1 Seattle Biomedical Research Institute, 307 Westlake Ave N, Suite 500, Seattle, WA 98109, USA
2 Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri-Columbia, Columbia, MO 65212, USA
3 Division of Infectious Diseases, Children's Hospital and Regional Medical Center, 4800 Sand Point Way, Seattle, WA 98105, USA
Correspondence
Arnold L. Smith
arnold.smith{at}sbri.org
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are AY568590, AY568591, AY568592, AY568593, AY568594, AY568595, AY568596 and AY568597.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hypermutable isolates of bacterial pathogens have been described among natural populations of Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Neisseria meningitidis and Staphylococcus aureus (Bucci et al., 1999; Denamur et al., 2002
; LeClerc et al., 1996
; Matic et al., 1997
; Oliver et al., 2000
, 2002
; Prunier et al., 2003
; Richardson et al., 2002
). The majority of mutators among natural bacterial populations have been found to have defects in one or more components of the methyl-directed mismatch repair (MMR) system, particularly mutS (LeClerc et al., 1996
; Li et al., 2003
; Matic et al., 1997
; Oliver et al., 2002
). The MMR system is responsible for correcting errors in newly replicated DNA and prevents recombination between homologous DNA sequences (Matic et al., 1995
; Modrich & Lahue, 1996
; Rayssiguier et al., 1989
). Mutants in MMR genes produce a global mutator phenotype with up to 1000-fold increased mutation rates, increased recombination between non-identical DNA sequences and increased rates of frameshift mutations, particularly in microsatellite regions with runs of repetitive sequences (Cox, 1976
; Glickman & Radman, 1980
; Matic et al., 1995
; Schaaper & Dunn, 1987
; Strauss, 1999
).
The prevalence of global mutators within some populations was estimated to be about 1 % of natural pathogenic and commensal isolates of E. coli and Sal. enterica, and considerably higher within specific conditions (i.e. up to 20 % of P. aeruginosa isolated from the sputum of patients with CF) (LeClerc et al., 1996; Matic et al., 1997
; Oliver et al., 2000
). It is thought that the reason mutators are not more prevalent is due to the decreased fitness of excess deleterious mutations associated with higher mutation frequencies (Dawson, 1999
). However, when a favourable mutation, such as antibiotic resistance, is acquired and selected for, the hypermutable phenotype can stabilize and be propagated by association with the selective mutation (hitch-hiking) (Chao & Cox, 1983
; Giraud et al., 2002
; Taddei et al., 1997a
). This has been demonstrated in a gnotobiotic mouse model in which antibiotic treatments not only selected for antibiotic-resistant bacteria, but also for MMR-defective mutators (Giraud et al., 2002
). Bacteria with high mutation frequencies are predicted to have a higher probability of survival in changing and unpredictable environments and have greater ability to adapt to new conditions, including antibiotic pressure (Giraud et al., 2002
; Ishii et al., 1989
; LeClerc et al., 1996
; Leigh, 1970
; Sniegowski et al., 1997
; Taddei et al., 1997b
). Therefore, mutator bacteria are clinically important as they can promote the emergence of strains resistant to one or more antibiotics which increases hospitalization costs as well as patient morbidity and mortality (Carmeli et al., 1999
). A positive correlation has been found between high mutation frequencies and antibiotic resistance in P. aeruginosa isolates from CF patients receiving long-term antibiotic therapy (Oliver et al., 2000
).
The present study defines the prevalence of global hypermutators within a panel of H. influenzae isolated from a variety of patients, and commensal isolates. We identified several hypermutable strains, with the majority of those being isolated from CF sputum. The hypermutable phenotype of these isolates was associated with polymorphisms in conserved regions of mutS. In addition, six hypermutable isolates were discovered to be clonal, originating from the same CF patient collected over a period of about 11 months. These clonal isolates were further characterized to examine the effect of the hypermutable phenotype on antibiotic resistance over time.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Five hundred H. influenzae isolates, both commensal and from a variety of diseases (Table 1) were screened for the hypermutable phenotype as described below. Isolates were obtained from 450 different patients, mostly from the area of Seattle, WA, USA, with 25 patients contributing two or more isolates over time. All isolates with the hypermutable phenotype on screening were confirmed as H. influenzae by Gram staining, oxidase testing and the requirement for X (haemin) and V (
-NAD+) factors for aerobic growth.
