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Published in: Gut Pathogens 1/2016

Open Access 01-12-2016 | Research

Inherent bacterial DNA contamination of extraction and sequencing reagents may affect interpretation of microbiota in low bacterial biomass samples

Authors: Angela Glassing, Scot E. Dowd, Susan Galandiuk, Brian Davis, Rodrick J. Chiodini

Published in: Gut Pathogens | Issue 1/2016

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Abstract

Background

The advent and use of highly sensitive molecular biology techniques to explore the microbiota and microbiome in environmental and tissue samples have detected the presence of contaminating microbial DNA within reagents. These microbial DNA contaminants may distort taxonomic distributions and relative frequencies in microbial datasets, as well as contribute to erroneous interpretations and identifications.

Results

We herein report on the occurrence of bacterial DNA contamination within commonly used DNA extraction kits and PCR reagents and the effect of these contaminates on data interpretation. When compared to previous reports, we identified an additional 88 bacterial genera as potential contaminants of molecular biology grade reagents, bringing the total number of known contaminating microbes to 181 genera. Many of the contaminants detected are considered normal inhabitants of the human gastrointestinal tract and the environment and are often indistinguishable from those genuinely present in the sample.

Conclusions

Laboratories working on bacterial populations need to define contaminants present in all extraction kits and reagents used in the processing of DNA. Any unusual and/or unexpected findings need to be viewed as possible contamination as opposed to unique findings.
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Literature
1.
go back to reference Pace NR. A molecular view of microbial diversity and the biosphere. Science. 1997;276(5313):734–40.CrossRefPubMed Pace NR. A molecular view of microbial diversity and the biosphere. Science. 1997;276(5313):734–40.CrossRefPubMed
2.
go back to reference Mojzsis SJ, Arrhenius G, McKeegan KD, Harrison TM, Nutman AP, Friend CR. Evidence for life on Earth before 3,800 million years ago. Nature. 1996;384(6604):55–9.CrossRefPubMed Mojzsis SJ, Arrhenius G, McKeegan KD, Harrison TM, Nutman AP, Friend CR. Evidence for life on Earth before 3,800 million years ago. Nature. 1996;384(6604):55–9.CrossRefPubMed
3.
go back to reference McAlister MB, Kulakov LA, O’Hanlon JF, Larkin MJ, Ogden KL. Survival and nutritional requirements of three bacteria isolated from ultrapure water. J Ind Microbiol Biotechnol. 2002;29(2):75–82.CrossRefPubMed McAlister MB, Kulakov LA, O’Hanlon JF, Larkin MJ, Ogden KL. Survival and nutritional requirements of three bacteria isolated from ultrapure water. J Ind Microbiol Biotechnol. 2002;29(2):75–82.CrossRefPubMed
4.
go back to reference McFeters GA, Broadaway SC, Pyle BH, Egozy Y. Distribution of bacteria within operating laboratory water purification systems. Appl Environ Microbiol. 1993;59(5):1410–5.PubMedPubMedCentral McFeters GA, Broadaway SC, Pyle BH, Egozy Y. Distribution of bacteria within operating laboratory water purification systems. Appl Environ Microbiol. 1993;59(5):1410–5.PubMedPubMedCentral
5.
go back to reference Shen H, Rogelj S, Kieft TL. Sensitive, real-time PCR detects low-levels of contamination by Legionella pneumophila in commercial reagents. Mol Cell Probes. 2006;20(3–4):147–53.CrossRefPubMed Shen H, Rogelj S, Kieft TL. Sensitive, real-time PCR detects low-levels of contamination by Legionella pneumophila in commercial reagents. Mol Cell Probes. 2006;20(3–4):147–53.CrossRefPubMed
