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. 2024 May 16;19(5):e0300733.
doi: 10.1371/journal.pone.0300733. eCollection 2024.

Metagenomic analysis of sewage for surveillance of bacterial pathogens: A release experiment to determine sensitivity

Simon Kohle  1 , Thomas N Petersen  1 , Håkan Vigre  1 , Markus Hans Kristofer Johansson  1 , Frank M Aarestrup  1
Affiliations

Metagenomic analysis of sewage for surveillance of bacterial pathogens: A release experiment to determine sensitivity

Simon Kohle et al. PLoS One. .

Abstract

Accurate monitoring of gastro-enteric and other diseases in large populations poses a challenge for public health management. Sewage represents a larger population, is freely obtainable and non-subject to ethical approval. Metagenomic sequencing offers simultaneous, multiple-target analysis. However, no study has demonstrated the sensitivity of metagenomics for detecting bacteria in sewage. In this study, we spot-released 1013 colony-forming units (CFU) of Staphyloccus hyicus (non-pathogenetic strain 842J-88). The strain was 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 WWTP's inlet over 30- and 60-minute intervals for a total period of seven hours. The experiment was conducted twice with one week in-between release days and under comparable weather conditions. For the metagenomics analyses, the pure single isolate of S. hyicus was sequenced, assembled and added to a large database of bacterial reference sequences. All sewage samples were analyzed by shotgun metagenome sequencing and mapped against the reference database. S. hyicus was identified in duplicate samples at both of two release days and these sequence fragment counts served as a proxy to estimate the minimum number of sick people or sensitivity required in order to observe at least one sick person at 95% probability. We found the sensitivity to be in the range 41-140 and 16-36 sick people at release days 1 and 2, respectively. The WWTP normally serves 36,000 people giving a normalized sensitivity in the range of one in 257 to 2,250 persons.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1
Staphylococcus hyicus fragment counts on release day 1 (a) and 2 (b).
Fig 2
Fig 2. Binomial distribution using fragment counts all Staphylococcus hyicus samples.
The figure show the plot of Eq 5 where fragment counts R and r (Eq 2) are summed from all samples where S. hyicus are identified. a and b are for release days 1 and 2, respectively. The horizontal dashed line represents a 95% probability threshold and at intersecting points are the minimum number of people required to observe a sick person i.e. 41, 64 on day 1 and 16, 20 on day 2 for subsamples A and B in blue and red color.
Fig 3
Fig 3. Binomial distribution using fragment counts for highest Staphylococcus hyicus sample.
The figure show the plot of Eq 5 where fragment counts R and r (Eq 2) are from one samples where max S. hyicus was identified. a and b are for release days 1 and 2, respectively. The horizontal dashed line represents a 95% probability threshold and at intersecting points are the minimum number of people required to observe a sick person i.e. 107, 140 on day 1 and 23, 36 on day 2 for subsamples A and B in blue and red color.
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Grants and funding

This study was supported by the EU's Horizon H2020 grant VEO (874735) and Novo Nordisk Foundation (NNF16OC0021856). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.