Culture-Independent Identification of Nontuberculous Mycobacteria in Cystic Fibrosis Respiratory Samples.
Respiratory tract infections with nontuberculous mycobacteria (NTM) are increasing in prevalence and are a significant cause of lung function decline in individuals with cystic fibrosis (CF). NTM have been detected in culture-independent analyses of CF airway microbiota at lower rates than would be expected based on published prevalence data, likely due to poor lysing of the NTM cell wall during DNA extraction. We compared a standard bacterial lysis protocol with a modified method by measuring NTM DNA extraction by qPCR and NTM detection with bacterial 16S rRNA gene sequencing. The modified method improved NTM DNA recovery from spiked CF sputum samples by a mean of 0.53 log10 copies/mL for M. abscessus complex and by a mean of 0.43 log10 copies/mL for M. avium complex as measured by qPCR targeting the atpE gene. The modified method also improved DNA sequence based NTM detection in NTM culture-positive CF sputum and bronchoalveolar lavage samples; however, both qPCR and 16S rRNA gene sequencing remained less sensitive than culture for NTM detection. We highlight the limitations of culture-independent identification of NTM from CF respiratory samples, and illustrate how alterations in the bacterial lysis and DNA extraction process can be employed to improve NTM detection with both qPCR and 16S rRNA gene sequencing.
Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
Lindsay J
LJ
Caverly
Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
Lisa A
LA
Carmody
Department of Civil & Environmental Engineering, University of Michigan, Ann Arbor, Michigan, United States of America.
Sarah-Jane
SJ
Haig
Department of Civil & Environmental Engineering, University of Michigan, Ann Arbor, Michigan, United States of America.
Nadine
N
Kotlarz
Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
Linda M
LM
Kalikin
Department of Civil & Environmental Engineering, University of Michigan, Ann Arbor, Michigan, United States of America.
Lutgarde
L
Raskin
Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.
John J
JJ
LiPuma
D003550:Cystic Fibrosis / Q000382:microbiology*; D004269:DNA, Bacterial / Q000235:genetics; D006801:Humans; D009165:Mycobacterium Infections, Nontuberculous / Q000382:microbiology*; D009170:Nontuberculous Mycobacteria / Q000235:genetics*; D015995:Prevalence; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012141:Respiratory Tract Infections / Q000382:microbiology*; D013183:Sputum / Q000382:microbiology*
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S