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

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.

本研究针对接种单核细胞增生李斯特菌（L. monocytogenes）后的菠菜叶片，在欧盟参考实验室（EURL）挑战条件下储存9天，实验期间于PCR阶段采用两种叶绿体共扩增抑制方案（COMPETE、BLOCK），并增设PCR后叶绿体序列过滤方案（CONTROL）。尽管三种方案均证实细菌群落随时间发生显著演替，基于肽核酸（pPNA）的BLOCK方案与CONTROL方案的群落多样性相似度最高。采用特异性引物阻断叶绿体扩增的COMPETE方案，虽实现了最低水平的叶绿体共扩增，但因所选引物区域的固有特性，对浮霉菌门（Planctomycetota）存在显著的丰度低估，同时对绿弯菌门（Chloroflexi）与疣微菌门（Verrucomicrobiota）的丰度低估程度相对较轻。单核细胞增生李斯特菌在第7天至第9天的生长潜力显著升高，同期伴随假单胞菌科（Pseudomonadaceae）相对丰度上升与乳杆菌目（Lactobacillales）丰度下降，这表明特定系统发育类群的菌群可能促进单核细胞增生李斯特菌的定植与增殖。本研究中菠菜的叶绿体共扩增程度相对温和，仅依靠序列过滤即可满足去除叶绿体干扰的需求，但其他叶菜类蔬菜可能需要采用本次测试的共扩增抑制方案之一。