Validation of microbial source tracking markers through PCR-based molecular analysis and microbial genome database

• Molecular markers displaying cross-reactivity that risks misidentification in MST. • Integrated approaches in MST analysis help select the best-performing marker. • Geographical location significantly influences the interpretation of MST results. Microbial Source Tracking (MST) uses molecular markers targeting host-associated gut microorganisms to identify fecal pollution. However, MST faces significant challenges in fecal source identification, particularly due to the markers’ poor specificity and shared genomic areas among microorganisms from different host sources. This study addresses these challenges by using host-specific Escherichia coli genetic markers, originally developed through a novel, library-independent approach, to detect sources of fecal pollution. A total of 563 E.coli isolates from chicken, cow, and pig feces were isolated and assessed by nine reported host-associated E. coli genetic markers (Chicken: CH7, CH9, CH12, CH13; Cow: CO2, CO3; Pig: P1, P3, P4) through PCR. Marker possession patterns, sensitivity, specificity, and accuracy were calculated. The NCBI Microbial Genome database was searched for sequences homologous to genome regions of studied genetic markers and evaluated by finding the percentage of host sources and sequence location in the genome. Homology evaluation with binary PCR results was used to predict the best-performing marker. PCR results exhibited that the most effective markers were chicken CH7 (67% sensitivity, 77.9% specificity, 74.4% accuracy) and CH9 (55% sensitivity, 99.4% specificity, 84.7% accuracy). However, a homology search in the database narrowed the selection of the top-performing marker to CH7, which showed homology with E.coli from chicken hosts, while other markers exhibited higher homology with E.coli from Humans. Furthermore, sequences from the database homologous to the CH9 and CO2 markers were found on a plasmid, while those for CH12, CO3, P1, and P4 were on the chromosome, and CH7, CH13, and P3 were on both. This study highlights the critical need for integrated approaches to assess molecular markers in MST assays, emphasizing their significance in advancing research within the field.