Validation of Host-associated Bacteroidales Gene Marker Assays for Microbial Source Tracking in Urban Tropical Environment of Singapore

NSHIMYIMANA, JEAN PIERRE; CRUZ, MERCEDES CECILIA; THOMPSON, JANELLE; WUERTZ, STEFAN

BOSTON

Congreso; 116th General Meeting of American Society for Microbiology; 2016

AMERICAN SOCIETY FOR MICROBIOLOGY

Background: Microbial source tracking (MST) is a widely applied tool in many temperateregions of Europe and North America as well as Australia, New Zealand and Japan, but has notseen much use yet in tropical urban environments. This study applied qPCR to validate the use ofhost-associated Bacteroidales marker assays based on the 16S rRNA gene to identify humanfecal pollution in the urban tropical environment of Singapore.Methodology: We collected atotal of 285 animal and human stool as well as sewage samples. Animals tested included cats,dogs, rabbits, chicken, birds, monkeys, and wild boars. Following DNA extraction, samples wereanalysed by qPCR using seven assays targeting human-associated Bacteroidales (HF183-SYBR,HF183, BacHum, BacH and B. thetaiotaomicron (B. theta)), dog-associated Bacteroidales(BacCan), and total Bacteroidales (BacUni). Results were used to compare the sensitivity andspecificity of assays.Results: The five human-associated assays (HF183-SYBR; HF183,BacHum, BacH and B. theta) and the total Bacteroidales assay BacUni had 100% sensitivity forsewage. The overall sensitivity for human stool samples ranged from 50 to 70% and B. theta andBacHum displayed the highest human stool sensitivity at 68.6% and 65.7%, respectively. Inaddition, these two assays had the highest specificity (98.6% for B. theta and 91.4% forBacHum). The HF183 assay recently recommended for MST in California, USA, had aspecificity of 90% and sensitivity of 60% and 100%, respectively, for human stool and sewagesamples. BacCan assay sensitivity and specificity for dog fecal samples were 80% and 97.3%,respectively.Conclusion: We confirmed the applicability of previously developed humanassociated(B.theta and BacHum), dog-associated (BacCan) and total (BacUni) Bacteroidalesgene markers for MST inthe tropical urban environment of Singapore. Such validation studiesare needed before applying any existing host-associated Bacteroidales assays under newenvironmental conditions and at different geographical locations. Source tracking could beimproved by identifying a specific human-associated gene marker for Singapore.