obtained. Clearly, the magnitudes of these measured free energies reflect the total change in the energetics of the system during binding (including changes in solvation, entropy, bond enthalpies, etc.). Therefore, they cannot be directly compared to the calculated gas-phase enthalpies of binding shown in Table 2
, which simply reflect the relative changes in bond enthalpy between substrate and enzyme resulting from substrate binding. Nevertheless, the reduced affinity of the shortened alkyl chain variant for IRS compared to that of mupirocin is consistent with our present findings concerning the effect of mutations on the region of the protein bordering the binding pocket of the alkyl chain of mupirocin.
Molecular modeling of isoleucyl-AMP binding.
The variation in fitness costs associated with the various mutants could result from changes in the binding of the enzyme to the reactive isoleucyl-AMP intermediate generated during acylation of the tRNA. We have therefore probed the structural details within a series of models that examine the binding energies of the reactive isoleucyl-AMP intermediate (Fig. 6
, structure 2) generated during acylation of the tRNA. The models of this reactive complex were constructed first by docking a model of structure 2 into the enzyme active site by using the automated molecular docking program AutoDock (19
). The lowest-energy complex was then used to create the various mutant structures, which were then subjected to energy minimization as described above. To date, there is no available X-ray structural data for the S. aureus
IRS containing the reactive intermediate, with which direct comparison of IRS/inhibitor models could be made.
However, the X-ray crystal structure of a sulfonamide-based inhibitor (Fig. 6
, structure 3), designed to mimic a reactive intermediate (Fig. 6
, structure 2) with an IRS, has been reported (21
). Comparison of the models with this structure reveals remarkable similarity with essentially all of the observed inhibitor/enzyme contacts present within the active complex/enzyme models (Fig. 7
). In particular, hydrogen bonds are predicted between the 2′-OH and backbone N of Q554
; the 3′-OH and side chain of Q554
; a phosphate oxygen and backbone NH of Y58
, and between the leucyl NH3
and the carbonyl of P56
. Similarly, the purine ring takes part in H bonding involving N5 and the backbone NH of V588
, and between N7 and H585
). In addition, the reactive intermediate and mupirocin bind in very similar regions within the enzyme (Fig. 8
), with the epoxide ring of mupirocin occupying the same space as the phosphate unit in the intermediate. The central sugar core of both systems is also seen to overlap. The close similarity in binding regions for mupirocin and the reactive intermediate may further explain why major conformational changes that result in higher-level resistance also incur significant fitness reductions. However, an important difference occurs in the region of the extended hydrophobic side chain of mupirocin, which is essentially unoccupied in the reactive intermediate-containing models.
lists the calculated binding energies of the model of the reactive intermediate within the mutant enzymes. Interestingly, the binding energies within all of the first-step mutants are essentially the same as that calculated for the wild-type system. In particular, as outlined above, the G593
V mutation occurs in a region several angstroms away from the phosphorylated intermediate binding cavity. Similar observations apply for the V631
F and V588
F first-step mutations (Fig. 9
). These mutations appear to have little effect upon the binding of the acylated intermediate due to the absence of any part of the ligand in these regions.
For the second-step mutants the calculated binding affinities show a marked decrease relative to the first-step mutants, with that from V588
L exhibiting the lowest value (Table 2
). Inspection of the models reveals the origins of the decreased binding energies. For the G593
F mutant, the presence of the V631
F mutation in particular appears to induce a small movement in the adjacent peptide strand at V588
which in turn induces a further movement (by ∼0.55 Å) in the neighboring peptide loop at position M65
. This widens the hydrophobic pocket holding the purine ring of the substrate, and results in movement of the substrate to better fit the wider pocket. This in turn alters the hydrogen bond distances between the substrate and enzyme in the region of the isoleucine fragment, most noticeably involving Y58
NH and one of the phosphate oxygens, which is lengthened by ∼0.1 Å and also that between the NH3
of the substrate and the side chain of D95
, which increases in length by 0.2 Å. This widening of the pocket binding the purine ring is also seen in the V631
F mutant and analogous side chain interactions appear to induce the same type of distortion in substrate binding and H-bond lengths.
