Wastewater Surveillance for Identifying SARS-CoV-2 Infections in Long-Term Care Facilities, Kentucky, USA, 2021-2022.

Persons living in long-term care facilities (LTCFs) were disproportionately affected by COVID-19. We used wastewater surveillance to detect SARS-CoV-2 infection in this setting by collecting and testing 24-hour composite wastewater samples 2-4 times weekly at 6 LTCFs in Kentucky, USA, during March 2021-February 2022. The LTCFs routinely tested staff and symptomatic and exposed residents for SARS-CoV-2 using rapid antigen tests. Of 780 wastewater samples analyzed, 22% (n = 173) had detectable SARS-CoV-2 RNA. The LTCFs reported 161 positive (of 16,905) SARS-CoV-2 clinical tests. The wastewater SARS-CoV-2 signal showed variable correlation with clinical test data; we observed the strongest correlations in the LTCFs with the most positive clinical tests (n = 45 and n = 58). Wastewater surveillance was 48% sensitive and 80% specific in identifying SARS-CoV-2 infections found on clinical testing, which was limited by frequency, coverage, and rapid antigen test performance.

Stabilization of SARS-CoV-2 RNA in wastewater via rapid RNA extraction.

Wastewater-based Epidemiology (WBE) has contributed to surveillance of SARS-CoV-2 in communities across the world. Both symptomatic and asymptomatic patients with COVID-19 can shed the virus through the gastrointestinal tract, enabling the quantification of the virus in stool and ultimately in wastewater (WW). Unfortunately, instability of SARS-CoV-2 RNA in wastewater limits the utility of WBE programs, particularly in remote/rural regions where reliable cold storage and/or rapid shipping may be unavailable. This study examined whether rapid SARS-CoV-2 RNA extraction on the day of sample collection could minimize degradation. Importantly, the extraction technology used in these experiments, termed exclusion-based sample preparation (ESP), is lightweight, portable, and electricity-free, making it suitable for implementation in remote settings. We demonstrated that immediate RNA extraction followed by ambient storage significantly increased the RNA half-life compared to raw wastewater samples stored at both 4 °C or ambient temperature. Given that RNA degradation negatively impacts both the sensitivity and precision of WBE measurements, efforts must be made to mitigate degradation in order to maximize the potential impact of WBE on public health.

Development and Validation of a Simplified Method for Analysis of SARS-CoV-2 RNA in University Dormitories.

Over the course of the COVID-19 pandemic, wastewater surveillance has become a useful tool for describing SARS-CoV-2 prevalence in populations of varying size, from individual facilities (e.g., university residence halls, nursing homes, prisons) to entire municipalities. Wastewater analysis for SARS-CoV-2 RNA requires specialized equipment, expensive consumables, and expert staff, limiting its feasibility and scalability. Further, the extremely labile nature of viral RNA complicates sample transportation, especially in regions with limited access to reliable cold chains. Here, we present a new method for wastewater analysis, termed exclusion-based sample preparation (ESP), that substantially simplifies workflow (at least 70% decrease in time; 40% decrease in consumable usage compared with traditional techniques) by targeting the labor-intensive processing steps of RNA purification and concentration. To optimize and validate this method, we analyzed wastewater samples from residence halls at the University of Kentucky, of which 34% (44/129) contained detectible SARS-CoV-2 RNA. Although concurrent clinical testing was not comprehensive, student infections were identified in the 7 days following a positive wastewater detection in 68% of samples. This pilot study among university residence halls validated the performance and utility of the ESP method, laying the foundation for future studies in regions of the world where wastewater testing is not currently feasible.

Co-Carriage of Metal and Antibiotic Resistance Genes in Sewage Associated Staphylococci.

Controlling spread of resistance genes from wastewater to aquatic systems requires more knowledge on how resistance genes are acquired and transmitted. Whole genomic sequences from sewage-associated staphylococcus isolates (20 S. aureus, 2 Staphylococcus warneri, and 2 Staphylococcus delphini) were analyzed for the presence of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs). Plasmid sequences were identified in each isolate to investigate co-carriage of ARGs and MRGs within. BLASTN analysis showed that 67% of the isolates carried more than one ARG. The carriage of multiple plasmids was observed more in CC5 than CC8 S. aureus strains. Plasmid exchange was observed in all staphylococcus species except the two S. delphini isolates that carried multiple MRGs, no ARGs, and no plasmids. 85% of S. aureus isolates carried the blaZ gene, 76% co-carried blaZ with cadD and cadX, with 62% of these isolates carrying blaZ, cadD, and cadX on the same plasmid. The co-carriage of ARGs and MRGs in S. warneri isolates, and carriage of MRGs in S. delphini, without plasmids suggests non-conjugative transmission routes for gene acquisition. More studies are required that focus on the transduction and transformation routes of transmission to prevent interspecies exchange of ARGs and MRGs in sewage-associated systems.

