MUTATOR STRAINS

4. MUTATOR STRAINS

Mutator strains are those that have acquired high rates of mutagenesis due to

a breakdown in the normal proofreading processes engaged during DNA repli- cation and repair. Throughout evolution there has been a drive to maintain the fidelity of the genome sequence. Mutation is essential for evolution to take place, but the rate must be controlled to ensure the overall viability of the organ- ism. Changes that occur naturally are minimised by repair systems that identify mismatched bases. In E. coli, four genetic loci (mutS, mutL, mutH, and uvrD) are associated with increased mutation rates due to loss of the mismatch repair system (Chopra et al., 2003; Gross and Siegel, 1981; Matic et al., 1997; Radman et al., 2000). Similarly mutD (dnaQ) codes for the ε (proofreading) subunit of DNA polymerase III and ensures the fidelity of DNA replication and mutations in this locus alter the rates of mutation due to overload of the mis- match repair system (Chopra et al., 2003; Oller et al., 1993). Altering the metabolism of cells can also predispose them to exhibit high rates of mutagen- esis. Cells that have undergone oxidative damage accumulate oxidised guanine bases, both as the free 8-oxo-dGTP and incorporated into DNA. Three gene products, MutT, MutM, and MutY eliminate these oxidised bases. A less obvi- ous example is that of nucleotide diphosphate kinase (ndk), loss of which causes increases in the pools of dGTP and dCTP that in turn appears to favour A:T to G:C changes (Oller et al., 1993). The mutation is synergistic with a mutS mutation since mismatch repair normally undoes the mutations caused by unbalanced pools of deoxynucleotide triphosphates. This study also found that a breakdown in the normal proofreading processes engaged during DNA repli- cation and repair. Throughout evolution there has been a drive to maintain the fidelity of the genome sequence. Mutation is essential for evolution to take place, but the rate must be controlled to ensure the overall viability of the organ- ism. Changes that occur naturally are minimised by repair systems that identify mismatched bases. In E. coli, four genetic loci (mutS, mutL, mutH, and uvrD) are associated with increased mutation rates due to loss of the mismatch repair system (Chopra et al., 2003; Gross and Siegel, 1981; Matic et al., 1997; Radman et al., 2000). Similarly mutD (dnaQ) codes for the ε (proofreading) subunit of DNA polymerase III and ensures the fidelity of DNA replication and mutations in this locus alter the rates of mutation due to overload of the mis- match repair system (Chopra et al., 2003; Oller et al., 1993). Altering the metabolism of cells can also predispose them to exhibit high rates of mutagen- esis. Cells that have undergone oxidative damage accumulate oxidised guanine bases, both as the free 8-oxo-dGTP and incorporated into DNA. Three gene products, MutT, MutM, and MutY eliminate these oxidised bases. A less obvi- ous example is that of nucleotide diphosphate kinase (ndk), loss of which causes increases in the pools of dGTP and dCTP that in turn appears to favour A:T to G:C changes (Oller et al., 1993). The mutation is synergistic with a mutS mutation since mismatch repair normally undoes the mutations caused by unbalanced pools of deoxynucleotide triphosphates. This study also found that

It seems probable that mutation rates will rise due to changes in the envi- ronment once a pathogenic organism has entered a macrophage or another environment in which it is nutritionally disadvantaged. The impact of oxida- tive damage has been alluded to above. In starving colonies of E. coli, an increase in error-prone repair has been suggested to cause increased genetic heterogeneity (Taddei et al., 1995, 1997). Recent work has shown that Brucella and Salmonella undergo modified gene expression when they enter macrophages that is consistent with them resisting the mutagenic effects of the natural electrophile methylglyoxal (MG) (Eriksson et al., 2003; Eskra et al., 2001; Kohler et al., 2002). MG is produced by cells either in response to phosphate limitation or when the balance of carbon metabolism is per- turbed leading to the accumulation of sugar phosphates (Booth et al., 2003). Overproduction of this metabolite by cells is known to be mutagenic and is countered by repair mechanisms and by detoxification of MG by a glu- tathione-dependent glyoxalase pathway. Salmonella cells that have been engulfed by macrophages exhibit induction both of phosphate scavenging pathways and systems for protection against MG (Eriksson et al., 2003). These data point to the potential for the intracellular environment to increase the rate of mutagenesis.