The V588F/F563L mutation also appears to disrupt the H bonding and perhaps also the hydrophobic interactions at the isoleucyl end of the substrate. This seems to result from loss of the edge-to-face interaction between the original aromatic ring present at F563 and the phenolic ring of Y559. This appears to widen the hydrophobic cavity carrying the isoleucyl chain of the substrate and also lengthens the H bonds within this region, particularly that between the NH3 of the substrate and the side chain of D95 (by 0.23 Å).
This same distortion of the isoleucyl binding pocket also appears to be responsible for the decreased affinity observed for the substrate in the V588F/H67Q mutant. In this case, however, it originates from loss of the original face-to-face interaction involving the imidazole rings of H67 and H64. In the mutant, the amide side chain at Q67 is forced to lie nearer the substrate resulting in a steric repulsion, which again lengthens the H bond at the NH3 and phosphate positions of the substrate by 0.1 and 0.2 Å, respectively.
The general trend within the binding energies broadly correlates with the fitness cost measurements and appears to show that the fitness within the various mutant IRS enzymes is a direct result of changes in the efficiency of binding of the key isoleucyl phosphate ester intermediate.
Mutations conferring antibiotic resistance with only low fitness costs may survive for very long periods in natural populations since there will be no counterselection favoring loss of resistance. The fitness studies reported here suggest that first-step V588
F and V631
mutations conferring low-level mupirocin resistance are low-cost mutations both in vitro and in vivo, a finding consistent with the occurrence of these mutations in clinical isolates (2
We observed in the laboratory that higher levels of mupirocin resistance could be generated by selection of second-step mutants from first-step hosts. Such second-step mutants exhibited fitness costs associated with the accumulation of a second mutation in ileS
. Studies on fitness costs imposed by other antibiotic resistance mutations (1
) do not necessarily preclude the clinical appearance of mupirocin-resistant mutants with more than one ileS
mutation because compensatory mutations to alleviate fitness might occur. Indeed, the studies presented here show that fitness burdens imposed by two mutations in ileS
are more frequently relieved by independent compensatory mutations rather than back mutations in the specific allele conferring resistance, at least in vitro. Nevertheless, there is currently no evidence that mupirocin-resistant mutants with more than one mutation in ileS
arise in the clinic (2
). Furthermore, although several mutations of uncertain significance have been located in the ileS
of clinical isolates (2
), none of them corresponds to the intragenic compensatory mutations we report here in laboratory strains. We therefore conclude that clinical selection of mupirocin-resistant strains possessing two mutations in ileS
, if it occurs, must be a rare event. There are a number of possible explanations for this situation. In the first instance the probability of a strain simultaneously acquiring two mutations in ileS
appears to be very low since combination of the individual mutation frequencies for generation of first- and second-step mutants provides a theoretical simultaneous two-step mutation frequency of ∼10−18
. Nevertheless, stepwise selection of two mutations conferring resistance could occur as a result of poor compliance or suboptimal application of mupirocin. Consequently, lack of fitness is the most likely explanation for the absence of resistant double mutants in clinical isolates.
In addition to the clinical implications of the work reported here, we provide a structural understanding of the mutations conferring mupirocin resistance and their effects upon bacterial fitness. For example, there was a correlation between binding energies for mupirocin-IRS interactions and degree of resistance to the antibiotic. Furthermore, the lack of fitness in some mupirocin-resistant mutants could be explained by decreased binding of the reactive intermediate Ile-AMP in the IRS enzyme. Unfortunately, it was not possible to extend the structural studies to the third-site fitness-compensated mutants since these were generally located at positions outside the 12-Å sphere centered on mupirocin within which the energy calculations could be performed.