6.
go back to reference Rand KH, Houck H. Taq polymerase contains bacterial DNA of unknown origin. Mol Cell Probes. 1990;4(6):445–50.CrossRefPubMed Rand KH, Houck H. Taq polymerase contains bacterial DNA of unknown origin. Mol Cell Probes. 1990;4(6):445–50.CrossRefPubMed
7.
go back to reference Corless CE, Guiver M, Borrow R, Edwards-Jones V, Kaczmarski EB, Fox AJ. Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. J Clin Microbiol. 2000;38(5):1747–52.PubMedPubMedCentral Corless CE, Guiver M, Borrow R, Edwards-Jones V, Kaczmarski EB, Fox AJ. Contamination and sensitivity issues with a real-time universal 16S rRNA PCR. J Clin Microbiol. 2000;38(5):1747–52.PubMedPubMedCentral
8.
go back to reference Chiodini RJ, Dowd SE, Chamberlin WM, Galandiuk S, Davis B, Glassing A. Microbial population differentials between mucosal and submucosal intestinal tissues in advanced Crohn’s disease of the ileum. PLoS ONE. 2015;10(7):e0134382.CrossRefPubMedPubMedCentral Chiodini RJ, Dowd SE, Chamberlin WM, Galandiuk S, Davis B, Glassing A. Microbial population differentials between mucosal and submucosal intestinal tissues in advanced Crohn’s disease of the ileum. PLoS ONE. 2015;10(7):e0134382.CrossRefPubMedPubMedCentral
9.
10.
go back to reference Patel P, Garson JA, Tettmar KI, Ancliff S, McDonald C, Pitt T, Coelho J, Tedder RS. Development of an ethidium monoazide-enhanced internally controlled universal 16S rDNA real-time polymerase chain reaction assay for detection of bacterial contamination in platelet concentrates. Transfusion. 2012;52(7):1423–32.CrossRefPubMed Patel P, Garson JA, Tettmar KI, Ancliff S, McDonald C, Pitt T, Coelho J, Tedder RS. Development of an ethidium monoazide-enhanced internally controlled universal 16S rDNA real-time polymerase chain reaction assay for detection of bacterial contamination in platelet concentrates. Transfusion. 2012;52(7):1423–32.CrossRefPubMed
11.
go back to reference Hein I, Schneeweiss W, Stanek C, Wagner M. Ethidium monoazide and propidium monoazide for elimination of unspecific DNA background in quantitative universal real-time PCR. J Microbiol Methods. 2007;71(3):336–9.CrossRefPubMed Hein I, Schneeweiss W, Stanek C, Wagner M. Ethidium monoazide and propidium monoazide for elimination of unspecific DNA background in quantitative universal real-time PCR. J Microbiol Methods. 2007;71(3):336–9.CrossRefPubMed
12.
go back to reference Salter SJ, Cox MJ, Turek EM, Calus ST, Cookson WO, Moffatt MF, Turner P, Parkhill J, Loman NJ, Walker AW. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 2014;12:87.CrossRefPubMedPubMedCentral Salter SJ, Cox MJ, Turek EM, Calus ST, Cookson WO, Moffatt MF, Turner P, Parkhill J, Loman NJ, Walker AW. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 2014;12:87.CrossRefPubMedPubMedCentral
13.
go back to reference Xuan C, Shamonki JM, Chung A, Dinome ML, Chung M, Sieling PA, Lee DJ. Microbial dysbiosis is associated with human breast cancer. PLoS ONE. 2014;9(1):e83744.CrossRefPubMedPubMedCentral Xuan C, Shamonki JM, Chung A, Dinome ML, Chung M, Sieling PA, Lee DJ. Microbial dysbiosis is associated with human breast cancer. PLoS ONE. 2014;9(1):e83744.CrossRefPubMedPubMedCentral
14.
go back to reference Bhatt AS, Freeman SS, Herrera AF, Pedamallu CS, Gevers D, Duke F, Jung J, Michaud M, Walker BJ, Young S, et al. Sequence-based discovery of Bradyrhizobium enterica in cord colitis syndrome. N Engl J Med. 2013;369(6):517–28.CrossRefPubMedPubMedCentral Bhatt AS, Freeman SS, Herrera AF, Pedamallu CS, Gevers D, Duke F, Jung J, Michaud M, Walker BJ, Young S, et al. Sequence-based discovery of Bradyrhizobium enterica in cord colitis syndrome. N Engl J Med. 2013;369(6):517–28.CrossRefPubMedPubMedCentral