-Lactamase production was detected with the nitrocefin dry slide test (Becton Dickinson). Encapsulation of strains was assessed by PCR for the presence of the capsule export locus (bexA) and, if present, PCR for the capsular gene cluster using primers specific for capsular types af (Falla et al., 1994
). Strains Rd KW20 (R652) (Wilcox & Smith, 1975
) and Rd KW20 mutS : : TSTE (R3544) (Watson et al., 2004
) were used as negative and positive controls, respectively, for determination of mutation frequency.
|
Determination of mutation frequency.
All 500 isolates were initially screened by assessment of the mutation frequency to rifampicin resistance and were considered potential hypermutators if they yielded 10 rifampicin-resistant c.f.u. per 109 viable cells. This value was chosen as it is approximately 10 times the mutation frequency of strain Rd KW20 and is similar to the mutation frequency of a constructed mutS : : TSTE mutant of Rd KW20 (R3544) (Watson et al., 2004
). Potential mutators were confirmed with additional measurement of mutation frequency to rifampicin, nalidixic acid and spectinomycin resistance assessed on sBHI agar plates. Fluctuation assays (Luria & Delbruck, 1943
) were performed by inoculating 10 ml sBHI broth in a 125 ml flask to achieve an initial density of 107 c.f.u. ml1 of H. influenzae and growing for 810 h shaking at 200 r.p.m. in 37 °C in room air. For unclear reasons, H. influenzae requires a relatively high inoculum density to achieve growth in sBHI liquid medium. The culture density after growth was approximately 1010 c.f.u. ml1. Cultures were pelleted and resuspended in 500 µl PBSg. From these concentrated cultures 100 µl was spread in duplicate onto sBHI plates containing rifampicin, nalidixic acid or spectinomycin to determine the number of drug-resistant mutants per ml of culture. Another 100 µl of the same culture was serially diluted in PBSg and plated in duplicate on sBHI plates without antibiotics to determine the viable cell count per ml of concentrated culture. Growth was allowed for 48 h at 37 °C in room air prior to quantification. Mutation frequency was calculated as the number of drug-resistant cells per ml of culture divided by the total number of viable cells per ml of culture. Because the mutation frequency fluctuates between individual cultures, at least four independent measurements were sampled in duplicate for each strain with the median mutation frequency reported. Using the median frequency method of Drake (1991)
an estimate was made of the mutation rate, defined as antibiotic-resistant bacteria produced per cell per division. It should be noted that due to the relatively high inoculum density required to achieve growth in broth media, some experiments could possibly have contained pre-existing mutants in strains with the highest mutation rate which would tend to lead to an overestimation of the mutation frequency. The combined effect of multiple replicates and reporting the median mutation frequency rather than the mean was used to minimize the effect of these artificial jackpots.
Enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting.
Random amplification PCR analysis was performed using primers ERIC 1 or ERIC 2. Primer sequences and reaction conditions were as described by Gomez-De-Leon et al. (2000). A negative control containing all reaction ingredients except for template DNA was run in parallel to each experiment and results were analysed only if the negative control failed to yield amplified DNA. The amplification products (8·5 µl) were separated by 2·0 % agarose gel electrophoresis at 5 V cm1 for 57 h with subsequent staining by ethidium bromide. Amplification reactions were repeated a minimum of three times to verify the reproducibility of the ERIC-PCR banding profiles.
Ribotyping of bacterial isolates.
Genomic DNA was prepared for each isolate and 10 µg DNA was digested with 10 units EcoRI. Digested DNA was separated by 0·8 % agarose gel electrophoresis and subsequently transferred to nylon membranes. Membranes were blotted with a DIG-labelled probe for H. influenzae 16S rRNA genes (rrnABCDEF) generated from the 740 bp PCR product of primers rrnB F and rrnB R. Hybridization was detected by chemiluminescence using an alkaline phosphatase antibody against digoxigenin.
MLST.
