Use of propidium monoazide followed by 16S rDNA sequencing in order to discriminate live and dead cells in surface and food samples from collective kitchens

Classical microbiological analyses have been the “Gold standard” in food microbiology in order to detect and quantify bacteria in food and surface samples. However, these methods bear the limitation of the ability of bacteria to grow on the used media. Advances in sequencing methods have allowed analysing the bacterial DNA in different types of samples, but these techniques do not evaluate if the bacteria are viable. In order to detect viable bacteria by sequencing methods, the interest in sample treatment with propidium monoazide (PMA) prior to DNA extraction is growing (Li et al., 2017; Nocker et al., 2010). This study addresses the possible benefit of this method for surface and food samples taken in collective kitchen. Samples have been collected aseptically, stored at 4°C and analysed by classical microbiological methods and 16S rDNA sequencing (with and without prior treatment by PMA).
The bacterial counts in many samples were low or under the detection limit (< 1 CFU/mL or < 10 CFU/g) and did rarely exceed the 5 log CFU/surface (mostly for sinks). This fact implied problems for the 16S rDNA sequencing, as a minimal bacterial load of 100-1000 CFU/mL is required in order to achieve reliable results. In addition, PMA-treated samples did generally generate fewer sequences. However, variations of relative population abundances are rendered visible and allow performing correlations between the bacterial populations and their occurrence in untreated and treated samples. This could give hints about residual bacterial DNA and bacteria that colonise kitchen surfaces, which have not been described previously.

References:
• Li, R. et al. (2017). Comparison of DNA-, PMA-, and RNA-based 16S rRNA Illumina sequencing for detection of live bacteria in water. Scientific reports, 7(1), 5752.
• Nocker, A., et al. (2010). Discrimination between live and dead cells in bacterial communities from environmental water samples analyzed by 454 pyrosequencing. Int Microbiol, 13(2), 59-65.