Antimicrobial Research Centre and Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Received 30 July 2001; returned 11 October 2001; revised 21 December 2001; accepted 28 February 2002.
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Abstract |
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Introduction |
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Although endogenous resistance to most antibiotic classes has been reported in clinical isolates,3 current concepts on the ability of bacteria to adapt and survive in the presence of antibiotics are based primarily on the assumption that resistance arises in bacteria that show normal mutation frequencies. However, naturally occurring strains that exhibit elevated mutation frequencies have recently been reported amongst populations of pathogenic Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa and Neisseria meningitidis.68 In some cases hypermutators exhibit frequencies of mutation to endogenous antibiotic resistance that are 1000-fold higher than normal strains and such hypermutators can be found at frequencies of up to 20% of clinical isolates.8 The naturally occurring E. coli and S. enterica hypermutators identified by LeClerc et al.6 contained defects in methyl-directed mismatch repair (MMR), a post-replicative repair system that corrects errors on newly synthesized DNA strands to ensure the fidelity of chromosome replication. Amongst the hypermutators described by LeClerc et al.6 variants defective in the mutS allele were the most common, followed by strains with deficiencies in mutH and mutU (uvrD).
Elevated mutation rates displayed by hypermutators are likely to benefit the organisms by increasing the frequency with which antibiotic resistance arises and enhancing the opportunity for compensatory mutations to reduce fitness costs sometimes associated with the acquisition of endogenous antibiotic resistance.9 Nevertheless, despite the potential importance of hypermutable strains with respect to the emergence of endogenous resistance, their response to antimicrobial selection pressure has not been examined in detail. A recent report by Tanabe et al.10 describes the in vitro development of resistance to ampicillin and ofloxacin by an E. coli dnaQ hypermutator strain. However, the significance of these observations for the emergence of resistance in the clinical setting is uncertain since variants with dnaQ deficiencies have not been reported amongst natural E. coli isolates.6 We therefore examined the response of several E.coli hypermutators, including those with MMR defects found in naturally occurring strains, to a variety of antibiotics with different endogenous resistance potentials.2 This included selection with polymyxin B, cefotaxime and D-cycloserine (low endogenous resistance potentials), rifampicin (high resistance potential) and ciprofloxacin under conditions of both high and low resistance potential.
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Materials and methods |
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Several E. coli strains displaying mutator phenotypes were used (Table 1). The wild-type parent of the isogenic series 14121419 is strain 1411 and the parent of the isogenic series CSH114117 is strain CSH109.
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Antibiotics were from Sigma-Aldrich, Poole, UK, with the exception of ciprofloxacin, which was a gift from Bayer AG, Leverkusen, Germany. MuellerHinton broth (MHB) and agar (MHA) were from Fisher, Loughborough, UK.
Determination of susceptibility to antibiotics
MICs were determined by either two-fold serial antibiotic dilutions or narrower increments in MHA with an inoculum in MHB of 104 cfu/spot.11 Plates were incubated aerobically for 18 h at 37°C and the MIC was defined as the lowest concentration that produced no visible growth. At least three, and up to five, replicate MIC determinations were carried out for all starting strains and mutants. The quoted MICs were reproducible on all occasions.
Determination of mutation frequencies for resistance to antibiotics
This was performed essentially as described by ONeill et al.12 using MH growth media. Both standard and concentrated cell techniques were used, whereby mutation frequencies as low as 1 in 1011 can be detected.12 In each case, cultures were started from single colonies of the test strain that were shown to be sensitive to the antibiotic under investigation. In most cases, antibiotic-resistant mutants were identified on medium containing the selective antibiotic at a concentration that was four-fold higher than the respective MIC for an individual strain. Frequencies of second-step mutations to ciprofloxacin resistance were determined following selections with ciprofloxacin at levels four-fold higher than the respective MICs for the primary mutants. Mutation frequencies were expressed as the number of resistant mutants recovered as a fraction of total viable bacteria.13
Determination of bacterial growth rates
The growth rates (doubling times) of bacteria cultured in MHB were determined from absorbance readings at 600 nm according to the method of Koch.14 Absorbance readings were taken automatically in a Molecular Devices Spectra Max Plus 384 microplate reader (Sunnyvale, CA, USA). Microplates were shaken and incubated at 37°C within the instrument.