MLST was performed for H. influenzae as described by Meats et al. (2003). The sequences were aligned and allele assignments were produced at the MLST website (www.mlst.net). Novel allele sequences identified in this work were submitted to the H. influenzae MLST database and assigned numbers by the database curator, Emma Meats.
Antibiotic susceptibility testing.
The close-interval MIC was determined using agar dilution and a Steer's replicator (Steers et al., 1959). Plates of sBHI with antibiotic concentrations of 0, 0·125, 0·25, 0·5, 1, 2, 4, 8, 16, 32 and 64 µg amoxycillin, ampicillin, cefaclor, ceftriaxone, cephalexin, chloramphenicol, ciprofloxacin, tetracycline, ticarcillin and tobramycin ml1 were prepared. Bacteria were harvested from fresh overnight growth at 37 °C in room air on chocolate agar and resuspended in PBSg to an OD600 of 0·2, which corresponds to approximately 1x108 c.f.u. ml1. This suspension was diluted 1 : 10 in PBSg and 150 µl was loaded into the wells of the multi-pin Steer's replicator; the multi-pin head was dipped into the wells and then gently applied to the surface of the agar plates. The pins deposit a volume of approximately 10 µl to the plate surface from each well which results in 105 c.f.u. delivered per spot. After drying for 30 min, the plates were incubated at 37 °C for 36 h in room air before reading. Inocula with less than 10 % growth relative to the antibiotic-free plate were considered negative for growth at that antibiotic concentration (Syriopoulou et al., 1979
). H. influenzae strain ATCC 49766 was used as a control for ampicillin susceptibility testing with MICs interpreted using the recommendations in NCCLS standard M100-S11 (NCCLS, 2001
). Antibiotic susceptibility tests were repeated three times and the mean MIC value reported.
Sequencing of mutS alleles from hypermutable isolates.
Genomic DNA from each strain was prepared to sequence mutS using overlapping primer sets listed in Table 2. Each primer set yields a product of about 5001000 bp and the products were sequenced in both forward and reverse directions with the same primers used for amplification. PCR products were amplified using the Expand High Fidelity Polymerase (Roche) for maximal accuracy and were subsequently visualized by 0·8 % agarose gel electrophoresis, purified and sequenced at the SBRI DNA core facility. Sequences were aligned with the Bioedit Sequence Alignment Editor for Windows which is available online as freeware at www.mbio.ncsu.edu/BioEdit/bioedit.html. The mutS sequences from H. influenzae strains Rd KW20 (Fleischmann et al., 1995
), Strain-12 (R2846; unpublished data), INT-1 (R2866; unpublished data) and E. coli strain K-12 (Blattner et al., 1997
) were used as a template for general alignment of mutS sequences. Multisequence alignments were also performed using CLUSTAL W available at http://clustalw.genome.ad.jp/ (Thompson et al., 1994
).
|
Methods for statistical analysis.
Statistical analyses were performed using the statistical functions of Microsoft Excel 1997 or by VassarStats, a website for Statistical Computation by Richard Lowry http://faculty.vassar.edu/lowry/VassarStats.html. The non-parametric, two-tailed MannWhitney test was used to test the significance of mutation frequencies, with P<0·05 indicating statistically significant differences.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The region upstream of mutS was PCR-amplified using the MutS promoter F-R primer pair or the MutS Front F and the MutS promoter R primer pair. The PCR products of strains C2677 and C2382 were of equal size (about 530 bp) to the Rd KW20 PCR product. The PCR products of strains C2696, C2372, C2394, C2202, C2475 and R465, were each about 3·7 kb. Partial sequencing of these large amplicons identified the presence of the tryptophanase operon (tnaCAB); these strains were all indole-positive (data not shown). This operon has been previously shown to be widespread (7075 %) among commensal respiratory isolates but more prevalent (94100 %) in disease isolates (Martin et al., 1998). All strains examined were nearly identical in their DNA sequence in the first 70 bp upstream of mutS. Further upstream sequences shared much less identity due to the presence of the tnaCAB operon in six of the strains. The hypermutable strain C2382 has a deletion of five conserved nucleotides about 30 bp upstream of the mutS ATG codon within the putative promoter region.
