Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water

Abstract

The limitation of 16S rRNA gene sequencing (DNA-based) for microbial community analyses in water is the inability to differentiate live (dormant cells as well as growing or non-growing metabolically active cells) and dead cells, which can lead to false positive results in the absence of live microbes. Propidium-monoazide (PMA) has been used to selectively remove DNA from dead cells during downstream sequencing process. In comparison, 16S rRNA sequencing (RNA-based) can target live microbial cells in water as both dormant and metabolically active cells produce rRNA. The objective of this study was to compare the efficiency and sensitivity of DNA-based, PMA-based and RNA-based 16S rRNA Illumina sequencing methodologies for live bacteria detection in water samples experimentally spiked with different combination of bacteria (2 gram-negative and 2 gram-positive/acid fast species either all live, all dead, or combinations of live and dead species) or obtained from different sources (First Nation community drinking water; city of Winnipeg tap water; water from Red River, Manitoba, Canada). The RNA-based method, while was superior for detection of live bacterial cells still identified a number of 16S rRNA targets in samples spiked with dead cells. In environmental water samples, the DNA- and PMA-based approaches perhaps overestimated the richness of microbial community compared to RNA-based method. Our results suggest that the RNA-based sequencing was superior to DNA- and PMA-based methods in detecting live bacterial cells in water.

Clinical metagenomics

“…or parasites; a possible paradoxi cal increase in exogenous background contamination by use of additional reagents 101 ; and the inability to detect free nucleic acid from dead organisms that are lysed in vivo by human host immune cells or antibiotic treatment. The importance of retaining the ability for cell free DNA detection from culture negative samples from dead organisms is also why incorporation of a propidium monoazide treatment step to select for DNA from live organisms may not be clinically useful as an enrichment method for mNGS 102 . In general, both the differential lysis and propidium monoazide approaches would also be cumbersome to implement in a highly reproducible fashion, which is needed for clinical laboratory validation.…”

Characterization of the bacterial microbiome in first‐pass meconium using propidium monoazide ( PMA ) to exclude nonviable bacterial DNA

“…PMA is cell membrane impermeable; thus, in a population of live and dead cells, only DNA from live cells with intact cell membranes will be amplified by subsequent PCR. This method has previously been used to selectively amplify DNA from intact bacterial cells in the adult human gut (Chu et al 2017), the preterm infant gut (Young et al 2017), water (Li et al 2017), and even space dust (Checinska et al 2015).…”

The microbiome of translocated bacterial populations in patients with and without inflammatory bowel disease

“…In contrast, bacterial RNA can be used as a marker of living and viable bacteria. However, as this has been associated with a moderate false positive rate, 25 RNA detection is preferred in tissues with low bacterial abundances. 16 rRNA and tRNA, which make up 98% of total cellular RNA, can be stable over several weeks but can also undergo intracellular digestion (by endonucleases).…”

“…24 PMA covalently binds and damages DNA in cells with damaged membranes, preventing amplification of DNA from non-viable bacteria. However, as this has been associated with a moderate false positive rate, 25 RNA detection is preferred in tissues with low bacterial abundances. 26,27 The aims of our study were: to investigate the existence of viable organisms in the lymph nodes of subjects with and without IBD using molecular techniques and to compare the bacteria present in the lymph nodes with the same patient's gut microbiome.…”