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

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.

Inactivation of hepatitis A virus and murine norovirus on surfaces of plastic, steel and raspberries using steam-ultrasound treatment.

The leading causes of foodborne viral disease outbreaks are human norovirus and hepatitis A virus (HAV). Their environmental persistence enables contamination of kitchen surfaces and crops often consumed raw, such as berries. Many decontamination procedures are inefficient and unsuitable for surfaces of industrial kitchen environments and soft fruits. In this study, we investigated the efficiency of a novel surface decontamination technology, combining steam and ultrasound (steam-ultrasound). Plastic, steel or raspberry surfaces were spiked with the norovirus surrogate, murine norovirus (MNV), and HAV, and steam-ultrasound treated at 85, 90 and 95 °C for 0-5 s. Post treatment viruses were titrated for survival by plaque assay and for genome stability by real-time quantitative PCR (RT-qPCR) of nucleic acid extracts. Survival of viruses were estimated in a log-linear model and the treatment time requirements for each decimal reduction (D value) in viral survival were calculated. The estimated D values of MNV or HAV were 0.4-0.2 or 1.1-0.8 s on plastic, 0.9-0.7 or 1.4-0.8 s on steel and 1.6-1.7 or 3.2-4.7 s on raspberries. No clear trend of genome reduction was observed with tested treatment parameters. Raspberries treated up to 4 s retained its natural texture and visual appeal similar to untreated controls whilst monitored for 7 days. In conclusion, steam-ultrasound treatment can within seconds reduce the titre of foodborne viruses on surfaces of plastic, steel and raspberries. This may particularly benefit industrial scale production of soft fruits for raw consumption and for swift non-hazardous decontamination of industrial kitchen surfaces.