Sputum Metagenomics

Metagenomics of Cystic Fibrosis Sputum

Metagenomics addresses the genetic potential of a sample irrespective of whether the microorganisms can be cultured. To date, approximately 3100 culturable species are known. However, this only comprises between 1 to 5 % of the potential microbial genomes in nature. This is due to a lack of knowledge as how to cultivate the unseen microbes with existing traditional culturing techniques that succeed only in recovering the same, well-characterized microbial species.

The respiratory tract has a normal resident flora. However, cystic fibrosis (CF) pulmonary infections introduce additional bacterial pathogens into the lower respiratory tract that eventually lead to chronic microbial colonization of the airways. The patients suffer from episodes of severe debilitating exacerbations as a result of both bacterial and viral infections superimposed upon progressive lung disease (2, 3). It appears that the airways of patients with CF are predisposed to infection by some bacteria more than by others. The most common microbial pathogens that have been isolated include Staphylococcus aureus, Haemophilus influenzae and Pseudomonas aeruginosa (1, 3, 5). However, there has been the emergence of other opportunistic pathogens such as Burkholderia cepacia, Alcaligenes xylosoxidans, Ralstonia pickettii, Burkholderia gladioli and Stenotrophomonas maltophilia (1, 3, 5). Susceptibility to a particular pathogen tends to be age related. S. aureus is the predominant pathogen in children, reaching a prevalence rate of nearly 50% by the age of 10 years (1, 3). S. aureus is followed by H. influenzae during early childhood and P. aeruginosa becomes the predominant pathogen in adolescence. P. aeruginosa approaches a prevalence rate of 80% in adults. The occurrence of the more recent emerging organisms increases with advancing age and increasing severity of lung disease (1, 3, 4). Once these infections are established, they almost invariably recur. Therefore, bacterial colonization of the CF lung is a dynamic process and discernible by the abundance of microorganisms in the sputum. To date, the identity of the microbes that infect the CF lung is based on cultured methods and it is likely that the majority of the microbes that colonize the CF lung are nonculturable. This is because the routine culture for bacterial pathogens from CF sputa yields limited microbiological information since it frequently fails to identify a number of pathogenic bacterial species that are potentially present in the CF lung (4). This project proposes to use select evolutionarily conserved molecular targets for the direct detection of specific organisms in the heterogeneous CF sputum samples. Microbial communities will be analyzed using amplicon length heterogeneity (ALH) DNA fingerprinting. Cloning and sequencing of the molecular targets such as 16S rDNA will identify indigenous and novel organisms.

References cited:

Beringer, P. M., and M. D. Appleman. 2000. Unusual respiratory bacterial flora in cystic fibrosis: microbiologic and clinical features. Curr Opin Pulm Med 6:545-550.

Høiby, N. 1982. Microbiology of lung infections in cystic fibrosis patients. Acta Paediatr Scan Supp 301:33-54.

Rajan, S., and L. Saiman. 2002. Pulmonary infections in patients with cystic fibrosis. Semin Respir Infect 17:47-56.

van Belkum, A., N. H. Renders, S. Smith, S. E. Overbeek, and H. A. Verbrugh. 2000. Comparison of conventional and molecular methods for the detection of bacterial pathogens in sputum samples from cystic fibrosis patients. FEMS Immunol Med Microbiol 27:51-57.

Whittier, S. 2001. Update on the microbiology of cystic fibrosis: traditional and emerging pathogens. Clinical Microbiol Newsletter 23:67-71.

Mathee Publications

S.Z. Miller. (2003) Molecular identification of microbes in the sputum of cystic fibrosis patients. Masters in Forensics thesis.

Students and Postdoctoral fellows involved:

Visiting Scientist Professor Chong-Lek Koh Sabbatical Visitor

Post-doc Dr. DeEtta Mills

Graduate Students Ms. Melissa Doud

Past Students Ms. Sasha Miller (Fall 2003 Past)
Ms. Sheria King (Summer 2004)