Comparing solid-based concentration methods for rapid and efficient recovery of SARS-CoV-2 for wastewater surveillance.

As wastewater-based surveillance of SARS-CoV-2 attracts interest globally, there is a need to evaluate and identify rapid and efficient methods for concentrating enveloped viruses in wastewater. When comparing five precipitation/flocculation-based concentration methods (including aluminum hydroxide adsorption-precipitation, AHAP; zinc acetate precipitation, ZAP; skimmed milk flocculation, SMF; FeCl3 precipitation, FCP; and direct centrifugation, DC), AHAP was found to be the most efficient method in terms of seeded BCoV recovery (50.2 %). Based on the BCoV recovery efficiency and turnaround time, the AHAP and DC methods were selected and tested on five additional wastewater samples containing both seeded BCoV and indigenous wastewater SARS-CoV-2 RNA. The BCoV recovery (DC: average=30.1 %, sx =14.7 %; AHAP: average=33.0 %, sx =14.2 %) and SARS-CoV-2 based on the N2 gene assay (DC: average=3.6 ×103 gene copies or GC/mL, sx =1.9 × 103 GC/mL; AHAP: average=3.0 ×103 GC/mL, sx =2.0 ×103 GC/mL) of both methods were not significantly different in solid fraction (p = 0.89). This study showed significant higher BCoV recovery and SARS-CoV-2 viral RNA in wastewater solid fraction (p = 0.006) than liquid fraction. Our result suggests that the solid fraction of wastewater samples is more suitable for recovering enveloped viruses from wastewater, and the DC and AHAP methods equally provide suitably rapid, cost-effective, and significantly higher recovery of SARS-CoV-2 viral RNA in wastewater samples.

Diversity of blaPOM in carbapenem-resistant opportunistic pathogenic Pseudomonas otitidis in municipal wastewater.

Metallo-ß-lactamases (MBLs) encoding carbapenem resistance in wastewater are a well-known serious threat to human health. Twelve Pseudomonas otitidis isolates obtained from a municipal wastewater treatment plant (WWTP) in Hawaii were found to possess a subclass B3 MBL - POM (P. otitidis MBL), with a minimum inhibition concentration (MIC) range of 8-16 mg/L. The unrooted neighbor-joining phylogenetic tree showed that these blaPOM genes isolated in wastewater samples (n = 12) were distinctly different from other reference genes isolated from clinical, freshwater, animal, and soil samples except for isolates MR7, MR8, and MR11. MR7, MR8, and MR11 were found to have 4, 3, and 3 amino acid substitutions when compared to the type strain MC10330T and were closely clustered to the clinical reference genes. The meropenem hydrolysis experiment showed that isolates with multiple amino acid substitutions completely hydrolyzed 64 mg/L of meropenem in 7 h. The emergence of the opportunistic pathogen P. otitidis chromosomally encoding blaPOM in the treated municipal wastewater is an alarming call for the spread of this MBL in the environment. Further studies are required to understand the mechanism and regulation of this carbapenem-resistant ß-lactamase in order to fill in the knowledge gap.

Fine-Scale Temporal Dynamics of SARS-CoV-2 RNA Abundance in Wastewater during A COVID-19 Lockdown.

Wastewater is a pooled sampling instrument that may provide rapid and even early disease signals in the surveillance of COVID-19 disease at the community level, yet the fine-scale temporal dynamics of SARS-CoV-2 RNA in wastewater remains poorly understood. This study tracked the daily dynamics of SARS-CoV-2 RNA in the wastewater from two wastewater treatment plants (WWTPs) in Honolulu during a rapidly expanding COVID-19 outbreak and a responding four-week lockdown that resulted in a rapid decrease of daily clinical COVID-19 new cases. The wastewater SARS-CoV-2 RNA concentration from both WWTPs, as measured by three quantification assays (N1, N2, and E), exhibited both significant inter-day fluctuations (101.2-105.1 gene copies or GC/L in wastewater liquid fractions, or 101.4-106.2 GC/g in solid fractions) and an overall downward trend over the lockdown period. Strong and significant correlation was observed in measured SARS-CoV-2 RNA concentrations between the solid and liquid wastewater fractions, with the solid fraction containing majority (82.5%-92.5%) of the SARS-CoV-2 RNA mass and the solid-liquid SARS-CoV-2 RNA concentration ratios ranging from 103.6 to 104.3 mL/g. The measured wastewater SARS-CoV-2 RNA concentration was normalized by three endogenous fecal RNA viruses (F+ RNA coliphages Group II and III, and pepper mild mottle virus) to account for variations that may occur during the multi-step wastewater processing and molecular quantification, and the normalized abundance also exhibited similar daily fluctuations and overall downward trend over the sampling period.