Bacterial community structure analysis on Listeria monocytogenes inoculated spinach leaves is affected by PCR based methods to exclude chloroplast co-amplification

Consumption of ready-to-eat leafy vegetables has increased in popularity due to their anticipated health benefits, but their consumption also poses a potential health risk in the form of foodborne pathogens. Listeria monocytogenes is a ubiquitous pathogen that has been regularly found on leafy vegetables including spinach. Growth determining factors go beyond plant species and cultivation practice and may include the phyllosphere bacteriome to affect the growth potential of L. monocytogenes. This study investigated the bacteriome of spinach leaves, stored under EURL challenge conditions for 9 days after inoculation with L. monocytogenes using two methods of excluding chloroplast co-amplification (COMPETE, BLOCK) at the PCR step as well as a post-PCR chloroplast sequence filter option (CONTROL). While all three approaches have demonstrated a change of bacterial communities over time, the pPNA based BLOCK approach resulted in greater diversity similarities to the CONTROL option. The COMPETE solution with a specifically designed primer to prevent chloroplast amplification had a strong underrepresentation of the Planctomycetota phylum and to a lesser extend underrepresentation of Chloroflexi and Verrucomicrobiota due to the inheritance of the selected primer region that allowed to deselect chloroplast co-amplification. However, the COMPETE approach had the lowest level of chloroplast co-amplification. Higher growth potential of L. monocytogenes from day 7 to 9 co-occurred with higher relative abundances of Pseudomonadaceae and lower numbers of Lactobacillales, suggesting that particular phylogenetic groups may support growth of L. monocytogenes. While chloroplast co-amplification with spinach in the present study was relatively modest and a purely filter based chloroplast removal was sufficient, other leafy vegetables may require one of the tested co-amplification prevention solutions. While the COMPETE solution in the present study was linked to some amplification bias, the approach may be useful when otherwise co-amplification is very high and the demonstrated BLOCK approach with pPNA is insufficient.