Molecular identification of bacteria in bronchoalveolar lavage fluid from children with cystic fibrosis

Abstract

Culture of bronchoalveolar lavage fluid (BALF) is the gold standard for detection of pathogens in the lower airways in cystic fibrosis (CF). However, current culture results do not explain all clinical observations in CF, including negative culture results during pulmonary exacerbation and inflammation in the absence of pathogens. We hypothesize that organisms not routinely identified by culture occur in the CF airway and may contribute to disease. To test this hypothesis we used a culture-independent molecular approach, based on use of rRNA sequence analysis, to assess the bacterial composition of BALF from children with CF and disease controls (DC). Specimens from 42 subjects (28 CF) were examined, and approximately 6,600 total clones were screened to identify 121 species of bacteria. In general, a single rRNA type dominated clone libraries from CF specimens, but not DC. Thirteen CF subjects contained bacteria that are not routinely assessed by culture. In four CF subjects, candidate pathogens were identified and include the anaerobe Prevotella denticola, a Lysobacter sp., and members of the Rickettsiales. The presumptive pathogens Tropheryma whipplei and Granulicatella elegans were identified in cases from the DC group. The presence of unexpected bacteria in CF may explain inflammation without documented pathogens and consequent failure to respond to standard treatment. These results show that molecular techniques provide a broader perspective on airway bacteria than do routine clinical cultures and thus can identify targets for further clinical evaluation.

Fig. 1.

Flow chart of specimens and PCR results for CF and DC specimens. Not all specimens were positive for PCR. For each specimen one to three processing types were examined. In 22 cases multiple libraries were constructed from the same specimen. Fifteen specimens were positive for PCR with both the hard- and soft-spin pellets and are labeled P1/P2. Replicate libraries were made in the remaining seven cases and are labeled replicates. Specimens with a single library constructed are labeled single.

Fig. 2.

Bacterial community comparisons of BALF-derived PCR libraries were determined with weighted UniFrac, and the relationships are displayed by the unweighted pair group method with arithmetic mean clustering algorithm. The numbers in the dendogram represent the jackknife support for the labeled nodes. Cohort and patient numbers were used to label each subject. Asterisks are used to mark CF subjects with samples that did not yield any pathogenic isolates. The bars represent the percentage of clones indicative of aerobic organisms (open) and anaerobic organisms (filled). The number of samples, specimens, clones identified by sequence (including RFLP counts), organisms, and prevalence of the most common organism identified are also given. The tree is labeled by phylogenetic identity (genus and division) of the most common organism identified in each subject.

Fig. 3.

Distribution of the 10 most prevalent organisms observed by subject groups. (A) Summary of the CF cohort. (B) The same comparisons as in A for the DC cohort. Organism names are abbreviated as follows: Sau, S. aureus; Sma, S. maltophilia; Pae, P. aeruginosa; Smg, Streptococcus mitis group; Hin, H. influenzae; Len, Lysobacter enzymogenes; Axy, A. xylosoxidans; Mab, Mycobacterium abscessus; CW, Porphyromonas sp. CW034;, Pme, Prevotella melaninogenica; Gad, Granulicatella adiacens; IK, Prevotella sp. IK062; Ssa; Streptococcus sanguis; Twh, Tropheryma whipplei; Fnu, Fusobacterium nucleatum; Cle, Capnocytophaga leadbetteri. The numbers in parentheses give the numbers of subjects in which each organism was observed.

Fig. 4.

Pie charts of the distribution of sequences from selected libraries that contained unexpected sequences. (A–D) Subjects with CF. (E and F) DC subjects. Dark gray portions represent sequences that were not expected based on traditional culture results. Light gray portions represent sequences representative of pathogens detected by both molecular and culture methods.

Fig. 5.

Summary of major CF pathogens detected by both culture and molecular analysis with all-bacteria primers. Dark bars represent detection by both methods, gray bars represent detection by molecular methods only, and empty bars represent detection by culture only.