Development of a dual-internal-reference technique to improve accuracy when determining bacterial 16S rRNA:16S rRNA gene ratio with application to Escherichia coli liquid and aerosol samples

Details

• Personal Author:
  Fennell DE ; Krumins V ; Mainelis G ; Zhen H
• Description:
  Accurate enumeration of rRNA content in microbial cells, e.g. by using the 16S rRNA:16S rRNA gene ratio, is critical to properly understand its relationship to microbial activities. However, few studies have considered possible methodological artifacts that may contribute to the variability of rRNA analysis results. In this study, a technique utilizing genomic DNA and 16S rRNA from an exogenous species (Pseudomonas fluorescens) as dual internal references was developed to improve accuracy when determining the 16S rRNA:16S rRNA gene ratio of a target organism, Escherichia coli. This technique was able to adequately control the variability in sample processing and analysis procedures due to nucleic acid (DNA and RNA) losses, inefficient reverse transcription of RNA, and inefficient PCR amplification. The measured 16S rRNA:16S rRNA gene ratio of E. coli increased by 2-3 fold when E. coli 16S rRNA gene and 16S rRNA quantities were normalized to the sample-specific fractional recoveries of reference (P. fluorescens) 16S rRNA gene and 16S rRNA, respectively. In addition, the intra-sample variation of this ratio, represented by coefficients of variation from replicate samples, decreased significantly after normalization. This technique was applied to investigate the temporal variation of 16S rRNA:16S rRNA gene ratio of E. coli during its non-steady-state growth in a complex liquid medium, and to E. coli aerosols when exposed to particle-free air after their collection on a filter. The 16S rRNA:16S rRNA gene ratio of E. coli increased significantly during its early exponential phase of growth; when E. coli aerosols were exposed to extended filtration stress after sample collection, the ratio also increased. In contrast, no significant temporal trend in E. coli 16S rRNA:16S rRNA gene ratio was observed when the determined ratios were not normalized based on the recoveries of dual references. The developed technique could be widely applied in studies of relationship between cellular rRNA abundance and bacterial activity. [Description provided by NIOSH]
• Subjects:
  Aerosols Genetics Microbiology Occupational Health Safety
• Keywords:
  16S RRNA:16S RRNA Gene Ratio Analytical Processes Author Keywords: Bacterial 16S RRNA Bacteria Bioaerosols Genes Internal Reference Microorganisms QPCR
• ISSN:
  0167-7012
• Document Type:
  Text
• Funding:
  Grant
• Genre:
  Journal Article
• Place as Subject:
  New Jersey ; OSHA Region 2
• CIO:
  National Institute for Occupational Safety and Health (NIOSH)
• Topic:
  Workplace Safety & Health
• Location:
  North America
• Pages in Document:
  113-121
• Volume:
  117
• NIOSHTIC Number:
  nn:20047498
• Citation:
  J Microbiol Methods 2015 Oct; 117:113-121
• Contact Point Address:
  Gediminas Mainelis, Rutgers University, Department of Environmental Sciences, 14 College Farm Rd., New Brunswick, NJ 08901, United States
• Email:
  mainelis@envsci.rutgers.edu
• Federal Fiscal Year:
  2016
• Performing Organization:
  Rutgers, The State University of New Jersey, Piscataway
• Peer Reviewed:
  True
• Start Date:
  20100801
• Source Full Name:
  Journal of Microbiological Methods
• End Date:
  20190731
• Collection(s):
  National Institute for Occupational Safety and Health
• Main Document Checksum:
  urn:sha-512:09e44f1d6d5a2693bbce4704b95b959e0b47904bed226be570214facf54beee5f71a3f25be83afafd17d29ccd7745db49cbeae95096f5a73a4a9c91ed33e5c60
• Download URL:
  https://stacks.cdc.gov/view/cdc/202551/cdc_202551_DS1.pdf
• File Type:
  [PDF - 833.71 KB ]