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Culture-Independent Identification of Nontuberculous Mycobacteria in Cystic Fibrosis Respiratory Samples.

Caverly LJ, Carmody LA, Haig SJ, Kotlarz N, Kalikin LM, Raskin L, LiPuma JJ - PLoS ONE (2016)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Improvement in NTM DNA extraction from spiked sputum samples with the modified as compared to the standard lysis protocol.Log10atpE gene copies/mL in DNA extracted from sputum spiked with either (A) MABSC or (B) MAC using the standard (blue circles) or the modified (red squares) lysis protocols. Error bars indicate mean and SD.
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pone.0153876.g001: Improvement in NTM DNA extraction from spiked sputum samples with the modified as compared to the standard lysis protocol.Log10atpE gene copies/mL in DNA extracted from sputum spiked with either (A) MABSC or (B) MAC using the standard (blue circles) or the modified (red squares) lysis protocols. Error bars indicate mean and SD.

Mentions: Based on qPCR of the mycobacterial atpE gene, the use of the modified lysis protocol significantly increased the recovery of NTM DNA from the NTM-spiked sputum samples for each of the four NTM strains (Fig 1). The mean (SD) increases in log10atpE gene copies/mL using the modified protocol compared to the standard protocol were 0.56 (0.19) for MABSC ATCC 19977 (p = 0.001, paired t-test), 0.51 (0.03) for the MABSC clinical isolate (p<0.001, paired t-test), 0.45 (0.23) for MAC ATCC 25291 (p = 0.005, paired t-test), and 0.4 (0.1) for the MAC clinical isolate (p = 0.004, paired t-test).


Culture-Independent Identification of Nontuberculous Mycobacteria in Cystic Fibrosis Respiratory Samples.

Caverly LJ, Carmody LA, Haig SJ, Kotlarz N, Kalikin LM, Raskin L, LiPuma JJ - PLoS ONE (2016)

Improvement in NTM DNA extraction from spiked sputum samples with the modified as compared to the standard lysis protocol.Log10atpE gene copies/mL in DNA extracted from sputum spiked with either (A) MABSC or (B) MAC using the standard (blue circles) or the modified (red squares) lysis protocols. Error bars indicate mean and SD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836755&req=5

pone.0153876.g001: Improvement in NTM DNA extraction from spiked sputum samples with the modified as compared to the standard lysis protocol.Log10atpE gene copies/mL in DNA extracted from sputum spiked with either (A) MABSC or (B) MAC using the standard (blue circles) or the modified (red squares) lysis protocols. Error bars indicate mean and SD.
Mentions: Based on qPCR of the mycobacterial atpE gene, the use of the modified lysis protocol significantly increased the recovery of NTM DNA from the NTM-spiked sputum samples for each of the four NTM strains (Fig 1). The mean (SD) increases in log10atpE gene copies/mL using the modified protocol compared to the standard protocol were 0.56 (0.19) for MABSC ATCC 19977 (p = 0.001, paired t-test), 0.51 (0.03) for the MABSC clinical isolate (p<0.001, paired t-test), 0.45 (0.23) for MAC ATCC 25291 (p = 0.005, paired t-test), and 0.4 (0.1) for the MAC clinical isolate (p = 0.004, paired t-test).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics and Communicable Diseases, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus