Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Waine, D. J. Articles by Dowson, C. G. Search for Related Content PubMed PubMed Citation Articles by Waine, D. J. Articles by Dowson, C. G.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, October 2008, p. 3491-3493, Vol. 46, No. 10
0095-1137/08/$08.00+0     doi:10.1128/JCM.00357-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Association between Hypermutator Phenotype, Clinical Variables, Mucoid Phenotype, and Antimicrobial Resistance in Pseudomonas aeruginosa

David J. Waine,^1,2* David Honeybourne,^1,2 E. Grace Smith,³ Joanna L. Whitehouse,² and Chris G. Dowson¹

Department of Biological Sciences, Warwick University, Coventry CV4 7AL, United Kingdom,¹ West Midlands Adult CF Unit, Heart of England Foundation Trust, Birmingham B9 5SS, United Kingdom,² Department of Microbiology, Heart of England Foundation Trust, Birmingham B9 5SS, United Kingdom³

Received 21 February 2008/ Returned for modification 29 March 2008/ Accepted 26 July 2008

Top ABSTRACT TEXT REFERENCES

 
The presence of hypermutator Pseudomonas aeruginosa was associated with poorer lung function in patients at the Adult West Midlands CF Unit. Mucoid isolates were more likely to be hypermutators. The presence of resistant mutant subpopulations was associated with hypermutator phenotype but was not good enough to be used as a test for this phenotype.

Top ABSTRACT TEXT REFERENCES

 
Patients with cystic fibrosis (CF) usually become infected with P. aeruginosa (5), which accelerates decline in lung function and increases mortality (4). Patients with CF commonly harbor hypermutator isolates of this bacterium (3, 16), the prevalence of which increases with duration of infection (3), but no other clinical correlates of hypermutator presence have been determined. Hypermutators are more resistant to antimicrobials in vitro (3, 11) and in vivo (13) and may increase the fitness of bacterial populations during environmental stress (17).

We examined 153 isolates from 40 adult patients with CF who had been infected with P. aeruginosa for at least 12 months. From their sputum samples (one to nine per patient, mean of three), isolates identified as P. aeruginosa by growth on isolation agar were tested against a panel of nine antimicrobials (ciprofloxacin, ceftazidime, piperacillin-tazobactam, gentamicin, meropenem, tobramycin, colistin, amikacin, and aztreonam) using the standard BSAC agar disk diffusion technique (2). Different morphotypes were tested separately (one to two per sample). Hypermutators were identified by using growth on rifampin-containing agar as described previously (11) but using the Miles-Misra dilution, counting colonies in 10-µl spots of serial dilutions (14). Ethical approval was granted by the local Research Ethics Committee.

A total of 47 isolates (30.7%) were hypermutators, and 16 patients (40.0%) had at least one hypermutator, in keeping with previous studies (3, 16). Patients with hypermutators had significantly worse lung function (Table 1), for which there are several possible explanations. Hypermutator prevalence increases with duration of infection (3), and it is possible that worse lung function is merely a marker of longer infection. Patients with worse lung function might also be expected to undergo more frequent antimicrobial courses, which might select for hypermutator development, although we did not find an association between frequency of intravenous courses and hypermutator phenotype. A further possibility is that patients with poorer lung function seek medical advice more frequently and therefore provide more sputum samples, thus increasing the likelihood of detection of hypermutators. Consistent with this possibility, patients with hypermutators provided more sputum samples during the period of the present study (Table 1).

View this table: [in this window] [in a new window]

TABLE 1. Patient demographics and clinical dataa

There were 76 nonmucoid isolates (mean, 1.9 per patient), of which 13 (17.1%) were hypermutators. There were 77 mucoid isolates (mean, 1.9 per patient), of which 34 (44.2%) were hypermutators (² test, P < 0.001). Alhough this is the first time this association has been demonstrated, it is not surprising because both of these phenotypes increase with duration of infection in the CF lung (3, 10). The mucoid phenotype is thought to protect bacteria against certain antimicrobials (1, 7), innate immune factors (9), and reactive oxygen species (8), and hypermutators increase the fitness of a bacterial population undergoing stress and increase the likelihood of antimicrobial resistance (11, 13, 15, 17). There may also be an interaction between the two phenotypes; hypermutators undergo mutations at a higher rate and are therefore more likely to acquire a mutation resulting in the mucoid phenotype.

