1) Evolution of antibiotic resistance during cystic fibrosis
(i). Hoffman, L.R., Déziel, E., D’Argenio, D. A., Lépine, F., Emerson, J., McNamara, S., Gibson, R.L., Ramsey, B.W., and Miller, S. I. Selection for Staphylococcus aureus small-colony variants due to growth in the presence of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 2006 Dec 26;103(52):19890-5. PMID: 17172450.
- Pseudomonas aeruginosa and Staphylococcus aureus are the two bacterial species isolated most frequently from CF airways. We present evidence that the interspecies interaction between these bacteria selects for antibiotic-resistant S. aureus adapted mutants, known as small-colony variants, that are common in CF patients. These results demonstrate that interspecies interactions can drive adaptation during chronic, polymicrobial infections.
(ii). Hayden, H.S., Gillett, W., Saenphimmachak, C., Lim, R., Zhou, Y., Jacobs, M.A., Chang, J., Rohmer, L., D’Argenio, D.A., Palmieri, A., Levy, R., Haugen, E., Wong, G. K., Brittnacher, M.J., Burns, J.L., Miller, S.I., Olson, M.V., and Kaul, R. Large-insert genome analysis technology detects structural variation in Pseudomonas aeruginosa clinical strains from cystic fibrosis patients. Genomics. 2008 Jun;91(6):530-7. PMID: 18445516.
- Large-insert genome analysis (LIGAN) is a broadly applicable, high-throughput technology designed to characterize genome-scale structural variation. Clonal pairs of Pseudomonas aeruginosa isolates from four cystic fibrosis patients were used to validate the LIGAN technology. Approximately 1.5 Mb of inserted sequences were identified, including 743 kb containing 615 ORFs that are absent from published P. aeruginosa genomes. Six rearrangement breakpoints and 220 kb of deleted sequences were also identified. Our study expands the “genome universe” of P. aeruginosa and validates a technology that complements emerging, short-read sequencing methods that are better suited to characterizing single-nucleotide polymorphisms than structural variation.
(iii). Beckmann, C., M. Brittnacher, R. Ernst, N. Mayer-Hamblett, S. I. Miller, and J. L. Burns. 2005. Use of phage display to identify potential Pseudomonas aeruginosa gene products relevant to early cystic fibrosis airway infections. Infect. Immun. 73:444-52. PMID: 15618183.
- We identified 76 P. aeruginosa proteins recognized by antibodies in CF patients generated during the first year of P. aeruginosa infection. Such proteins, expressed early in infection, may provide supporting evidence for candidate phenotypic markers of early bacterial adaptation to the CF airway.
(iv). Wu, M., T. Guina, M. Brittnacher, H. Nguyen, J. Eng, and S. I. Miller. 2005. The Pseudomonas aeruginosa proteome during anaerobic growth. J. Bacteriol. 187:8185-90. PMID: 16291692 .
- We used quantitative proteomic technology to identify the physiological responses of P. aeruginosa to low oxygen, conditions likely to be relevant for the bacterium growing in mucus-filled regions of the CF airway.
(v). Ernst, R. K., K. N. Adams, S. M. Moskowitz, G. M. Kraig, K. Kawasaki, C. M. Stead, M. S. Trent, and S. I. Miller. 2006. The Pseudomonas aeruginosa lipid A deacylase: selection for expression and loss within the cystic fibrosis airway. J. Bacteriol. 188:191-201. PMID: 16352835.
- We showed that the evolution of P. aeruginosa in CF includes stereotypical patterns of change in expression of an enzyme involved in bacterial lipopolysaccharide modification.
(vi). Ernst RK, Moskowitz SM, Emerson JC, Kraig GM, Adams KN, Harvey MD, Ramsey B, Speert DP, Burns JL, and S.I. Miller. 2007. Unique lipid A modifications in Pseudomonas aeruginosa isolated from the airways of patients with cystic fibrosis. J Infect Dis. 196(7):1088-92. PMID:17763333
- The results of this study extend our initial finding that isolates from CF patients have a specific lipopolysaccharide structure, and show that the relevant structural modifications are both highly prevalent and unique to these isolates.