mutS alleles are defective in trans
To determine if mutS alleles sequenced in this study were defective, complementation analysis was used: we cloned mutS alleles into the plasmid pSU2718 and then tested the ability of these plasmids to suppress the mutator phenotype of Rd KW20 mutS : : TSTE (R3544). Table 4 lists the mutation frequency to rifampicin resistance for the Rd KW20 mutS : : TSTE (R3544) mutator strain transformed with various plasmid constructs. Strain R3544 has a mutation rate about eightfold greater than the parental strain R652; the addition of the pSU2718 vector by itself did not significantly alter the R3544 mutation frequency (two-tailed MannWhitney test, P=0·144). Strain R3544 carrying the pMW058 plasmid with the R652 wild-type mutS allele had a significantly reduced mutation frequency (P<0·0005). Strain R3544 with either pMW059, pMW060 or pMW064 did not have a significantly reduced mutation frequency compared to R3544 alone (P=0·168 for each). Strain R3544 with the pMW065 plasmid had an intermediate mutation frequency that was significantly reduced from the R3544 parent strain (P<0·001), but remained significantly greater than R3544 with plasmid pMW058 (P<0·01).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hypermutable bacteria commonly have defects in genes involved with DNA proofreading, such as mutS of the MMR system (LeClerc et al., 1996; Li et al., 2003
; Oliver et al., 2002
). As a result of lesions that inactivate the MMR system, the global mutation rate increases (up to 1000-fold greater in E. coli) and the barrier to genetic recombination between divergent DNA sequences is lowered (Matic et al., 1995
; Rayssiguier et al., 1989
). Mutants of mutS are more abundant than isolates with mutations in other MMR genes in natural populations of E. coli (LeClerc et al., 1996
; Matic et al., 1997
). In contrast, in the laboratory environment mutants of other E. coli MMR genes, including mutL, are readily isolated, suggesting certain mutations may be counterselected against in nature due to deleterious effects that are not compensated by advantageous randomly generated mutations (Denamur et al., 2000
). We have found several mutations in mutS alleles from our hypermutator panel that were in regions associated with defective MutS activity in E. coli. Four of these mutS alleles were cloned and these failed to completely complement the mutator phenotype of strain Rd KW20 mutS : : TSTE (R3544), suggesting that these mutS alleles are defective and are likely to be responsible for a mutator phenotype. The mutS allele of strain C2394 in plasmid pMW065 partially complemented the R3544 mutator phenotype (Table 4
). C2394 is a moderate-level mutator with at least two mutations at conserved positions, and even though its MutS is defective, it still seems to retain some functions capable of partial complementation. In H. influenzae, mutants of mutS may be particularly common as mutS is an isolated ORF; in contrast, mutL and mutH are located within operons with several other genes involved in DNA metabolism, protein synthesis and cell envelope functions, and therefore only mutations which do not produce polar effects are more likely to be selected, potentially limiting the frequency of mutL and mutH mutations in nature. A similar explanation has been previously offered to account for the overrepresentation of natural E. coli mutS mutators compared to mutL mutators, with mutL in E. coli being part of an operon subject to polar mutations (Denamur et al., 2000
). In addition, in H. influenzae point mutations inactivating MMR activity may be preferred over gene deletions as the remaining DNA sequence following a point mutation may facilitate genetic recombination at a later time to reacquire a functional mutS allele from the environment by lateral transfer. However, it should be noted that no evidence exists to suggest deletions inhibit lateral transfer. The mutS region of E. coli is highly polymorphic and phylogenetic analysis between mutS alleles and their respective genomes suggests that mutS may have been transferred by horizontal exchange multiple times throughout E. coli populations to rescue defective mutS alleles (Brown et al., 2001
). The mutS sequences among our isolates were polymorphic and further comparison of these sequences to other housekeeping genes is required to determine if a similar mechanism is likely to occur in H. influenzae.
