Metagenomic Analysis of Sewage for Surveillance of Bacterial Pathogens: A Release Experiment to Determine Sensitivity

Accurate monitoring of orally-faecally transmitted, gastro-enteric diseases in a population poses a challenge in public health management. Sewage represents a larger population, is freely obtainable and non-subject to approval. In addition, metagenomic sequencing offers simultaneous, multiple-target analysis. However, no study has ever demonstrated the sensitivity of metagenomics applied in this fashion before, in terms of number of ill persons.In this study, we spot-released 1013 colony-forming units (CFU) of Staphyloccus hyicus (S. hyicus, non-pathogenetic strain 842J-88) and 1014 CFU of Thalassobacillus hwangdonensis (T. hwangdonensis). The analytes were flushed down a toilet into the sewer in the catchment area of a public wastewater treatment plant (WWTP), serving a population of 36,000 people. Raw sewage was continuously sampled at the WWTPs inlet over 30- and 60-minute intervals and a total period of eight hours. The experiment was conducted twice with one week in-between release days and under comparable weather conditions.For the metagenomics analyses, a pure single isolate of S. hyicus was grown, sequenced, assembled and added to a large database of reference sequences. The sewage samples were mapped against the reference database. S. hyicus was identified on both release days. The overall mean sensitivity was 52 diarrhoeic persons, computed as simultaneously shedding 200 g of faeces at 106 CFU of analyte. That equalled 0.15 percent of the population of the WWTPs catchment area. No T. hwangdonensis was detected in any of the samples. We successfully demonstrated a repeatable release of a bacterial agent into the sewage system, retrieving and detecting it in a quantitative fashion. Sewage may be used to monitor the transmission of some faecally shed bacteria in a population. However, our results also show that suitability and sensitivity may have to be assessed a new for every analyte and experimental setup.