Solar disinfection of turbid hygiene waters in Lexington, KY, USA.

Solar disinfection (SODIS) could be a key to providing a clean, hygiene water for birthing uses, but the recommended climate zone is limited, the microbial indicators are related to gastrointestinal illness and not wound infections. SODIS feasibility was investigated to remove Escherichia coli from turbid water at temperatures less than 50 °C in Lexington, KY. Increasing turbidity from 0 to 200 NTU decreased E. coli inactivation from 5 to 1 log. With the same experimental protocol, more than 4-log inactivation of Staphylococcus aureus and Staphylococcus epidermidis (common human-skin microorganisms related to serious post-partum infections of both mother and child) was achieved at different turbidity levels with a maximum, in-bottle temperature of 49.2 °C after 5.5 h. The thermal inactivation of the bacterial indicators was assessed without UV radiation and turbidity in water at 37 and 47 °C. Skin bacteria were inactivated completely after 9.5 h at 47 °C, but only 58% removal happened for thermo-tolerant E. coli. These results suggest that SODIS application may be expanded geographically to treat water for hygiene purposes. However, as E. coli is also capable of causing wound infections, UV with thermal inactivation may be required to produce safe hygiene water by SODIS outside of recommended latitudes.

Thiol-Affinity Immobilization of Casein-Coated Silver Nanoparticles on Polymeric Membranes for Biofouling Control.

Silver nanoparticles (AgNPs) have been widely studied for the control of biofouling on polymeric membranes due to their antimicrobial properties. However, nanoparticle leaching has posed a significant impediment against their widespread use. In this study, a one-step method of chemically embedding AgNPs on cellulose acetate (CA) membranes via their affinity to thiol group chemistry was investigated. The operational efficiency of the membranes was then determined via filtration and biofouling experiments. During filtration study, the average flux values of pure CA membranes was determined to be 11 ± 2 L/(m2·hr) (LMH), while membranes embedded with AgNPs showed significant increases in flux to 18 ± 2 LMH and 25 ± 9 LMH, with increasing amounts of AgNPs added, which is likely due to the NPs acting as pore formers. Leaching studies, performed both in dead-end and crossflow filtration, showed approximately 0.16 mg/L leaching of AgNPs after the first day of filtration, but afterwards the remaining chemically-attached AgNPs did not leach. Over 97% of AgNPs remained on the membranes after seven days of crossflow leaching filtration studies. Serratia marcescens were then used as target microorganisms in biofouling studies. It was observed that membranes embedded with AgNPs effectively suppressed the growth of Serratia marcescens, and specifically, membranes with AgNPs displayed a decrease in microbial growth by 59% and 99% as the amount of AgNP increased.

Prevalence and characterization of Staphylococcus aureus in wastewater treatment plants by whole genomic sequencing.

Infections with Staphylococcus aureus are being spread through contact with the community environment, but the role of wastewater treatment plants in the transmission routes is not defined. This study investigated the prevalence, types, genetic elements, and potential for transmission of S. aureus by these engineered systems. Synchronized sampling events at two wastewater treatment plants were conducted with isolates of S. aureus obtained by a selective enrichment method using acriflavine that suppressed Staphylococcus epidermidis growth. DNA was extracted from a subset of the S. aureus isolates, checked by PCR to assure the absence of S. epidermidis, and sequenced to determine the multilocus sequence type, spa type, and carriage of the methicillin resistance and Panton-Valentine leukocidin genetic elements. Sequences were analyzed for single nucleotide polymorphism differences in pairwise comparison of isolates. There were two dominant S. aureus clonal complexes identified in the isolates, one commonly identified as hospital-related (CC5) and one community-related (CC8). Both types of isolates were found at both treatment facilities, even though only one facility had significant hospital sewage inputs. The presence of S. aureus persisted through treatment, with some isolates recovered from the final processes showing genetic diversity. The presence of the Panton-Valentine leukocidin genetic element was greater than the 1-5% expected from global reports. Our results suggest that treatment provides an opportunity for genetic shift, while the persistence and release of evolved strains of S. aureus may provide an environmentally relevant pathway to new hosts in the environment.