Antibiotic resistance has been previously correlated with the presence of the mutator phenotype in P. aeruginosa and Sta. aureus isolates from CF patients receiving long-term antibiotic therapy (Oliver et al., 2000; Prunier et al., 2003
). Among our panel of mutators we found one BLNAR CF sputum isolate (R465) out of 12 (8·3 %) non-
-lactamase-producing mutators, and seven of 12 (58·3 %) non-
-lactamase-producing mutator isolates were resistant to the second-generation cephalosporin cefaclor (MIC
32 µg ml1). H. influenzae BLNAR isolates are relatively rare in the United States with estimates ranging from <0·1 to 2·5 % of all isolates investigated (Doern et al., 1997
). The higher prevalence of hypermutable H. influenzae in patients receiving long-term antibiotic therapy, as in CF, may select and increase the frequency of BLNAR isolates recovered from those populations.
We have been able to identify six hypermutable CF sputum isolates from the same patient over a period of about 11 months that were determined by three independent genetic methods to have at least originated from the same ancestral strain, providing us with the ability to track the effects of the hypermutable phenotype on patterns of antibiotic resistance. Examination of antibiotic susceptibilities for these isolates with close-interval MIC testing showed increases in resistance for multiple antibiotics for some strains, mostly to the antibiotics the patient received as treatment for chronic respiratory infection. The significance of the MIC for tobramycin being unstable in these isolates is unknown, but may indicate that although these isolates are clonal in origin they may not be direct descendants of each other. These isolates may have originated from the same hypermutable ancestral strain and then underwent parallel evolution in different microenvironments of the CF lung. Parallel evolution of serial E. coli isolates with different antibiotic susceptibilities has been described (Low et al., 2001). We believe these increases in resistance demonstrate the ability of a persistent hypermutable strain to evolve antimicrobial resistance over time and illustrate the difficulty of eradicating pathogens in diseases such as CF where small variations in antibiotic accessibility may create a microenvironment where resistant bacterial strains will emerge (Pennington, 1981
).
In summary, hypermutable clinical isolates of H. influenzae with mutations in mutS exist at relatively high frequencies, particularly in certain environments such as CF sputum. These isolates are capable of causing persistent infections and are likely to be selected for and maintained within natural populations by the benefits associated with an increased mutation frequency and a relaxed barrier to genetic recombination, producing isolates with new traits including antibiotic resistance.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bayliss, C. D., Sweetman, W. A. & Moxon, E. R. (2004). Mutations in Haemophilus influenzae mismatch repair genes increase mutation rates of dinucleotide repeat tracts but not dinucleotide repeat-driven pilin phase variation rates. J Bacteriol 186, 29282935.
Blattner, F. R., Plunkett, G., 3rd, Bloch, C. A. & 14 other authors (1997). The complete genome sequence of Escherichia coli K-12. Science 277, 14531474.
Brown, E. W., LeClerc, J. E., Li, B., Payne, W. L. & Cebula, T. A. (2001). Phylogenetic evidence for horizontal transfer of mutS alleles among naturally occurring Escherichia coli strains. J Bacteriol 183, 16311644.
Bucci, C., Lavitola, A., Salvatore, P., Del Giudice, L., Massardo, D. R., Bruni, C. B. & Alifano, P. (1999). Hypermutation in pathogenic bacteria: frequent phase variation in meningococci is a phenotypic trait of a specialized mutator biotype. Mol Cell 3, 435445.[Medline]
Carmeli, Y., Troillet, N., Karchmer, A. W. & Samore, M. H. (1999). Health and economic outcomes of antibiotic resistance in Pseudomonas aeruginosa. Arch Intern Med 159, 11271132.
Chao, L. & Cox, E. C. (1983). Competition between high and low mutating strains of Escherichia coli. Evolution 37, 125134.
Cox, E. C. (1976). Bacterial mutator genes and the control of spontaneous mutation. Annu Rev Genet 10, 135156.[CrossRef][Medline]
Dawson, K. J. (1999). The dynamics of infinitesimally rare alleles, applied to the evolution of mutation rates and the expression of deleterious mutations. Theor Popul Biol 55, 122.[CrossRef][Medline]
Denamur, E., Lecointre, G., Darlu, P. & 9 other authors (2000). Evolutionary implications of the frequent horizontal transfer of mismatch repair genes. Cell 103, 711721.[Medline]
Denamur, E., Bonacorsi, S., Giraud, A. & 8 other authors (2002). High frequency of mutator strains among human uropathogenic Escherichia coli isolates. J Bacteriol 184, 605609.