Selection of mutants by continuous subculture in the presence of sub-inhibitory concentrations of antibiotic
These experiments were only performed with cefotaxime, D-cycloserine and polymyxin B. Bacteria were grown overnight at 37°C in MHB in the absence of antibiotics and 5 µL aliquots of these cultures were used to inoculate 9 mL of fresh broth containing the appropriate antibiotic at one-quarter of the respective MIC for the strain. These cultures were incubated overnight (18 h). Control experiments, involving determination of viable cell numbers, established that such cultures progressed through 13 generations. After overnight incubation a 5 µL aliquot of the culture was used to repeat the passage through further cycles, continuously in the presence of 0.25 x MIC of the antibiotic. Each day, samples of undiluted culture were plated on to MHA containing the selective antibiotic at 4x the original MIC, and plates were incubated at 37°C to identify the point at which resistant mutants emerged during the sequential passage.
Sequencing of the quinolone resistance-determining regions in gyrA and parC
Chromosomal DNA was prepared from bacteria according to Sambrook & Russell.15 Primers described by Weigel et al.16 were used to amplify the QRDRs (quinolone resistance-determining regions) of gyrA and parC. The PCR products were purified using Microcon PCR Centrifugal Filter Devices (Millipore, Bedford, MA, USA) according to the manufacturers instructions. DNA sequencing was performed using an Applied Biosystems 377 DNA sequencer.
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Results and discussion |
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Endogenous resistance to antibiotics usually results from mutations that affect drug uptake into the cell or alter target structure, thereby decreasing drug binding at the molecular site of action. The mutational response of hypermutators to selection with antibiotics with a range of endogenous resistance potentials has not been determined previously. We carried out such studies with rifampicin, ciprofloxacin, D-cycloserine, cefotaxime and polymyxin B.
Rifampicin has a single molecular target, the RpoB subunit of RNA polymerase, and no specific mechanism of entry into bacteria.17 Consequently, rifampicin has high endogenous resistance potential, and single-step point mutations in only one gene, rpoB, can confer high-level resistance in E. coli.18
In E. coli, fluoroquinolones, including ciprofloxacin, primarily inhibit DNA gyrase (GyrA) with topoisomerase IV (ParC) as a secondary target.19 Single-site mutations at codon 83 or 87 of the gyrA QRDR are sufficient to confer low-level ciprofloxacin resistance, whereas higher levels of resistance either require double (codons 83 and 87) mutations in gyrA, or gyrA mutations in combination with parC or marA mutations, the latter enhancing drug efflux via the AcrABTolC system and reducing influx by suppressing expression of the outer membrane protein OmpF.20,21 Thus, at low concentrations ciprofloxacin has a high endogenous resistance potential, but at higher selective concentrations there is a requirement to generate simultaneous mutations in two or more loci4 and the agent consequently displays low endogenous resistance potential.
D-Cycloserine and cefotaxime are antibiotics that have multiple lethal targets, i.e. D-alanyl-D-alanine ligases A and B (DdlA, B) and alanine racemase (Alr) for D-cycloserine,22 and PBPs 1a (MrcA), 1b (MrcB) and 3 (FtsI) for cefotaxime.23 These antibiotics are predicted to have low endogenous resistance potentials, since at the level of target site interaction it is expected that independent mutations in more than one gene will be required to express a resistance phenotype.4 However, cefotaxime uses the OmpF porin pathway for uptake across the E. coli outer membrane24 and D-cycloserine is transported into the cell by the D-alanine permease system (Cyc).25 In the case of cefotaxime, point mutations conferring resistance could also potentially occur in the ampC-encoded ß-lactamase,26 or marA, the latter both enhancing AcrAB-mediated ß-lactam efflux and decreasing drug uptake through OmpF porin channels.20,27 Therefore, single-site mutations conferring resistance to these antibiotics could in principle arise in genes other than those encoding the drug targets, thereby making the antibiotics prone to endogenous resistance development.
Membrane-active agents, such as polymyxin B, have low endogenous resistance potentials because they have no specific routes of entry and it is difficult to alter fundamental bacterial membrane composition by mutation.28,29 Polymyxin-resistant mutants of E. coli have been isolated.30 However, multiple outer membrane protein and lipopolysaccharide alterations were detected, indicating that several mutations are required to express a resistant phenotype.