15.
go back to reference Nadkarni MA, Martin FE, Jacques NA, Hunter N. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology. 2002;148(Pt 1):257–66.CrossRefPubMed Nadkarni MA, Martin FE, Jacques NA, Hunter N. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology. 2002;148(Pt 1):257–66.CrossRefPubMed
16.
go back to reference Horz HP, Vianna ME, Gomes BP, Conrads G. Evaluation of universal probes and primer sets for assessing total bacterial load in clinical samples: general implications and practical use in endodontic antimicrobial therapy. J Clin Microbiol. 2005;43(10):5332–7.CrossRefPubMedPubMedCentral Horz HP, Vianna ME, Gomes BP, Conrads G. Evaluation of universal probes and primer sets for assessing total bacterial load in clinical samples: general implications and practical use in endodontic antimicrobial therapy. J Clin Microbiol. 2005;43(10):5332–7.CrossRefPubMedPubMedCentral
17.
go back to reference Chiodini RJ, Dowd SE, Davis B, Galandiuk S, Chamberlin WM, Kuenstner JT, McCallum RW, Zhang J. Crohn’s disease may be differentiated into 2 distinct biotypes based on the detection of bacterial genomic sequences and virulence genes within submucosal tissues. J Clin Gastroenterol. 2013;47(7):612–20.CrossRefPubMed Chiodini RJ, Dowd SE, Davis B, Galandiuk S, Chamberlin WM, Kuenstner JT, McCallum RW, Zhang J. Crohn’s disease may be differentiated into 2 distinct biotypes based on the detection of bacterial genomic sequences and virulence genes within submucosal tissues. J Clin Gastroenterol. 2013;47(7):612–20.CrossRefPubMed
18.
go back to reference Garson JA, Patel P, McDonald C, Ball J, Rosenberg G, Tettmar KI, Brailsford SR, Pitt T, Tedder RS. Evaluation of an ethidium monoazide-enhanced 16S rDNA real-time polymerase chain reaction assay for bacterial screening of platelet concentrates and comparison with automated culture. Transfusion. 2014;54(3 Pt 2):870–8.CrossRefPubMedPubMedCentral Garson JA, Patel P, McDonald C, Ball J, Rosenberg G, Tettmar KI, Brailsford SR, Pitt T, Tedder RS. Evaluation of an ethidium monoazide-enhanced 16S rDNA real-time polymerase chain reaction assay for bacterial screening of platelet concentrates and comparison with automated culture. Transfusion. 2014;54(3 Pt 2):870–8.CrossRefPubMedPubMedCentral
19.
20.
go back to reference Glassing A, Dowd SE, Galandiuk S, Davis B, Jorden JR, Chiodini RJ. Changes in 16S RNA gene microbial community profiling by concentration of prokaryotic DNA. J Microbiol Methods. 2015;119:239–42.CrossRefPubMed Glassing A, Dowd SE, Galandiuk S, Davis B, Jorden JR, Chiodini RJ. Changes in 16S RNA gene microbial community profiling by concentration of prokaryotic DNA. J Microbiol Methods. 2015;119:239–42.CrossRefPubMed
21.
go back to reference Willerslev E, Hansen AJ, Poinar HN. Isolation of nucleic acids and cultures from fossil ice and permafrost. Trends Ecol Evol. 2004;19(3):141–7.CrossRefPubMed Willerslev E, Hansen AJ, Poinar HN. Isolation of nucleic acids and cultures from fossil ice and permafrost. Trends Ecol Evol. 2004;19(3):141–7.CrossRefPubMed
22.
go back to reference Kearney MF, Spindler J, Wiegand A, Shao W, Anderson EM, Maldarelli F, Ruscetti FW, Mellors JW, Hughes SH, Le Grice SF, et al. Multiple sources of contamination in samples from patients reported to have XMRV infection. PLoS ONE. 2012;7(2):e30889.CrossRefPubMedPubMedCentral Kearney MF, Spindler J, Wiegand A, Shao W, Anderson EM, Maldarelli F, Ruscetti FW, Mellors JW, Hughes SH, Le Grice SF, et al. Multiple sources of contamination in samples from patients reported to have XMRV infection. PLoS ONE. 2012;7(2):e30889.CrossRefPubMedPubMedCentral
