Quantitative microbial risk assessment of the gastrointestinal risks to swimmers at Southeast Asian urban beaches using site-specific and combined autochthonous and fecal bacteria exposure data.

Recreational exposure to microbial pollution at urban beaches poses a health risk to beachgoers. The accurate quantification of such risks is crucial in managing beaches effectively and establishing warning guidelines. In this study, we employed a quantitative microbial risk assessment (QMRA) framework to assess marine water quality and estimate the risks associated with Vibrio parahaemolyticus, an autochthonous pathogen that causes gastrointestinal illnesses, and enterococci, a traditional fecal bacteria indicator. The microbial contamination levels of V. parahaemolyticus and enterococci were determined from 48 water samples collected at two beaches in Thailand during dry and wet seasons. The accidentally ingested water volumes were obtained through a survey involving 438 respondents. The probability of illness (Pill) was estimated using dose-response models and Monte Carlo simulation. The results revealed that enterococci posed a higher risk of illness than V. parahaemolyticus at all seven study sites. The median combined gastrointestinal (GI) risk from both bacteria at all sites met the US EPA risk benchmark of 0.036 and the 0.05 benchmark set by the WHO, but the 95th percentile risk data at all sites exceeded the benchmarks. This emphasizes the need for the continuous monitoring and management of microbial pollution at these sites. The site-specific exposure data showed higher estimated risks with increased variations compared to the WHO-referenced values, which highlights the significance of locally measured microbial concentrations and survey exposure data to avoid underestimation. Estimating the risks from recreational exposure to waterborne bacteria can inform beach management policies aimed at reducing public health risks to swimmers. The study findings improve the understanding of the risks associated with water recreation activities at Southeast Asian beaches and offer valuable insights for the development of water quality guidelines, which are crucial for the sustainable development of the blue economy.

Microbial source tracking using molecular and cultivable methods in a tropical mixed-use drinking water source to support water safety plans.

Microbial contamination deteriorates source water quality, posing a severe problem for drinking water suppliers worldwide and addressed by the Water Safety Plan framework to ensure high-quality and reliable drinking water. Microbial source tracking (MST) is used to examine different microbial pollution sources via host-specific intestinal markers for humans and different types of animals. However, the application of MST in tropical surface water catchments that provide raw water for drinking water supplies is limited. We analyzed a set of MST markers, namely, three cultivable bacteriophages and four molecular PCR and qPCR assays, together with 17 microbial and physicochemical parameters, to identify fecal pollution from general, human-, swine-, and cattle-specific sources. Seventy-two river water samples at six sampling sites were collected over 12 sampling events during wet and dry seasons. We found persistent fecal contamination via the general fecal marker GenBac3 (100 % detection; 2.10-5.42 log10 copies/100 mL), with humans (crAssphage; 74 % detection; 1.62-3.81 log10 copies/100 mL) and swine (Pig-2-Bac; 25 % detection; 1.92-2.91 log10 copies/100 mL). Higher contamination levels were observed during the wet season (p < 0.05). The conventional PCR screening used for the general and human markers showed 94.4 % and 69.8 % agreement with the respective qPCR results. Specifically, in the studied watershed, coliphage could be a screening parameter for the crAssphage marker (90.6 % and 73.7 % positive and negative predictive values; Spearman's rank correlation coefficient = 0.66; p < 0.001). The likelihood of detecting the crAssphage marker significantly increased when total and fecal coliforms exceeded 20,000 and 4000 MPN/100 mL, respectively, as Thailand Surface Water Quality Standards, with odds ratios and 95 % confidence intervals of 15.75 (4.43-55.98) and 5.65 (1.39-23.05). Our study confirms the potential benefits of incorporating MST monitoring into water safety plans, supporting the use of this approach to ensure high-quality drinking water supplies worldwide.

Wastewater monitoring in tourist cities as potential sentinel sites for near real-time dynamics of imported SARS-CoV-2 variants.

