Changes in cystic fibrosis sputum microbiology in the United States between 1995 and 2008

Pediatr Pulmonol. 2010 Mar 15.

Changes in cystic fibrosis sputum microbiology in the United States between 1995 and 2008.

Emerson J, McNamara S, Buccat AM, Worrell K, Burns JL.

Seattle Children's Hospital, Seattle, Washington.

STUDY OBJECTIVES: The study objective was to identify changes in cystic fibrosis (CF) sputum microbiology over 13 years.

METHODS: This study recruited a contemporary cohort of CF patients meeting similar eligibility criteria as the 520 subjects in the Phase 3 trials of inhaled tobramycin (historical cohort). Subjects submitted a single sputum specimen to a centralized laboratory for culture and susceptibility testing. Data were summarized and cross-sectional prevalence estimates were compared between cohorts. Exploratory analyses examined associations between recent antibiotic exposures and resistance prevalence.

RESULTS: Sputum samples from 267 subjects from 33 US CF centers were submitted for testing. A total of 656 Pseudomonas aeruginosa isolates were identified from 253 culture-positive subjects. Comparison between cohorts revealed an increase in the prevalence of subjects with tobramycin resistant (11.8% vs. 30.4%, P < 0.001) and amikacin resistant (24.2% vs. 42.7%, P < 0.001) P. aeruginosa. Prevalence of ciprofloxacin resistance was similar (34.4% vs. 33.6%, P = 0.81). Within the contemporary cohort, potential associations between recent antibiotic exposures and prevalence of P. aeruginosa resistance were examined; findings included that exposure to intravenous carbapenems was significantly associated with aztreonam resistance, meropenem resistance, and multidrug resistance (P = 0.0002, P = 0.0003, and P = 0.0002, respectively). Prevalences of Staphylococcus aureus, methicillin-resistant S. aureus, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans were also increased in the contemporary cohort.

CONCLUSIONS: We identified important changes in the patterns of CF airway microbiology, including increased aminoglycoside resistance and prevalence of other antibiotic resistant organisms. These changes are concerning to clinicians caring for individuals with CF because they impact treatment options. These data point to a critical need to develop new antimicrobials for CF. Pediatr Pulmonol. (c) 2010 Wiley-Liss, Inc.

Studying bacteria in respiratory specimens by using conventional and molecular microbiological approaches

Studying bacteria in respiratory specimens by using conventional and molecular microbiological approaches

Geraint B Rogers,¹ Thomas WV Daniels,² Andrew Tuck,³ Mary P Carroll,⁴ Gary J Connett,⁵ Gondi JP David,¹ and Kenneth D Bruce¹

Background

Drawing from previous studies, the traditional routine diagnostic microbiology evaluation of samples from chronic respiratory conditions may provide an incomplete picture of the bacteria present in airways disease. Here, the aim was to determine the extent to which routine diagnostic microbiology gave a different assessment of the species present in sputa when analysed by using culture-independent assessment.

Methods

Six different media used in routine diagnostic microbiology were inoculated with sputum from twelve patients. Bacterial growth on these plates was harvested and both RNA and DNA extracted. DNA and RNA were also extracted directly from the same sample of sputum. All nucleic acids served as templates for PCR and reverse transcriptase-PCR amplification of "broad range" bacterial 16S rRNA gene regions. The regions amplified were separated by Terminal Restriction Fragment Length Polymorphism (T-RFLP) profiling and compared to assess the degree of overlap between approaches.

Results

A mean of 16.3 (SD 10.0) separate T-RF band lengths in the profiles from each sputum sample by Direct Molecular Analysis, with a mean of 8.8 (SD 5.8) resolved by DNA profiling and 13.3 (SD 8.0) resolved by RNA profiling. In comparison, 8.8 (SD 4.4) T-RF bands were resolved in profiles generated by Culture-derived Molecular Analysis. There were a total of 184 instances of T-RF bands detected in the direct sputum profiles but not in the corresponding culture-derived profiles, representing 83 different T-RF band lengths. Amongst these were fifteen instances where the T-RF band represented more than 10% of the total band volume (with a mean value of 23.6%). Eight different T-RF band lengths were resolved as the dominant band in profiles generated directly from sputum. Of these, only three were detected in profiles generated from the corresponding set of cultures.

Conclusion

Due to their focus on isolation of a small group of recognised pathogens, the use of culture-dependent methods to analyse samples from chronic respiratory infections can provide a restricted understanding of the bacterial species present. The use of a culture-independent molecular approach here identifies that there are many bacterial species in samples from CF and COPD patients that may be clinically relevant.

Full text of the publication available here: http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=19368727