23.
25.
go back to reference Chiodini RJ, Chamberlin WM, Sarosiek J, McCallum RW. Crohn’s disease and the mycobacterioses: a quarter century later. Causation or simple association? Crit Rev Microbiol. 2012;38(1):52–93.CrossRefPubMed Chiodini RJ, Chamberlin WM, Sarosiek J, McCallum RW. Crohn’s disease and the mycobacterioses: a quarter century later. Causation or simple association? Crit Rev Microbiol. 2012;38(1):52–93.CrossRefPubMed
26.
go back to reference Jervis-Bardy J, Leong LE, Marri S, Smith RJ, Choo JM, Smith-Vaughan HC, Nosworthy E, Morris PS, O’Leary S, Rogers GB, et al. Deriving accurate microbiota profiles from human samples with low bacterial content through post-sequencing processing of Illumina MiSeq data. Microbiome. 2015;3:19.CrossRefPubMedPubMedCentral Jervis-Bardy J, Leong LE, Marri S, Smith RJ, Choo JM, Smith-Vaughan HC, Nosworthy E, Morris PS, O’Leary S, Rogers GB, et al. Deriving accurate microbiota profiles from human samples with low bacterial content through post-sequencing processing of Illumina MiSeq data. Microbiome. 2015;3:19.CrossRefPubMedPubMedCentral
27.
go back to reference Walker AW, Martin JC, Scott P, Parkhill J, Flint HJ, Scott KP. 16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice. Microbiome. 2015;3:26.CrossRefPubMedPubMedCentral Walker AW, Martin JC, Scott P, Parkhill J, Flint HJ, Scott KP. 16S rRNA gene-based profiling of the human infant gut microbiota is strongly influenced by sample processing and PCR primer choice. Microbiome. 2015;3:26.CrossRefPubMedPubMedCentral
28.
go back to reference Silva PE, Costa PS, Avila MP, Suhadolnik ML, Reis MP, Salgado AP, Lima MF, Chartone-Souza E, Nascimento AM. Leprous lesion presents enrichment of opportunistic pathogenic bacteria. Springerplus. 2015;4:187.CrossRefPubMedPubMedCentral Silva PE, Costa PS, Avila MP, Suhadolnik ML, Reis MP, Salgado AP, Lima MF, Chartone-Souza E, Nascimento AM. Leprous lesion presents enrichment of opportunistic pathogenic bacteria. Springerplus. 2015;4:187.CrossRefPubMedPubMedCentral
29.
go back to reference Burcelin R, Serino M, Chabo C, Garidou L, Pomie C, Courtney M, Amar J, Bouloumie A. Metagenome and metabolism: the tissue microbiota hypothesis. Diabetes Obes Metab. 2013;15(Suppl 3):61–70.CrossRefPubMed Burcelin R, Serino M, Chabo C, Garidou L, Pomie C, Courtney M, Amar J, Bouloumie A. Metagenome and metabolism: the tissue microbiota hypothesis. Diabetes Obes Metab. 2013;15(Suppl 3):61–70.CrossRefPubMed
30.
go back to reference Lluch J, Servant F, Paisse S, Valle C, Valiere S, Kuchly C, Vilchez G, Donnadieu C, Courtney M, Burcelin R, et al. The characterization of novel tissue microbiota using an optimized 16S metagenomic sequencing pipeline. PLoS ONE. 2015;10(11):e0142334.CrossRefPubMedPubMedCentral Lluch J, Servant F, Paisse S, Valle C, Valiere S, Kuchly C, Vilchez G, Donnadieu C, Courtney M, Burcelin R, et al. The characterization of novel tissue microbiota using an optimized 16S metagenomic sequencing pipeline. PLoS ONE. 2015;10(11):e0142334.CrossRefPubMedPubMedCentral
Metadata
Title
Inherent bacterial DNA contamination of extraction and sequencing reagents may affect interpretation of microbiota in low bacterial biomass samples
Authors
Angela Glassing
Scot E. Dowd
Susan Galandiuk
Brian Davis
Rodrick J. Chiodini
Publication date
01-12-2016
Publisher
BioMed Central
Published in
Gut Pathogens / Issue 1/2016
Electronic ISSN: 1757-4749
DOI
https://doi.org/10.1186/s13099-016-0103-7

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