Metagenomic Source Tracking of Microbial Contaminants from Manure to Lettuce

Abstract

Raw lettuce consumption poses food safety risks when its cultivated using animal manure, which serves as a reservoir for enteric microorganisms. This study used shotgun metagenomic sequencing to profile microbial communities in lettuce, chicken manure, and goat manure and identify potential contamination pathways along with its associated food safety risks. Shotgun metagenomic sequencing was conducted, followed by quality control and host DNA depletion, then directly to taxonomic classification. Lettuce showed lower bacterial diversity (Shannon 1.59) compared to chicken manure (Shannon 4.59) and goat manure (Shannon 5.07). Based on the classified reads, lettuce is dominated by phyla Pseudomonadota (87.91%) while manure samples are dominated by phyla Actinomycetota (58%). Spoilage-associated microorganisms predominated on lettuce, led by the genera Pseudomonas (31.39%) and Pectobacterium (25.81%). Furthermore, indicator microorganisms such as Enterobacteriaceae, Enterobacter, Klebsiella, Enterococcus, as well as foodborne pathogens Escherichia coli and Salmonella enterica are shared between lettuce and manure samples. These shared microorganisms highlight the potential microbial contamination pathway associated with animal manure fertilizer application in vegetables cultivation.