Assessment of Human Fecal Contamination Risks in Frozen Berries Using Human-Associated Bacteroides Marker

• RT-qPCR enhanced detection sensitivity for human-associated Bacteroides in berries. • Human fecal markers were found in 25% of frozen berry samples from retail markets. • The method distinguished human and animal fecal contamination sources in foods. Contaminated frozen berries have frequently been linked to outbreaks of enteric viruses, including hepatitis A virus (HAV) and norovirus (NoV), primarily because they are consumed without heat treatment. To evaluate the risk of fecal contamination beyond virus detection, a quantitative PCR (qPCR) assay targeting the human-associated Bacteroides marker BacH was applied, with enhanced sensitivity achieved by incorporating a reverse transcription (RT) step. The BacH RT-qPCR assay using human fecal specimens yielded cycle threshold values at an average of 15.94 cycles fewer than those obtained without RT, indicating markedly improved detection sensitivity. Among the 48 frozen berry and cherry samples collected from retail markets, neither HAV nor NoV RNA was detected. However, the BacH RT-qPCR assay followed by RT-nested PCR and sequencing identified Phocaeicola (Bacteroides) dorei and P. vulgatus in 4 and 8 samples, respectively, suggesting that 25.0% (95% confidence interval: 13.6–39.6%) of the samples were potentially contaminated with human fecal material. Additional detection of ruminant- and avian-associated Bacteroides species demonstrated that this method could distinguish animal sources of contamination. These results indicate that human-associated Bacteroides markers provide a sensitive and source-specific tool for assessing fecal contamination in frozen foods and can complement standard viral monitoring to enhance food safety management.