(vii). Smith, E. E., D. G. Buckley, Z. Wu, C. Saenphimmachak, L. R. Hoffman, D. A. D’Argenio, S. I. Miller, B. W. Ramsey, D. P. Speert, S. M. Moskowitz, J. L. Burns, R. Kaul, and M. V. Olson. 2006. Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc. Natl. Acad. Sci. USA 103:8487-92. PMID: 16687478.
- To define the adaptive changes undergone by P. aeruginosa during chronic CF infections (particularly early adaptive changes), we used both whole-genome and targeted sequencing to examine paired, genetically-related isolates from individual young children with CF. We showed that the P. aeruginosa lineage in one CF patient underwent extensive genetic adaptation, accumulating 68 mutations during 8 years of chronic infection. This adaptation included changes unique to the particular patient as well as highly conserved genotypes found among the P. aeruginosa isolates from 29 more patients, reflecting evolutionary fitness. Such conserved adaptations included inactivation of the global regulatory protein LasR (encoded by the lasR gene) and inactivation of a repressor of the MexXY aminoglycoside efflux system. Additionally, there were other conserved changes, resulting in the striking loss of most of the known acute virulence determinants, such as Type III secretion, exoenzymes, exotoxins, and the major siderophore. Among these changes, lasR mutation was the most common adaptive change among the isolates studied. This study provides a new perspective for delineating the traits that define opportunistic pathogens, such as P. aeruginosa. Furthermore, this study strongly suggests that to understand the factors underlying P. aeruginosa persistence during chronic infection, clinical isolates must be studied, in particular bacterial lineages from multiple patients beginning with sequential isolates sampled early in infection.
(viii). D’Argenio DA, Wu M, Hoffman LR, Kulasekara HD, Déziel E, Smith EE, Nguyen H, Ernst RK, Larson Freeman TJ, Spencer DH, Brittnacher M, Hayden HS, Selgrade S, Klausen M, Goodlett DR, Burns JL, Ramsey BW, Miller SI. Growth phenotypes of Pseudomonas aeruginosa lasR mutants adapted to the airways of cystic fibrosis patients. Mol Microbiol. 2007 Apr;64(2):512-33. PMID: 17493132.
- Pseudomonas aeruginosa undergoes genetic change during chronic infection of the airways of CF patients. The most common such adaptive change identified among 30 CF patients in Smith et al (above), was mutation of lasR. lasR encodes a transcriptional regulator that responds to one of the quorum sensing signals in P. aeruginosa, and regulates acute virulence factor expression, as well as central metabolic functions. P. aeruginosa lasR mutants emerged in the airways of CF patients early during chronic infection, and also during growth in the laboratory on Luria-Bertani agar. Both environments are rich in amino acids. Inactivating mutation in lasR in these isolates conferred a growth advantage with amino acids, a phenotype that could in part account for selection of lasR mutants both in vivo and in vitro. Furthermore, inactivating mutation in lasR led to increased expression of b-lactamase activity, resulting in resistance to b-lactam antibiotics, including ceftazidime. The high prevalence of lasR mutations, and the strong phenotypes predicted to be of clinical importance conferred by lasR mutation, have led to the current research focus on this adaptive change.
(ix). 10. Hoffman, L.R., H.D. Kulasekara, J. Emerson, L. Houston, J.L. Burns, B.W. Ramsey, and S.I. Miller. Pseudomonas aeruginosa lasR mutants are associated with cystic fibrosis lung disease progression
- Here, we performed an exploratory, cross-sectional, retrospective investigation of the prevalence of lasR mutant isolates among children attending the local pediatric CF clinic, and compared age and lung function values among the source patients.