It has been known for almost 50 years that mutator bacteria exist within the natural populations, but they do so at a low frequency (approx. 1%) (LeClerc et al., 1996). These observations have been repeated and extended at intervals, particularly during the investigation of the emergence of new pathogenic strains and the growth in interest in antibiotic-resistant isolates. Hypermutable Pseudomonas aeruginosa were isolated from a cystic fibrosis (CF) lung infec- tion (Oliver et al., 2000). From a range of perspectives, this is a very challeng- ing environment for a bacterial cell—both natural and man-made challenges are prevalent. In contrast to isolates from acute clinical infections, those from the CF patients were found to be quite diverse in appearance and in physiology (Oliver et al., 2000). Many of the CF isolates gave mucoid colonies, a property that is strongly associated with pathogenesis. It may affect adherence, resis- tance to antibiotics and to macrophages, quenching of oxygen radicals and hypochlorite. Mutations in the mucA gene, which forms part of the regulatory network governing algD that produces the immediate precursor of alginate, have been observed in P. aeruginosa isolates from CF patients. Loss of MucA leads to high levels of expression of AlgD and synthesis of alginate, which enhances survival of P. aeruginosa in the lung (Boucher et al., 1997; Govan and Deretic, 1996).

Evolution of Antibiotic Resistance within Patients 375

376 Ian R. Booth Such considerations led to the hypothesis that mutators might be more

frequent among CF patient isolates than among those from acute infections (Oliver et al., 2000). Mutator isolates were obtained from 11 of 30 CF patients and 19.5% of all isolates from all the patients exhibited the mutator phenotype (scored by the acquisition of resistance to either rifampicin or streptomycin at

a frequency 20 times that of the control strain PAO1; Oliver et al., 2000). Each patient who had mutator clones exhibited unique P. aeruginosa lineages with no suggestion that there had been cross-infection. In contrast, it was observed that no mutators were found among 50 blood isolates and among 25 respira- tory isolates from non-CF patients. The mutation rates were ⬃100 times greater in the mutators than in the non-mutator strains. In most cases, the origin of the mutator phenotype was a mutation in either mutS, which could be comple- mented by the cloned mutS gene or in the mutY gene (Oliver et al., 2000). The patients from whom these strains were obtained had received several courses of different antibiotics and thus the isolates were screened for their MIC values for a range of antimicrobial agents. As expected, all of the CF isolates exhib- ited a tendency to be more resistant to antibiotics that non-CF isolates, but for almost all drugs, a higher frequency of resistance was observed among the CF isolates (Oliver et al., 2000).

4.1. Persistence of mutators

There is a strong prediction that mutators pose only a transient advantage while the selective pressure is strong, but should be at a disadvantage in the long run when selective pressure is relieved (Chopra et al., 2003; Funchain et al., 2000; Giraud et al., 2001a). As a rule, significant frequencies of mutator strains have been found in clinical situations where the organism persists for extended periods and is probably subject to severe challenge from both host defences and antibiotic treatment. In the CF example, mutator bacteria sur- vived for long periods in the patients, with similar isolates being obtained from one patient after a 4-year interval. The constantly changing environment of the lung as the patient’s health deteriorates poses a continuing challenge to the colonising organisms, and consequently may act as a selective force in favour of mutators despite their potential to generate nonbeneficial mutations (Giraud et al., 2001b). Recent work has identified mutators among fluoroquinolone resistant clones of E. coli isolated from the urinary tract (Lindgren et al., 2003). The most resistant clones were found to have multiple mutations in gyrA and parC and were also found to exhibit mutation rates two orders of magnitude greater than the more sensitive clones that also possessed fewer mutations contributing to resistance (Lindgren et al., 2003). Similarly, in a comparison of 603 commensal and pathogenic isolates of E. coli and Shigella, the highest rates of mutation were found to be associated with uropathogenic

Evolution of Antibiotic Resistance within Patients 377 strains (Denamur et al., 2002). The incidence of mutators did not differ signif-

icantly between commensals and pathogenic organisms, but was found to be enhanced among strains recovered from urinary tract infections. Strains recov- ered from pus were found to have the lowest rate of mutation among the whole collection of strains, but the reasons for this are unknown.

4.2. Gram-positive bacteria

The role of mutator strains in the development of antibiotic resistance among Gram-positive organisms in the clinical setting is largely unknown. Analysis of the mutation rate of over 490 clinical Staphylococcus aureus iso- lates suggested that none were exceptional (O’Neill and Chopra, 2002). In contrast, mutS alleles of S. aureus have been created and they exhibit the expected increase in mutation rate and were used to select high level vancomycin-resistant clones that only arise from the acquisition of multiple mutations (Schaaff et al., 2002). Among 200 clinical isolates of Streptococcus pneumoniae, ⬃8% had mutation rates equivalent to those seen in mutS mutants of this organism (Morosini et al., 2003). However, these strains appeared to have normal hexA (mutS) and hexB (mutL) loci and the presence of the muta- tors did not appear to influence the level of resistance to penicillin. In labora- tory experiments, the potential of mutators has again been demonstrated. Low concentrations of cefotaxime led to enrichment of a mixed population (hexA/wild type) of S. pneumoniae cells for the hypermutable strain due to the higher rates of acquisition of mutations in pbp2x (Thr550Ala) (Negri et al., 2002). From these initial studies, it seems likely that mutators will play a role in the development of resistance among Gram positives in an appropriately selective environment.