Hypermutators were not resistant to more of the panel of nine antimicrobials than nonhypermutators (mean, 2.81 versus 2.86; Mann-Whitney U test, P = 0.971), although significantly fewer were pansensitive (13% versus 27%; ², P = 0.047). A receiver-operator-characteristic curve describing resistance as a potential test for hypermutator phenotype had an area under the curve of 0.502 (where 0.5 indicates the test is no better than chance), implying a very poor predictive power (Fig. 1A). This was surprising given previous findings that hypermutators had higher MICs than nonhypermutators (3, 11, 15, 16). Although the use of MICs would have given more detailed data, most clinical laboratories use the disk diffusion method, and so our results are of practical interest. While the unreliability of disk diffusion in chronic P. aeruginosa infection should be borne in mind (6), it is likely that, in this particular population, the association between resistance and hypermutator phenotype was weakened by another factor. Consistent with this explanation was the presence of epidemic strains in the data set; as determined by multilocus sequence typing, 50% of the typed isolates were double-locus variants of previously described epidemic strains (Clone C, Midlands-1, and Liverpool). Also, our patients were all adults and would therefore have been exposed to more antimicrobials and thus developed more antimicrobial resistance.

View larger version (11K): [in this window] [in a new window]

FIG. 1. Receiver-operator-characteristic curve for tests of hypermutator status using resistance (0 to 9) (A) and resistant mutant subpopulations (B).

The association between hypermutator phenotype and resistant mutant subpopulations (RMS) was tested in a subset of 40 isolates by recording the presence of RMS in the zones of five antimicrobial disks (ceftazidime, tobramycin, ciprofloxacin, meropenem, and imipenem) as described previously (12). The subset was chosen as follows: a group of 20 nonhypermutators and a group of 20 hypermutators were examined, and in each group there were 10 isolates with resistance to five or more of the panel of nine antimicrobials and 10 isolates with resistance to two or fewer of the panel of nine antimicrobials.

Nine (45%) hypermutators had RMS in three or more antimicrobial disk zones, compared to two (10%) nonhypermutators (² test, P = 0.013), replicating the association demonstrated by Macia et al. (12). In contrast to those results, the association noted here was not strong enough to allow RMS to be used as a test for hypermutators in our population. Macia et al.'s cutoff of three or more RMS to identify a hypermutator would give a sensitivity of 45% and a specificity of 90%, and the receiver-operator-characteristic curve for RMS as a test of hypermutator phenotype would have an area under the curve of 0.703 (Fig. 1B). This contradictory finding may be, as discussed for antimicrobial resistance, a result of differences between the patient and microbiological populations between the two studies. If so, it is important that laboratories considering the use of RMS as a screening test for hypermutators are aware of the problem.

In summary, hypermutators from this population of adult CF patients were not resistant to more antimicrobials than nonhypermutators, as tested by the disk diffusion method, but were significantly associated with the mucoid morphotype. In contrast to previous results, resistant mutant subpopulations could not be used to test for the hypermutator phenotype. Hypermutator presence was significantly associated with poorer lung function, although this variable was correlated with the number of sputum samples provided.

 
This research was funded by the Heart of England Foundation Trust and the Birmingham Heartlands Cystic Fibrosis Appeal Charity.

We thank Cerith Harries (Warwick University), Alan Jackson, Helen Charles, and the rest of the sputum lab and BSAC bench at the Heart of England Foundation Trust microbiology laboratory. We also thank Adam Baldwin, Frances Bolt, Anna Gao, and Eleanor Pinnock for their help and support with laboratory work.

 
* Corresponding author. Mailing address: West Midlands Adult CF Unit, Heart of England Foundation Trust, Birmingham B9 5SS, United Kingdom. Phone: 44(0)121 424 5670. Fax: 44(0)121 424 1661. E-mail: djwaine{at}doctors.org.uk

Published ahead of print on 6 August 2008.

Top ABSTRACT TEXT REFERENCES

 

