Whole microbial community viability is not quantitatively reflected by propidium monoazide sequencing approach

Abstract

Background: High-throughput sequencing provides a powerful window into the structural and functional profiling of microbial communities, but it is unable to characterize only the viable portion of microbial communities at scale. There is as yet not one best solution to this problem. Previous studies have established viability assessments using propidium monoazide (PMA) treatment coupled with downstream molecular profiling (e.g. qPCR or sequencing). While these studies have met with moderate success, most of them focused on the resulting “viable” communities without systematic evaluations of the technique. Here, we present our work to rigorously benchmark “PMA-seq” (PMA treatment followed by 16S rRNA gene amplicon sequencing) for viability assessment in synthetic and realistic microbial communities. Results: PMA-seq was able to successfully reconstruct simple synthetic communities comprising viable/heat-killed Escherichia coli and Streptococcus sanguinis . However, in realistically complex communities (computer screens, computer mice, soil and human saliva) with E. coli spike-in controls, PMA-seq did not accurately quantify viability, with its performance largely affected by community properties such as initial biomass, sample types and compositional diversity. We then applied this technique to environmental swabs from the Boston subway system. Several taxa differed significantly after PMA treatment, while not all microorganisms responded consistently. To elucidate the “PMA-responsive” microbes, we compared our results with previous PMA-based studies and found that PMA-responsiveness varied widely when microbes were sourced from different ecosystems but were reproducible within similar environments across studies. Conclusions: This study provides a comprehensive evaluation of PMA-seq exploring its quantitative accuracy in synthetic and complex microbial communities, where the technique was effective for semi-quantitative purposes in simple synthetic communities, but provided only qualitative assessments in realistically complex community samples.

In vitro metabolic capacity of carbohydrate degradation by intestinal microbiota of adults and pre-frail elderly

“…Quantification of total 16S rRNA gene copy numbers demonstrated lower fraction of microbes in the PMA treated samples (Table S 3 ), which is in line with a previous study reporting that up to one-third of microbes in the faeces consist of dead cells [ 32 ]. Although acknowledging the limitation of the PMA analyses [ 33 ], overall, our results demonstrated a good recovery of microbes with the anoxic cryo-conservation protocol used to transport and store the samples for incubation studies.…”

Toward Accurate and Robust Environmental Surveillance Using Metagenomics

“…Application of PMA treatment to environmental samples has been hindered partly because these samples contain a diverse microbial community in a complex matrix. As pointed out by Wang et al, PMA-seq with a universal protocol is not feasible to quantify viability of realistic communities, even with E. coli controls spiked in Wang et al (2020). To facilitate its application in surveillance, instead of insisting on differentiation of the viability for every community member, we should start by identifying sets of similar pathogens, as these groups will have the highest potential to fit in one protocol while maintaining relatively good efficiencies.…”

African fermented foods: overview, emerging benefits, and novel approaches to microbiome profiling

“…The propidium monoazide-based sequencing methods are getting better at resolving live/dead cell differentiation, while metatranscriptomics can add a further layer of information in terms of which taxa are metabolically active. Flow cytometry and quantitative PCR can also be employed to facilitate insights into counts 91 – 98 . As noted above, combining long and short read data can facilitate the complete assembly of genomes including plasmids, mitigating some of the issues related to ambiguous assignment and false positives.…”