Bacteroidales markers for microbial source tracking in Southeast Asia

The island city country of Singapore served as a model to validate the use of host-associated Bacteroidales 16S rRNA gene marker assays for identifying sources of fecal pollution in the urban tropical environment of Southeast Asia. A total of 295 samples were collected from sewage, humans, domesticated animals (cats, dogs, rabbits and chicken), and wild animals (birds, monkeys and wild boars). Samples were analyzed by real time PCR using five human-associated assays (HF183-SYBR Green, HF183, BacHum, BacH and B. thetaiotaomicron α-1-6, mannanase (B. theta), one canine-associated assay (BacCan), and a total Bacteroidales assay (BacUni). The best performing human-associated assay was B. theta with a diagnostic sensitivity of 69% and 100% in human stool and sewage, respectively, and a specificity of 98%. BacHum achieved the second highest sensitivity and specificity for human stool at 65% and 91%, respectively. The canine-associated Bacteroidales assay (BacCan) had a sensitivity and specificity above 80% and was validated for tracking fecal pollution from dogs. BacUni demonstrated a sensitivity and specificity of 100% for mammals, thus BacUni was confirmed for total Bacteroidales detection in the region. We showed for the first time that rabbit fecal samples cross-react with human-associated assays (HF183-SYBR Green, HF183, BacHum and BacH) and with BacCan. Our findings regarding the best performing human-associated assays differ from those reported in Bangladesh and India, which are geographically close to Southeast Asia, and where HF183 and BacHum were the preferred assays, respectively.