Doern, G. V., Brueggemann, A. B., Pierce, G., Holley, H. P., Jr & Rauch, A. (1997). Antibiotic resistance among clinical isolates of Haemophilus influenzae in the United States in 1994 and 1995 and detection of beta-lactamase-positive strains resistant to amoxicillin-clavulanate: results of a national multicenter surveillance study. Antimicrob Agents Chemother 41, 292297.[Abstract]
Drake, J. W. (1991). A constant rate of spontaneous mutation in DNA-based microbes. Proc Natl Acad Sci U S A 88, 71607164.[Abstract]
Falla, T. J., Crook, D. W., Brophy, L. N., Maskell, D., Kroll, J. S. & Moxon, E. R. (1994). PCR for capsular typing of Haemophilus influenzae. J Clin Microbiol 32, 23822386.[Abstract]
Fleischmann, R. D., Adams, M. D., White, O. & 37 other authors (1995). Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269, 496512.[Medline]
Foxwell, A. R., Kyd, J. M. & Cripps, A. W. (1998). Nontypeable Haemophilus influenzae: pathogenesis and prevention. Microb Mol Biol Rev 62, 294308.
Giraud, A., Matic, I., Radman, M., Fons, M. & Taddei, F. (2002). Mutator bacteria as a risk factor in treatment of infectious diseases. Antimicrob Agents Chemother 46, 863865.
Glickman, B. W. & Radman, M. (1980). Escherichia coli mutator mutants deficient in methylation-instructed DNA mismatch correction. Proc Natl Acad Sci U S A 77, 10631067.[Abstract]
Gomez-De-Leon, P., Santos, J. I., Caballero, J., Gomez, D., Espinosa, L. E., Moreno, I., Pinero, D. & Cravioto, A. (2000). Genomic variability of Haemophilus influenzae isolated from Mexican children determined by using enterobacterial repetitive intergenic consensus sequences and PCR. J Clin Microbiol 38, 25042511.
Ishii, K., Matsuda, H., Iwasa, Y. & Sasaki, A. (1989). Evolutionarily stable mutation rate in a periodically changing environment. Genetics 121, 163174.
LeClerc, J. E., Li, B., Payne, W. L. & Cebula, T. A. (1996). High mutation frequencies among Escherichia coli and Salmonella pathogens. Science 274, 12081211.
Leigh, E. G. (1970). Natural selection and mutability. Am Nat 104, 301305.[CrossRef]
Li, B., Tsui, H. C., LeClerc, J. E., Dey, M., Winkler, M. E. & Cebula, T. A. (2003). Molecular analysis of mutS expression and mutation in natural isolates of pathogenic Escherichia coli. Microbiology 149, 13231331.[CrossRef][Medline]
Low, A. S., MacKenzie, F. M., Gould, I. M. & Booth, I. R. (2001). Protected environments allow parallel evolution of a bacterial pathogen in a patient subjected to long-term antibiotic therapy. Mol Microbiol 42, 619630.[CrossRef][Medline]
Luria, S. & Delbruck, M. (1943). Mutations of bacteria from virus sensitivity to virus resistance. Genetics 28, 491.
Maiden, M. C., Bygraves, J. A., Feil, E. & 10 other authors (1998). Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A 95, 31403145.
Martin, K., Morlin, G., Smith, A., Nordyke, A., Eisenstark, A. & Golomb, M. (1998). The tryptophanase gene cluster of Haemophilus influenzae type b: evidence for horizontal gene transfer. J Bacteriol 180, 107118.
Martinez, E., Bartolome, B. & de la Cruz, F. (1988). pACYC184-derived cloning vectors containing the multiple cloning site and lacZ alpha reporter gene of pUC8/9 and pUC18/19 plasmids. Gene 68, 159162.[CrossRef][Medline]
Matic, I., Rayssiguier, C. & Radman, M. (1995). Interspecies gene exchange in bacteria: the role of SOS and mismatch repair systems in evolution of species. Cell 80, 507515.[Medline]
Matic, I., Radman, M., Taddei, F., Picard, B., Doit, C., Bingen, E., Denamur, E. & Elion, J. (1997). Highly variable mutation rates in commensal and pathogenic Escherichia coli. Science 277, 18331834.
