Performance of host-associated genetic markers for microbial source tracking in China

Numerous genetic markers have been developed to establish microbial source tracking (MST) assays in the last decade. However, the selection of suitable markers is challenging due to a lack of understanding of fundamental factors such as sensitivity, specificity, and concentration in target/nontarget hosts, especially in East Asia. In this study, a total of 506 faecal samples comprised of human and 12 nonhuman hosts were collected from 28 cities across China and tested for marker performance characteristics. We firstly tested 40 host-associated markers based on a binary (presence/absence) criterion. Here, 15 markers (7 human-associated, 4 pig-associated, 3 ruminant-associated, and 1 poultry-associated) showed potential applicability in our study area. The selected 15 markers were then tested using qualitative and quantitative methods to characterise their performance. Overall, Bacteroidales markers presented higher sensitivity and concentrations in target samples compared to other bacterial or viral markers, but their specificity was low. Among nontarget samples, pets accounted for 43.7% and 35.7% of cross-reactivity with human-associated and poultry-associated markers, respectively. Noncommon animals, including horse and donkey, contributed 61.3% of cross-reactivity with ruminant-associated markers. When considering the quantitative distribution of markers, their concentration in nontarget samples were 1-3 orders of magnitude lower than in target samples. Moreover, a novel classification method was proposed to classify the nontarget hosts into four groups spanning "no cross-reactivity", "weak cross-reactivity", "moderate cross-reactivity", and "strong cross-reactivity" animal hosts. There were 77.9% nontarget samples identified as no cross-reactivity and weak cross-reactivity hosts, suggesting that these nontarget hosts produce little interference for corresponding markers. Our findings elucidate the performance of host-associated markers around China in a qualitative and quantitative manner, and reveal the interference degree of cross-reactivity from nontarget animals to genetic markers, which will facilitate tracking of multiple faecal pollution sources and planning timely remedial strategies in China.