Screening for extended-spectrum beta-lactamase Escherichia coli in recreational beach waters in Singapore.

Aims: As part of Singapore's One Health antimicrobial resistance (AMR) management, this work was designed to understand the AMR burden in recreational beach waters using extended-spectrum beta-lactamase Escherichia coli (ESBL-EC) as an indicator. Materials & methods: A total of 90 water samples were collected from six different recreational beaches over three different time periods. Only 28/90 (31.3%) water samples yielded E. coli colonies ranging from 1 to 80 colony-forming units/100 ml. Results & conclusion: Screening of all colonies using CHROMID® ESBL agar and Luria-Bertani broth supplemented with ceftriaxone showed that none was ESBL-EC. Further monitoring is required to understand the prevalence of ESBL-EC spatiotemporally, contributing to the national AMR surveillance program and providing timely risk assessment for exposure to ESBL-EC.

Development of an efficient wastewater testing protocol for high-throughput country-wide SARS-CoV-2 monitoring.

Wastewater-based surveillance has been widely used as a non-intrusive tool to monitor population-level transmission of COVID-19. Although various approaches are available to concentrate viruses from wastewater samples, scalable methods remain limited. Here, we sought to identify and evaluate SARS-CoV-2 virus concentration protocols for high-throughput wastewater testing. A total of twelve protocols for polyethylene glycol (PEG) precipitation and four protocols for ultrafiltration-based approaches were evaluated across two phases. The first phase entailed an initial evaluation using a small sample set, while the second phase further evaluated five protocols using wastewater samples of varying SARS-CoV-2 concentrations. Permutations in the pre-concentration, virus concentration and RNA extraction steps were evaluated. Among PEG-based methods, SARS-CoV-2 virus recovery was optimal with 1) the removal of debris prior to processing, 2) 2 h to 24 h incubation with 8% PEG at 4 °C, 3) 4000 xg or 14,000 xg centrifugation, and 4) a column-based RNA extraction method, yielding virus recovery of 42.4-52.5%. Similarly, the optimal protocol for ultrafiltration included 1) the removal of debris prior to processing, 2) ultrafiltration, and 3) a column-based RNA extraction method, yielding a recovery of 38.2%. This study also revealed that SARS-CoV-2 RNA recovery for samples with higher virus concentration were less sensitive to changes in the PEG method, but permutations in the PEG protocol could significantly impact virus yields when wastewater samples with lower SARS-CoV-2 RNA were used. Although both PEG precipitation and ultrafiltration methods resulted in similar SARS-CoV-2 RNA recoveries, the former method is more cost-effective while the latter method provided operational efficiency as it required a shorter turn-around-time (PEG precipitation, 9-23 h; Ultrafiltration, 5 h). The decision on which method to adopt will thus depend on the use-case for wastewater testing, and the need for cost-effectiveness, sensitivity, operational feasibility and scalability.

Non-intrusive wastewater surveillance for monitoring of a residential building for COVID-19 cases.

Wastewater-based surveillance for SARS-CoV-2 has been used for the early warning of transmission or objective trending of the population-level disease prevalence. Here, we describe a new use-case of conducting targeted wastewater surveillance to complement clinical testing for case identification in a small community at risk of COVID-19 transmission. On 2 July 2020, a cluster of COVID-19 cases in two unrelated households residing on different floors in the same stack of an apartment building was reported in Singapore. After cases were conveyed to healthcare facilities and six healthy household contacts were quarantined in their respective apartments, wastewater surveillance was implemented for the entire residential block. SARS-CoV-2 was subsequently detected in wastewaters in an increasing frequency and concentration, despite the absence of confirmed COVID-19 cases, suggesting the presence of fresh case/s in the building. Phone interviews of six residents in quarantine revealed that no one was symptomatic (fever/respiratory illness). However, when nasopharyngeal swabs from six quarantined residents were tested by PCR tests, one was positive for SARS-CoV-2. The positive case reported episodes of diarrhea and the case's stool sample was also positive for SARS-CoV-2, explaining the SARS-CoV-2 spikes observed in wastewaters. After the case was conveyed to a healthcare facility, wastewaters continued to yield positive signals for five days, though with a decreasing intensity. This was attributed to the return of recovered cases, who had continued to shed the virus. Our findings demonstrate the utility of wastewater surveillance as a non-intrusive tool to monitor high-risk COVID-19 premises, which is able to trigger individual tests for case detection, highlighting a new use-case for wastewater testing.

Environmental Contamination of SARS-CoV-2 in a Non-Healthcare Setting.

Fomite-mediated transmission has been identified as a possible route for the spread of COVID-19 disease caused by SARS-CoV-2. In healthcare settings, environmental contamination by SARS-CoV-2 has been found in patients' rooms and toilets. Here, we investigated environmental presence of SARS-CoV-2 in non-healthcare settings and assessed the efficacy of cleaning and disinfection in removing virus contamination. A total of 428 environmental swabs and six air samples was taken from accommodation rooms, toilets and elevators that have been used by COVID-19 cases. By using a reverse transcription polymerase chain reaction assay, we detected two SARS-CoV-2 RNA positive samples in a room where a COVID-19 patient stayed prior to diagnosis. The present study highlights the risk of fomite-mediated transmission in non-healthcare settings and the importance of surface disinfection in spaces occupied by cases. Of note, neither air-borne transmission nor surface contamination of elevators, which were transiently exposed to infected individuals, was evident among samples analyzed.

Occurrence and Antimicrobial Resistance Traits of Escherichia coli from Wild Birds and Rodents in Singapore.

Antimicrobial resistance (AMR) in Escherichia coli (E. coli) poses a public health concern worldwide. Wild birds and rodents, due to their mobility, are potential vehicles for transmission of AMR bacteria to humans. Ninety-six wild birds' faecal samples and 135 rodents' droppings samples were collected and analysed in 2017. Forty-six E. coli isolates from wild birds and rodents were subjected to AMR phenotypic and genotypic characterisation. The proportion of E. coli isolates resistant to at least one of the antimicrobials tested from wild birds (80.8%) was significantly higher than that of isolates from rodents (40.0%). The proportion of E. coli isolates resistant to each antimicrobial class for wild birds was 3.8% to 73.1% and that for rodents was 5.0% to 35.0%. Six out of 26 E. coli isolates from wild birds (23.1%) and two out of 20 (10.0%) isolates from rodents were multi-drug resistant (MDR) strains. These MDR E. coli isolates were detected with various antimicrobial resistance genes such as blaTEM-1B and qnrS1 and could be considered as part of the environmental resistome. Findings in this study suggested that wild birds and rodents could play a role in disseminating antimicrobial resistant E. coli, and this underscores the necessity of environment management and close monitoring on AMR bacteria in wild birds and rodents to prevent spreading of resistant organisms to other wildlife animals and humans.