The susceptibilities of hypermutators and their parent strains to each of the antibiotics discussed above were determined (Table 1). The nalA (gyrA) genotypes of strains 1411, 1412, 1413, 1417, 1419 and CSH114A were consistent with the low-level ciprofloxacin-resistant phenotypes exhibited by these strains (Table 1). Sequence analysis of gyrA and parC from strains 14111419 indicated that they contain a single point mutation conferring a Ser-83Leu substitution in the QRDR of GyrA. This is a common single-step mutation that confers low-level resistance to fluoroquinolones, including ciprofloxacin, in E. coli.21
Attempts to select mutants resistant to rifampicin, ciprofloxacin, D-cycloserine, cefotaxime and polymyxin B from the hypermutator strains and their parent strains were carried out by plating bacteria on to MHA containing antibiotics at 4 x the respective MICs listed in Table 1. No resistant mutants were recovered, even for hypermutators, for selections at 4 x MIC with D-cycloserine, cefotaxime or polymyxin B (i.e. frequencies were <1011). These data are consistent with low endogenous resistance potentials for these antibiotics.
For rifampicin, resistant mutants were recovered from all strains (Table 2) and hypermutators demonstrated elevated mutation frequencies compared with that of the parent strain. Mutation frequencies varied from c. 108 for non-hypermutator strains to as high as 105 for strain CSH114 (mutT). These results demonstrate the ease with which resistance can arise in hypermutators to agents that have a single molecular target and no specific uptake mechanism, i.e. exhibiting high endogenous resistance potential.
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The QRDRs of gyrA and parC in strains CSH109, CSH114 and CSH115CSH117 were sequenced following PCR amplification of these regions. No mutations were detected. Using a low ciprofloxacin selection concentration, first-step ciprofloxacin-resistant mutants arose with a frequency of 109 with the parental strain (CSH109) and at elevated frequencies, up to 1000-fold higher, with the hypermutators (Table 2). Randomly selected, first-step ciprofloxacin-resistant mutants (strains KM1, KM2 and KM6KM11) (Table 3) derived from strains CSH109, CSH114 and CSH115CSH117 were chosen and their QRDRs of gyrA and parC were sequenced. KM1 and KM2 each demonstrated a single Ser-83Leu mutation in GyrA, whereas the other mutants had Asp-87
Gly mutations in GyrA. Since involvement of these mutations in the expression of low-level fluoroquinolone resistance is well established,21,31,32 it can be concluded that each of the first-step ciprofloxacin-resistant mutants KM1, KM2 and KM6KM11 contains a single-step point mutation at codon 83 or 87 in gyrA. The sequence data confirm that the ciprofloxacin-resistant mutants arose readily from the CSH hosts (Table 2) under conditions in which ciprofloxacin displays high endogenous resistance potential.
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By combining the individual mutation frequencies for ciprofloxacin resistance (Tables 2 and 3), it is possible to derive theoretical resistance mutation frequencies (Table 3) under conditions where ciprofloxacin has low endogenous resistance potential, i.e. 16 x MIC selective concentration. These values are extremely low for CSH109 and its mutT (CSH114), mutS (CSH115), mutD (CSH116) and mutY (CSH117) derivatives. Indeed, consistent with these theoretical values, it was not possible to select resistant mutants of strains CSH109CSH117 directly when the ciprofloxacin selective concentration was 16 x MIC, i.e. the experimental mutation frequency was <1011 (data not shown). This confirms the low endogenous resistance potential of ciprofloxacin when there is a requirement to acquire simultaneous mutations in two or more genes to express moderate- or high-level resistance.
