Limit of detection of ser. Enteritidis using culture-based versus culture-independent diagnostic approaches.

Unlabelled: To prevent the spread of foodborne illnesses, the presence of pathogens in the food chain is monitored by government agencies and food producers. The culture-based methods currently employed are sensitive but time- and labor-intensive, leading to increasing interest in exploring culture-independent diagnostic tests (CIDTs) for pathogen detection. However, few studies quantify the relative sensitivity and reliability of these CIDTs compared to current approaches. To address this issue, we conducted a comparison of the limit of detection (LOD) for between a culture-based method and three CIDTs: qPCR (targeting and ), metabarcode (16S) sequencing, and shotgun metagenomic sequencing. Samples of chicken feed and chicken caecal contents were spiked with . serovar Enteritidis and subjected to culture- and DNA-based detection methods. To explore the impact of non-selective enrichment on LOD, all samples underwent both immediate DNA extraction and overnight enrichment prior to gDNA extraction. In addition to this spike-in experiment, feed and caecal samples acquired from the field were tested with culturing, qPCR, and metabarcoding. In general, LOD was comparable between qPCR and shotgun sequencing methods. Overnight microbiological enrichment resulted in an improvement in LOD with up to a three-log decrease. However, reads were detected in some unspiked feed samples, suggesting false-positive detection of . In addition, the LOD in feeds was three logs lower than in caecal contents, underscoring the impact of background microbiota on detection using all methods.

Importance: The appeal of culture-independent diagnostic tests (CIDTs) is increased speed with lowered cost, as well as the potential to detect multiple pathogen species in a single analysis and to monitor other areas of concern such as antimicrobial resistance genes or virulence factors. This study provides quantitative data on the sensitivity of CIDTs relative to current approaches, which is essential for determining the feasibility of implementing these methods in pathogen surveillance programs.