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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

(A) Relative abundances of NTM OTUs and (B) total bacterial load in NTM culture-positive samples. (A) The mean relative abundance of NTM OTUs in the samples processed with the standard protocol was not significantly different from that observed when these samples were processed with the modified protocol (mean 0.098% and 1.21%, respectively; p = 0.08, paired t-test). (B) Total bacterial load in NTM culture-positive samples as measured by 16S rRNA gene qPCR did not significantly differ between lysis protocols. (p = 0.91, paired t-test). Error bars indicate mean and SD.
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pone.0153876.g002: (A) Relative abundances of NTM OTUs and (B) total bacterial load in NTM culture-positive samples. (A) The mean relative abundance of NTM OTUs in the samples processed with the standard protocol was not significantly different from that observed when these samples were processed with the modified protocol (mean 0.098% and 1.21%, respectively; p = 0.08, paired t-test). (B) Total bacterial load in NTM culture-positive samples as measured by 16S rRNA gene qPCR did not significantly differ between lysis protocols. (p = 0.91, paired t-test). Error bars indicate mean and SD.

Mentions: The relative abundances of NTM sequence reads in the eight samples in which NTM were detected by 16S rRNA gene sequencing after processing with the modified lysis protocol were compared to the relative abundances of NTM sequence reads detected in the same samples after processing with the standard lysis protocol. The mean relative abundance of NTM sequence reads increased from 0.098% (range 0.0%– 0.33%) with the standard lysis protocol to 1.21% (range 0.003% - 3.95%) with the modified lysis protocol; however, this difference was not statistically significant (p = 0.08, paired t-test) (Fig 2A). Based on qPCR targeting the bacterial 16S rRNA gene, total bacterial DNA did not differ between paired NTM culture-positive samples processed with the standard lysis protocol compared to the modified protocol (Fi 2B).


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)

(A) Relative abundances of NTM OTUs and (B) total bacterial load in NTM culture-positive samples. (A) The mean relative abundance of NTM OTUs in the samples processed with the standard protocol was not significantly different from that observed when these samples were processed with the modified protocol (mean 0.098% and 1.21%, respectively; p = 0.08, paired t-test). (B) Total bacterial load in NTM culture-positive samples as measured by 16S rRNA gene qPCR did not significantly differ between lysis protocols. (p = 0.91, paired t-test). Error bars indicate mean and SD.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153876.g002: (A) Relative abundances of NTM OTUs and (B) total bacterial load in NTM culture-positive samples. (A) The mean relative abundance of NTM OTUs in the samples processed with the standard protocol was not significantly different from that observed when these samples were processed with the modified protocol (mean 0.098% and 1.21%, respectively; p = 0.08, paired t-test). (B) Total bacterial load in NTM culture-positive samples as measured by 16S rRNA gene qPCR did not significantly differ between lysis protocols. (p = 0.91, paired t-test). Error bars indicate mean and SD.
Mentions: The relative abundances of NTM sequence reads in the eight samples in which NTM were detected by 16S rRNA gene sequencing after processing with the modified lysis protocol were compared to the relative abundances of NTM sequence reads detected in the same samples after processing with the standard lysis protocol. The mean relative abundance of NTM sequence reads increased from 0.098% (range 0.0%– 0.33%) with the standard lysis protocol to 1.21% (range 0.003% - 3.95%) with the modified lysis protocol; however, this difference was not statistically significant (p = 0.08, paired t-test) (Fig 2A). Based on qPCR targeting the bacterial 16S rRNA gene, total bacterial DNA did not differ between paired NTM culture-positive samples processed with the standard lysis protocol compared to the modified protocol (Fi 2B).

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