Selection of resistant mutants by continuous subculture in the presence of sub-inhibitory antibiotic concentrations
Experiments were also conducted with D-cycloserine, cefotaxime and polymyxin B to investigate the potential for the emergence of resistance in hypermutators during prolonged subculture. This involved attempts to select resistant mutants by continuous subculture in the presence of sub-inhibitory (0.25 x MIC) antibiotic concentrations. Separate experiments established that the antibiotics under these conditions imposed selection pressures. For example, the doubling time of strain CSH109 was increased 1.8-, 3.5- and 1.5-fold in the presence of 0.25 x MIC polymyxin B, cefotaxime and D-cycloserine, respectively. Repeated passage in the presence of D-cycloserine at 0.25 x MIC eventually led to the emergence of D-cycloserine-resistant mutants in both hypermutators and their parent strains (Figure 1). However, resistant mutants arose more frequently from hypermutators than their parental strains and resistance also emerged earlier from hypermutators (the majority between 26 and 39 generations) compared with the parents (3952 generations). Each of the 18 D-cycloserine-resistant mutants recovered (Figure 1) exhibited a four-fold increase in resistance to the antibiotic compared with the parent strain. The hypermutator status of D-cycloserine-resistant mutants arising in hosts CSH114ACSH117 was confirmed by demonstrating that these strains retained resistance to tetracycline (MIC > 128 mg/L), indicating mini-Tn10 insertions in the respective mut genes. Furthermore, the D-cycloserine-resistant mutants derived from strains 14121499 and CSH114CSH117 each retained elevated mutation frequencies for rifampicin resistance, consistent with retention of hypermutator status. We have not explored the mechanism of D-cycloserine resistance in these mutants. However, in mycobacteria, resistance to D-cycloserine can arise by mutations that either increase the expression of alanine racemase (alr),33 or alter the D-cycloserine uptake system (cyc).34 Since D-cycloserine-resistant mutants could not be selected by direct plating of E. coli (i.e. mutation frequency <1011) (see previous section) it is possible that both types of mutation are required simultaneously to express resistance in E. coli. The frequency with which double mutations arise is predicted to be low and such mutants might only be selected by sequential passage in the presence of D-cycloserine.
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Population analysis of antibiotic-resistant mutants
Population analyses were performed to determine whether there was a bias towards the generation of mutants with higher, or lower, levels of antibiotic resistance from hypermutator strains compared with non-hypermutators. For rifampicin, 20 resistant mutants derived from each of the 12 strains listed in Table 1 were picked at random from colonies appearing on selection plates containing rifampicin at 4 x MIC levels. The individual MICs of rifampicin were determined for these 240 strains. Rifampicin MICs for mutants were in the range 32256x that of the starting strain. For the majority (18 isolates, 90%) of rifampicin-resistant mutants from strain CSH109 (the parent of series CSH114CSH117) the MIC was 1024 mg/L, i.e. 128 x MIC of rifampicin for strain CSH109 itself (Figure 2). The two further mutants derived from strain CSH109 displayed rifampicin MICs of 512 mg/L, i.e. 64 x MIC (one strain, 5%) and 2048 mg/L, i.e. 256 x MIC (one strain, 5%) (Figure 2). Similar determinations were carried out for the other strains (Figure 2). Statistical analysis of the data by an R x C test of independence35 indicated significant differences in the MIC distribution of rifampicin-resistant mutants arising from the majority of the hypermutators when compared with those of the respective parent strains (Figure 2). A greater proportion of high-level rifampicin-resistant mutants was recovered from the mutD-, mutH-, mutL- and mutY-deficient strains compared with the non-hypermutator strains. Furthermore, the highest level of resistance displayed by mutants arising from hypermutators (>256 x MIC for the starting strain) was greater than that of any mutant recovered from non-hypermutator strains, which never exceeded 256 x MIC (Figure 2). The range of rifampicin resistance levels exhibited by the 240 mutants studied here presumably reflects variations in the nature and position of point mutations occurring within rpoB.18
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To our knowledge this situation has not previously been reported. Furthermore, our data indicate that the occurrence of these spontaneous mutations in the absence of ciprofloxacin selection pressure may be enhanced in hypermutable hosts.
2 Population analysis35 was carried out on the second-step ciprofloxacin-resistant mutants (160 mutants) derived from strains KM1, KM2 and KM6KM11 that had themselves been selected from the host strains CSH109 and CSH114CSH117. Compared with KM1, derived from non-hypermutator strain CSH109, no significant differences in second-step ciprofloxacin MIC distribution were obtained for mutD-, mutS- and mutY-deficient strains (Figure 4). However, second-step mutants derived from KM2 (mutT) demonstrated a trend towards generation of mutants with higher levels of ciprofloxacin resistance than those derived from KM1 (Figure 4).
