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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

Impact of lysis method on community structure.Bray-Curtis-based nonmetric multidimensional scaling (NMDS) plot showing pairwise comparison of samples processed with the standard (blue symbols) or modified (red symbols) lysis protocols. Solid symbols represent paired samples with greater separation.
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pone.0153876.g003: Impact of lysis method on community structure.Bray-Curtis-based nonmetric multidimensional scaling (NMDS) plot showing pairwise comparison of samples processed with the standard (blue symbols) or modified (red symbols) lysis protocols. Solid symbols represent paired samples with greater separation.

Mentions: The influence of the lysis protocols on bacterial community structure of the NTM culture-positive samples was assessed with a Bray-Curtis-based nonmetric multidimensional scaling (NMDS) plot (Fig 3). The majority of the sample pairs clustered closely together despite differences in lysis protocol. However, in five of the 15 sample pairs (solid symbols in Fig 3) some separation between samples was noted on the ordination plot. These 5 sample pairs tended to have higher levels of alpha diversity (higher Shannon diversity and evenness; blue and red symbols in Fig 4) than the 10 sample pairs that remained more tightly clustered despite differences in lysis protocol. Overall, the samples processed with the modified protocol had higher measures of richness and Shannon diversity than the samples processed with the standard protocol (Fig 4).


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)

Impact of lysis method on community structure.Bray-Curtis-based nonmetric multidimensional scaling (NMDS) plot showing pairwise comparison of samples processed with the standard (blue symbols) or modified (red symbols) lysis protocols. Solid symbols represent paired samples with greater separation.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153876.g003: Impact of lysis method on community structure.Bray-Curtis-based nonmetric multidimensional scaling (NMDS) plot showing pairwise comparison of samples processed with the standard (blue symbols) or modified (red symbols) lysis protocols. Solid symbols represent paired samples with greater separation.
Mentions: The influence of the lysis protocols on bacterial community structure of the NTM culture-positive samples was assessed with a Bray-Curtis-based nonmetric multidimensional scaling (NMDS) plot (Fig 3). The majority of the sample pairs clustered closely together despite differences in lysis protocol. However, in five of the 15 sample pairs (solid symbols in Fig 3) some separation between samples was noted on the ordination plot. These 5 sample pairs tended to have higher levels of alpha diversity (higher Shannon diversity and evenness; blue and red symbols in Fig 4) than the 10 sample pairs that remained more tightly clustered despite differences in lysis protocol. Overall, the samples processed with the modified protocol had higher measures of richness and Shannon diversity than the samples processed with the standard protocol (Fig 4).

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