Meats, E., Feil, E. J., Stringer, S., Cody, A. J., Goldstein, R., Kroll, J. S., Popovic, T. & Spratt, B. G. (2003). Characterization of encapsulated and noncapsulated Haemophilus influenzae and determination of phylogenetic relationships by multilocus sequence typing. J Clin Microbiol 41, 16231636.
Medeiros, A. A., Levesque, R. & Jacoby, G. A. (1986). An animal source for the ROB-1 beta-lactamase of Haemophilus influenzae type b. Antimicrob Agents Chemother 29, 212215.[Medline]
Mendelman, P. M., Roberts, M. C. & Smith, A. L. (1982). Mutation frequency of Haemophilus influenzae to rifampin resistance. Antimicrob Agents Chemother 22, 531533.[Medline]
Mendelman, P. M., Chaffin, D. O., Stull, T. L., Rubens, C. E., Mack, K. D. & Smith, A. L. (1984). Characterization of non-beta-lactamase-mediated ampicillin resistance in Haemophilus influenzae. Antimicrob Agents Chemother 26, 235244.[Medline]
Mendelman, P. M., Chaffin, D. O. & Kalaitzoglou, G. (1990a). Penicillin-binding proteins and ampicillin resistance in Haemophilus influenzae. J Antimicrob Chemother 25, 525534.[Abstract]
Mendelman, P. M., Chaffin, D. O., Krilov, L. R., Kalaitzoglou, G., Serfass, D. A., Onay, O., Wiley, E. A., Overturf, G. D. & Rubin, L. G. (1990b). Cefuroxime treatment failure of nontypable Haemophilus influenzae meningitis associated with alteration of penicillin-binding proteins. J Infect Dis 162, 11181123.[Medline]
Mitchell, M. A., Skowronek, K., Kauc, L. & Goodgal, S. H. (1991). Electroporation of Haemophilus influenzae is effective for transformation of plasmid but not chromosomal DNA. Nucleic Acids Res 19, 36253628.[Abstract]
Modrich, P. & Lahue, R. (1996). Mismatch repair in replication fidelity, genetic recombination, and cancer biology. Annu Rev Biochem 65, 101133.[CrossRef][Medline]
Moxon, E. R., Rainey, P. B., Nowak, M. A. & Lenski, R. E. (1994). Adaptive evolution of highly mutable loci in pathogenic bacteria. Curr Biol 4, 2433.[Medline]
NCCLS (2001). Performance Standards for Antimicrobial Susceptibility Testing. Approved standard M100-S11. Wayne, PA: National Committee for Clinical Laboratory Standards.
Oliver, A., Canton, R., Campo, P., Baquero, F. & Blazquez, J. (2000). High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 288, 12511254.
Oliver, A., Baquero, F. & Blazquez, J. (2002). The mismatch repair system (mutS, mutL and uvrD genes) in Pseudomonas aeruginosa: molecular characterization of naturally occurring mutants. Mol Microbiol 43, 16411650.[CrossRef][Medline]
Pennington, J. E. (1981). Penetration of antibiotics into respiratory secretions. Rev Infect Dis 3, 6773.[Medline]
Pettigrew, M. M., Foxman, B., Ecevit, Z., Marrs, C. F. & Gilsdorf, J. (2002). Use of pulsed-field gel electrophoresis, enterobacterial repetitive intergenic consensus typing, and automated ribotyping to assess genomic variability among strains of nontypeable Haemophilus influenzae. J Clin Microbiol 40, 660662.
Powell, M. & Williams, J. D. (1988). In-vitro activity of cefaclor, cephalexin and ampicillin against 2458 clinical isolates of Haemophilus influenzae. J Antimicrob Chemother 21, 2731.[Abstract]
Prunier, A. L., Malbruny, B., Laurans, M., Brouard, J., Duhamel, J. F. & Leclercq, R. (2003). High rate of macrolide resistance in Staphylococcus aureus strains from patients with cystic fibrosis reveals high proportions of hypermutable strains. J Infect Dis 187, 17091716.[CrossRef][Medline]
Rayssiguier, C., Thaler, D. S. & Radman, M. (1989). The barrier to recombination between Escherichia coli and Salmonella typhimurium is disrupted in mismatch-repair mutants. Nature 342, 396401.[CrossRef][Medline]
Richardson, A. R., Yu, Z., Popovic, T. & Stojiljkovic, I. (2002). Mutator clones of Neisseria meningitidis in epidemic serogroup A disease. Proc Natl Acad Sci U S A 99, 61036107.