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As noted in previous sections, elevated mutation frequencies for selection of second-step ciprofloxacin-resistant mutants in strains KM2 (GyrA, Ser-83Leu) and KM6KM11 (GyrA; Asp-87
Gly) were not observed (Table 3), even though these strains retained hypermutator status for the generation of rifampicin-resistant mutants. Second-step ciprofloxacin-resistant mutants were also selected from strains 14111419 (GyrA; Ser-83
Leu). Mutation frequencies for second-step ciprofloxacin resistance were again suppressed in the mutator hosts (Table 3), even though these strains exhibited elevated frequencies for selection of rifampicin-resistant mutants. In addition, it was noted that the MIC distribution of second-step ciprofloxacin-resistant mutants arising from strains KM6KM11 (Figure 4) and 14121419 (data not shown) did not differ significantly from that of second-step mutants arising from parent strains KM1 and 1411. These observations suggest that the nature of the second-step ciprofloxacin-resistant mutations arising from these strains is identical and that the locus responsible is not subject to elevated mutation frequencies despite the presence of defective MMR pathways in strains KM6KM11 and 14121419.
The possibility that second-step mutations to ciprofloxacin resistance in hypermutator and parent strains arise in the mar regulatory locus was suggested by the observation that out of 170 second-step mutants analysed, each displayed four-fold increases in resistance to chloramphenicol (MIC 16 mg/L) compared with their immediate parents (chloramphenicol MIC 4 mg/L) and the parents of the first-step ciprofloxacin-resistant mutants (chloramphenicol MIC 4 mg/L). As already noted, the chloramphenicol-resistant phenotype is characteristic of mar mutations.20,21
Conclusions
In recent years progress has been made in the discovery and development of new agents to address problems caused by bacterial resistance to existing antibiotics.3841 Clearly, one of the critical elements in the selection of lead compounds for development rests upon choosing new compounds with low endogenous resistance potentials.2,3,42 Since bacterial loads as high as 109 cells/mL can occur within infected tissues,43 new agents should ideally possess endogenous mutation rates of <109 at concentrations where they exhibit effective antibacterial action.
Recently, we42 and others4,44 advocated the use of bacterial hypermutators, i.e. strains with elevated mutation rates, as valuable tools for assessing the potential for the development of resistance to new antibiotics. Since hypermutators exist in the clinical setting,6,7,44 their use in the laboratory can indicate worst case scenarios for resistance development.42
Despite the potential value of hypermutators for antibiotic discovery research, their response to selection pressure with antibiotics has only partially been characterized.6,10 This paper demonstrates that the presence of hypermutators in the clinical setting presents an enhanced risk for the emergence of endogenous resistance to agents where mutations in single gene targets confer resistance phenotypes. Hypermutators could also present problems in the case of agents with multiple targets but single uptake routes, since resistance development may only need mutation in the uptake system. Although we did not characterize D-cycloserine-resistant mutants, we noted that they arose more readily from hypermutable hosts than from non-hypermutators, perhaps reflecting the enhanced ability of hypermutators to generate double mutations in both the transport system cyc and the alanine racemase (alr) promoter. In some cases hypermutators gave rise to mutants whose antibiotic resistance levels were greater than those recovered from non-hypermutator hosts. These results demonstrate that hypermutators possess superior genetic backgrounds for the selection of some antibiotic-resistant mutations and further emphasize the need to develop new drugs with minimal resistance potential.
We noted different relative mutation frequencies with some of the hypermutators for the selection of single-step rifampicin- and ciprofloxacin-resistant mutants. For instance, CSH116 (mutD) was relatively more proficient in generating rifampicin-resistant mutants than ciprofloxacin-resistant mutants under conditions where both agents were acting as single target drugs (Table 2). These observations probably reflect the outcome of several variables, including the preference for the introduction of transitions or transversions by the various hypermutators.45
Finally, we noted that the MICs for single-step rifampicin-resistant mutants, and in particular those arising in hypermutators, varied considerably. In contrast the MICs for single-step ciprofloxacin mutants fell into a more restricted MIC range that was not greatly influenced by hypermutator status. These differences probably reflect the fact that rifampicin resistance can be acquired by mutation at many sites in rpoB,3,46 whereas mutations conferring ciprofloxacin resistance are confined to a more limited number of sites in gyrA or parC.16,21,31,36,47
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Acknowledgements |
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Footnotes |
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References |
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