1
1. Alkawash, M. A., J. S. Soothill, and N. L. Schiller. 2006. Alginate lyase enhances antibiotic killing of mucoid Pseudomonas aeruginosa in biofilms. APMIS 114:131-138.[CrossRef][Medline]
2
2. Andrews, J. M. 2006. BSAC standardized disc susceptibility testing method (version 5). J. Antimicrob. Chemother. 58:511-529.[Free Full Text]
3
3. Ciofu, O., B. Riis, T. Pressler, H. E. Poulsen, and N. Hoiby. 2005. Occurrence of hypermutable Pseudomonas aeruginosa in cystic fibrosis patients is associated with the oxidative stress caused by chronic lung inflammation. Antimicrob. Agents Chemother. 49:2276-2282.[Abstract/Free Full Text]
4
4. Cystic Fibrosis Foundation. 1996. Cystic fibrosis foundation registry: 1996 annual data report. Cystic Fibrosis Foundation, Bethesda, MD.
5
5. Cystic Fibrosis Trust Clinical Standards and Accreditation Group. 2001. Standards for the clinical care of children and adults with cystic fibrosis in the UK. Cystic Fibrosis Trust Clinical Standards and Accreditation Group, London, United Kingdom.
6
6. Foweraker, J. E., C. R. Laughton, D. F. Brown, and D. Bilton. 2005. Phenotypic variability of Pseudomonas aeruginosa in sputa from patients with acute infective exacerbation of cystic fibrosis and its impact on the validity of antimicrobial susceptibility testing. J. Antimicrob. Chemother. 55:921-927.[Abstract/Free Full Text]
7
7. Hentzer, M., G. M. Teitzel, G. J. Balzer, A. Heydorn, S. Molin, M. Givskov, and M. R. Parsek. 2001. Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function. J. Bacteriol. 183:5395-5401.[Abstract/Free Full Text]
8
8. Learn, D. B., E. P. Brestel, and S. Seetharama. 1987. Hypochlorite scavenging by Pseudomonas aeruginosa alginate. Infect. Immun. 55:1813-1818.[Abstract/Free Full Text]
9
9. Leid, J. G., C. J. Willson, M. E. Shirtliff, D. J. Hassett, M. R. Parsek, and A. K. Jeffers. 2005. The exopolysaccharide alginate protects Pseudomonas aeruginosa biofilm bacteria from IFN--mediated macrophage killing. J. Immunol. 175:7512-7518.[Abstract/Free Full Text]
10
10. Li, Z., M. R. Kosorok, P. M. Farrell, A. Laxova, S. E. West, C. G. Green, J. Collins, M. J. Rock, and M. L. Splaingard. 2005. Longitudinal development of mucoid Pseudomonas aeruginosa infection and lung disease progression in children with cystic fibrosis. JAMA 293:581-588.[Abstract/Free Full Text]
11
11. Macia, M. D., D. Blanquer, B. Togores, J. Sauleda, J. L. Perez, and A. Oliver. 2005. Hypermutation is a key factor in development of multiple-antimicrobial resistance in Pseudomonas aeruginosa strains causing chronic lung infections. Antimicrob. Agents Chemother. 49:3382-3386.[Abstract/Free Full Text]
12
12. Macia, M. D., N. Borrell, J. L. Perez, and A. Oliver. 2004. Detection and susceptibility testing of hypermutable Pseudomonas aeruginosa strains with the Etest and disk diffusion. Antimicrob. Agents Chemother. 48:2665-2672.[Abstract/Free Full Text]
13
13. Macia, M. D., N. Borrell, M. Segura, C. Gomez, J. L. Perez, and A. Oliver. 2006. Efficacy and potential for resistance selection of antipseudomonal treatments in a mouse model of lung infection by hypermutable Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 50:975-983.[Abstract/Free Full Text]
14
14. Miles, A. A., S. S. Misra, and J. O. Irwin. 1938. The estimation of bactericidal power of blood. J. Hyg. 38:733-749.
15
15. Oliver, A., B. R. Levin, C. Juan, F. Baquero, and J. Blazquez. 2004. Hypermutation and the preexistence of antibiotic-resistant Pseudomonas aeruginosa mutants: implications for susceptibility testing and treatment of chronic infections. Antimicrob. Agents Chemother. 48:4226-4233.[Abstract/Free Full Text]
16
16. Oliver, A., R. Canton, P. Campo, F. Baquero, and J. Blazquez. 2000. High frequency of hypermutable Pseudomonas aeruginosa in cystic fibrosis lung infection. Science 288:1251-1254.[Abstract/Free Full Text]
17
17. Taddei, F., M. Radman, J. Maynard-Smith, B. Toupance, P. H. Gouyon, and B. Godelle. 1997. Role of mutator alleles in adaptive evolution. Nature 387:700-702.[CrossRef][Medline]

---

Journal of Clinical Microbiology, October 2008, p. 3491-3493, Vol. 46, No. 10
0095-1137/08/$08.00+0     doi:10.1128/JCM.00357-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Louie, A., Fregeau, C., Liu, W., Kulawy, R., Drusano, G. L. (2009). Pharmacodynamics of Levofloxacin in a Murine Pneumonia Model of Pseudomonas aeruginosa Infection: Determination of Epithelial Lining Fluid Targets. Antimicrob. Agents Chemother. 53: 3325-3330 [Abstract] [Full Text]  
• Mena, A., Smith, E. E., Burns, J. L., Speert, D. P., Moskowitz, S. M., Perez, J. L., Oliver, A. (2008). Genetic Adaptation of Pseudomonas aeruginosa to the Airways of Cystic Fibrosis Patients Is Catalyzed by Hypermutation. J. Bacteriol. 190: 7910-7917 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Waine, D. J. Articles by Dowson, C. G. Search for Related Content PubMed PubMed Citation Articles by Waine, D. J. Articles by Dowson, C. G.