Schaaper, R. M. & Dunn, R. L. (1987). Spectra of spontaneous mutations in Escherichia coli strains defective in mismatch correction: the nature of in vivo DNA replication errors. Proc Natl Acad Sci U S A 84, 62206224.[Abstract]
Sharma, A., Kaur, R., Ganguly, N. K., Singh, P. D. & Chakraborti, A. (2002). Subtype distribution of Haemophilus influenzae isolates from north India. J Med Microbiol 51, 399404.
Sniegowski, P. D., Gerrish, P. J. & Lenski, R. E. (1997). Evolution of high mutation rates in experimental populations of E. coli. Nature 387, 703705.[CrossRef][Medline]
Steers, E. E., Foltz, L., Graves, B. S. & Riden, J. (1959). An inocula replicating apparatus for routine testing of bacterial susceptibility of antibiotics. Antibiot Chemother 9, 307311.
Strauss, B. S. (1999). Frameshift mutation, microsatellites and mismatch repair. Mutat Res 437, 195203.[CrossRef][Medline]
Syriopoulou, V. P., Scheifele, D. W., Sack, C. M. & Smith, A. L. (1979). Effect of inoculum size on the susceptibility of Haemophilus influenzae b to beta-lactam antibiotics. Antimicrob Agents Chemother 16, 510513.[Medline]
Tachiki, H., Kato, R., Masui, R., Hasegawa, K., Itakura, H., Fukuyama, K. & Kuramitsu, S. (1998). Domain organization and functional analysis of Thermus thermophilus MutS protein. Nucleic Acids Res 26, 41534159.
Taddei, F., Radman, M., Maynard-Smith, J., Toupance, B., Gouyon, P. H. & Godelle, B. (1997a). Role of mutator alleles in adaptive evolution. Nature 19, 700702.
Taddei, F., Vulic, M., Radman, M. & Matic, I. (1997b). Genetic variability and adaptation to stress. In Environmental Stress, Adaptation, and Evolution, pp. 271290. Edited by R. Bijlsma & V. Loeschcke. Basel, Switzerland: Birkhauser.
Tang, C. M., Hood, D. W. & Moxon, E. R. (2001). Pathogenesis of Haemophilus influenzae Infections. In Principles of Bacterial Pathogenesis, pp. 675716. Edited by E. A. Groisman. St Louis, MO: Academic Press.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.[Abstract]
van Belkum, A., Duim, B., Regelink, A., Moller, L., Quint, W. & van Alphen, L. (1994). Genomic DNA fingerprinting of clinical Haemophilus influenzae isolates by polymerase chain reaction amplification: comparison with major outer-membrane protein and restriction fragment length polymorphism analysis. J Med Microbiol 41, 6368.[Abstract]
Vega, R., Sadoff, H. L. & Patterson, M. J. (1976). Mechanisms of ampicillin resistance in Haemophilus influenzae type B. Antimicrob Agents Chemother 9, 164168.[Medline]
Watson, M. E., Jr, Jarisch, J. & Smith, A. L. (2004). Inactivation of deoxyadenosine methyltransferase (dam) attenuates Haemophilus influenzae virulence. Mol Microbiol 53, 651664.[CrossRef][Medline]
Wilcox, K. & Smith, H. (1975). Isolation and characterization of mutants of Haemophilus influenzae deficient in an adenosine 5'-triphosphate-dependent deoxyribonuclease activity. J Bacteriol 122, 443453.[Medline]
Wu, T. H. & Marinus, M. G. (1994). Dominant negative mutator mutations in the mutS gene of Escherichia coli. J Bacteriol 176, 53935400.[Abstract]
Wu, T. H. & Marinus, M. G. (1999). Deletion mutation analysis of the mutS gene in Escherichia coli. J Biol Chem 274, 59485952.
Received 10 April 2004;
revised 17 June 2004;
accepted 30 June 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |