A viability-linked metagenomic analysis of cleanroom environments: eukarya, prokaryotes, and viruses

BackgroundRecent studies posit a reciprocal dependency between the microbiomes associated with humans and indoor environments. However, none of these metagenome surveys has considered the viability of constituent microorganisms when inferring impact on human health.ResultsReported here are the results of a viability-linked metagenomics assay, which (1) unveil a remarkably complex community profile for bacteria, fungi, and viruses and (2) bolster the detection of underrepresented taxa by eliminating biases resulting from extraneous DNA. This approach enabled, for the first time ever, the elucidation of viral genomes from a cleanroom environment. Upon comparing the viable biomes and distribution of phylotypes within a cleanroom and adjoining (uncontrolled) gowning enclosure, the rigorous cleaning and stringent control countermeasures of the former were observed to select for a greater presence of anaerobes and spore-forming microflora. Sequence abundance and correlation analyses suggest that the viable indoor microbiome is influenced by both the human microbiome and the surrounding ecosystem(s).ConclusionsThe findings of this investigation constitute the literature’s first ever account of the indoor metagenome derived from DNA originating solely from the potential viable microbial population. Results presented in this study should prove valuable to the conceptualization and experimental design of future studies on indoor microbiomes aimed at inferring impact on human health.

[1]  S. Kingsmore,et al.  Comprehensive human genome amplification using multiple displacement amplification , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S. Kim,et al.  Using propidium monoazide to distinguish between viable and nonviable bacteria, MS2 and murine norovirus , 2012, Letters in applied microbiology.

[3]  M. Stieglmeier,et al.  Cultivation of Anaerobic and Facultatively Anaerobic Bacteria from Spacecraft-Associated Clean Rooms , 2009, Applied and Environmental Microbiology.

[4]  Robert A. Edwards,et al.  Quality control and preprocessing of metagenomic datasets , 2011, Bioinform..

[5]  A. Nocker,et al.  Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology , 2007, Applied and Environmental Microbiology.

[6]  Daniel H Huson,et al.  Microbial community analysis using MEGAN. , 2013, Methods in enzymology.

[7]  G. Peñuela,et al.  Discrimination of infectious bacteriophage T4 virus by propidium monoazide real-time PCR. , 2010, Journal of virological methods.

[8]  Christine Moissl-Eichinger,et al.  Archaea in artificial environments: Their presence in global spacecraft clean rooms and impact on planetary protection , 2011, The ISME Journal.

[9]  K. Venkateswaran,et al.  Microbial Monitoring of Spacecraft and Associated Environments , 2004, Microbial Ecology.

[10]  J. Miller,et al.  Mycotoxins as harmful indoor air contaminants , 2004, Applied Microbiology and Biotechnology.

[11]  Kasthuri Venkateswaran,et al.  Isolation and Characterization of Bacteria Capable of Tolerating the Extreme Conditions of Cleanroom Environments , 2007 .

[12]  Alexander Mahnert,et al.  Cleanroom Maintenance Significantly Reduces Abundance but Not Diversity of Indoor Microbiomes , 2015, PloS one.

[13]  Paramvir S. Dehal,et al.  FastTree 2 – Approximately Maximum-Likelihood Trees for Large Alignments , 2010, PloS one.

[14]  J. Banfield,et al.  Community structure and metabolism through reconstruction of microbial genomes from the environment , 2004, Nature.

[15]  Alexander J. Probst,et al.  Archaea on Human Skin , 2013, PloS one.

[16]  C. Dock,et al.  PCR-Based Method Using Propidium Monoazide To Distinguish Viable from Nonviable Bacillus subtilis Spores , 2009, Applied and Environmental Microbiology.

[17]  T La DucMyron,et al.  A Genetic Inventory of Spacecraft and Associated Surfaces , 2014 .

[18]  A. Nocker,et al.  Discrimination between live and dead cellsin bacterial communities from environmental water samples analyzed by 454 pyrosequencing. , 2010, International microbiology : the official journal of the Spanish Society for Microbiology.

[19]  Brian C. Thomas,et al.  Microbes in the neonatal intensive care unit resemble those found in the gut of premature infants , 2014, Microbiome.

[20]  Siu-Ming Yiu,et al.  IDBA-UD: a de novo assembler for single-cell and metagenomic sequencing data with highly uneven depth , 2012, Bioinform..

[21]  J. Morató,et al.  Viable quantitative PCR for assessing the response of Candida albicans to antifungal treatment , 2012, Applied Microbiology and Biotechnology.

[22]  A. Nocker,et al.  Effect of air drying on bacterial viability: A multiparameter viability assessment. , 2012, Journal of microbiological methods.

[23]  Yongan Zhao,et al.  RAPSearch2: a fast and memory-efficient protein similarity search tool for next-generation sequencing data , 2011, Bioinform..

[24]  K. Venkateswaran,et al.  Evaluation of Procedures for the Collection, Processing, and Analysis of Biomolecules from Low-Biomass Surfaces , 2011, Applied and Environmental Microbiology.

[25]  M. Stieglmeier,et al.  Abundance and diversity of microbial inhabitants in European spacecraft-associated clean rooms. , 2012, Astrobiology.

[26]  Kasthuri Venkateswaran,et al.  Archaeal diversity analysis of spacecraft assembly clean rooms , 2008, The ISME Journal.

[27]  I. Andorrà,et al.  Determination of viable wine yeast using DNA binding dyes and quantitative PCR. , 2010, International journal of food microbiology.

[28]  Jiajie Zhang,et al.  PEAR: a fast and accurate Illumina Paired-End reAd mergeR , 2013, Bioinform..

[29]  Kasthuri Venkateswaran,et al.  Molecular bacterial community analysis of clean rooms where spacecraft are assembled. , 2007, FEMS microbiology ecology.

[30]  F. Raymond,et al.  which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ray Meta: scalable de novo metagenome assembly and profiling , 2012 .

[31]  Kasthuri Venkateswaran,et al.  Pyrosequencing-Derived Bacterial, Archaeal, and Fungal Diversity of Spacecraft Hardware Destined for Mars , 2012, Applied and Environmental Microbiology.

[32]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[33]  N. Perna,et al.  progressiveMauve: Multiple Genome Alignment with Gene Gain, Loss and Rearrangement , 2010, PloS one.

[34]  Gabriele Berg,et al.  The ignored diversity: complex bacterial communities in intensive care units revealed by 16S pyrosequencing , 2013, Scientific Reports.

[35]  Petra Rettberg,et al.  Lessons learned from the microbial analysis of the Herschel spacecraft during assembly, integration, and test operations. , 2013, Astrobiology.

[36]  E. Birney,et al.  Velvet: algorithms for de novo short read assembly using de Bruijn graphs. , 2008, Genome research.

[37]  Jillian F Banfield,et al.  Cultivating the uncultivated: a community genomics perspective. , 2005, Trends in microbiology.

[38]  A. Nocker,et al.  Molecular monitoring of disinfection efficacy using propidium monoazide in combination with quantitative PCR. , 2007, Journal of microbiological methods.

[39]  N. Gross-Camp,et al.  Multiple Diverse Circoviruses Infect Farm Animals and Are Commonly Found in Human and Chimpanzee Feces , 2009, Journal of Virology.

[40]  Rob Knight,et al.  Longitudinal analysis of microbial interaction between humans and the indoor environment , 2014, Science.

[41]  Robert C. Edgar,et al.  MUSCLE: a multiple sequence alignment method with reduced time and space complexity , 2004, BMC Bioinformatics.

[42]  Sandhya U. Parshionikar,et al.  Use of Propidium Monoazide in Reverse Transcriptase PCR To Distinguish between Infectious and Noninfectious Enteric Viruses in Water Samples , 2010, Applied and Environmental Microbiology.

[43]  Alexander Sczyrba,et al.  Decontamination of MDA Reagents for Single Cell Whole Genome Amplification , 2011, PloS one.

[44]  P. Elizaquível,et al.  Discrimination of Infectious Hepatitis A Viruses by Propidium Monoazide Real-Time RT-PCR , 2011, Food and Environmental Virology.

[45]  Kasthuri Venkateswaran,et al.  Microbial characterization of the Mars Odyssey spacecraft and its encapsulation facility. , 2003, Environmental microbiology.

[46]  K. Venkateswaran,et al.  A genetic inventory of spacecraft and associated surfaces. , 2014, Astrobiology.

[47]  Allyson L. Byrd,et al.  Biogeography and individuality shape function in the human skin metagenome , 2014, Nature.

[48]  Jeff Kline,et al.  Architectural design influences the diversity and structure of the built environment microbiome , 2012, The ISME Journal.

[49]  Brian C. Thomas,et al.  Accurate, multi-kb reads resolve complex populations and detect rare microorganisms , 2015, Genome research.

[50]  Kasthuri Venkateswaran,et al.  Comprehensive Census of Bacteria in Clean Rooms by Using DNA Microarray and Cloning Methods , 2009, Applied and Environmental Microbiology.

[51]  Kasthuri Venkateswaran,et al.  New perspectives on viable microbial communities in low-biomass cleanroom environments , 2012, The ISME Journal.

[52]  Kasthuri Venkateswaran,et al.  Diversity of Anaerobic Microbes in Spacecraft Assembly Clean Rooms , 2010, Applied and Environmental Microbiology.

[53]  Marcel Martin Cutadapt removes adapter sequences from high-throughput sequencing reads , 2011 .