Wastewater-based epidemiology (WBE) complements the clinical surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants' distribution in populations. Many developed nations have established national and regional WBE systems; however, governance and budget constraints could be obstacles for low- and middle-income countries. An urgent need thus exists to identify hotspots to serve as sentinel sites for WBE. We hypothesized that representative wastewater treatment plants (WWTPs) in two international gateway cities, Bangkok and Phuket, Thailand, could be sentineled for SARS-CoV-2 and its variants to reflect the clinical distribution patterns at city level and serve as early indicators of new variants entering the country. Municipal wastewater samples (n = 132) were collected from eight representative municipal WWTPs in Bangkok and Phuket during 19 sampling events from October 2021 to March 2022, which were tested by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) using the US CDC N1 and N2 multiplex and variant (Alpha, Delta, and Omicron BA.1 and BA.2) singleplex assays. The variant detection ratios from Bangkok and Phuket followed similar trends to the national clinical testing data, and each variant's viral loads agreed with the daily new cases (3-d moving average). Omicron BA.1 was detected in Phuket wastewater prior to Bangkok, possibly due to Phuket's WWTPs serving tourist communities. We found that the Omicron BA.1 and BA.2 viral loads predominantly drove the SARS-CoV-2 resurgence. We also noted a shifting pattern in the Bangkok WBE from a 22-d early warning in early 2021 to a near real-time pattern in late 2021. The potential application of tourist hotspots for WBE to indicate the arrival of new variants and re-emerging or unprecedented infectious agents could support tourism-dependent economies by complementing the reduced clinical regulations while maintaining public health protection via wastewater surveillance.

SARS-CoV-2 RNA surveillance in large to small centralized wastewater treatment plants preceding the third COVID-19 resurgence in Bangkok, Thailand.

Wastewater surveillance for SARS-CoV-2 RNA has been a successful indicator of COVID-19 outbreaks in populations prior to clinical testing. However, this has been mostly conducted in high-income countries, which means there is a dearth of performance investigations in low- and middle-income countries with different socio-economic settings. This study evaluated the applicability of SARS-CoV-2 RNA monitoring in wastewater (n = 132) to inform COVID-19 infection in the city of Bangkok, Thailand using CDC N1 and N2 RT-qPCR assays. Wastewater influents (n = 112) and effluents (n = 20) were collected from 19 centralized wastewater treatment plants (WWTPs) comprising four large, four medium, and 11 small WWTPs during seven sampling events from January to April 2021 prior to the third COVID-19 resurgence that was officially declared in April 2021. The CDC N1 assay showed higher detection rates and mostly lower Ct values than the CDC N2. SARS-CoV-2 RNA was first detected at the first event when new reported cases were low. Increased positive detection rates preceded an increase in the number of newly reported cases and increased over time with the reported infection incidence. Wastewater surveillance (both positive rates and viral loads) showed strongest correlation with daily new COVID-19 cases at 22-24 days lag (Spearman's Rho = 0.85-1.00). Large WWTPs (serving 432,000-580,000 of the population) exhibited similar trends of viral loads and new cases to those from all 19 WWTPs, emphasizing that routine monitoring of the four large WWTPs could provide sufficient information for the city-scale dynamics. Higher sampling frequency at fewer sites, i.e., at the four representative WWTPs, is therefore suggested especially during the subsiding period of the outbreak to indicate the prevalence of COVID-19 infection, acting as an early warning of COVID-19 resurgence.

Persisting antibiotic resistance gene pollution and its association with human sewage sources in tropical marine beach waters.

Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) are pollutants of worldwide concern that threaten human health and ecosystems. Anthropogenic activities and wastewater could be ARB and ARG pollution sources; however, research on ARG abundance and microbial source tracking (MST) of contamination in tropical marine waters is limited. This study examined spatiotemporal variations of six ARGs (blaNDM, blaTEM, blaVIM, mcr-1, sul1, and tetQ) against the widely used antibiotic groups and a class 1 integron-integrase gene (intI1) at two Thai tropical recreational beaches (n = 41). Correlations between ARGs and sewage-specific MST markers (i.e., crAssphage and human polyomaviruses [HPyVs]) and fecal indicator bacteria (i.e., total coliforms, fecal coliforms, and enterococci) were also investigated. BlaTEM, intI1, sul1, and tetQ were ubiquitous at both beaches (85.4-100% detection rate); intI1 was the most abundant (3-6 orders in log10 copies/100 mL), followed by blaTEM (2-4 orders), sul1 (2-3 orders), and tetQ (2-4 orders). BlaNDM was found in 7.3% (up to 4 orders), and no mcr-1 was detected. Interestingly, blaVIM was prevalent at one beach (2-5 orders; n = 17), but found in only one sample at the other (4 orders). Temporal, but not spatial, differences were noticed; blaTEM was at higher levels in the wet season. IntI1 correlated with sul1 and tetQ (Spearman's rho = 0.47-0.97), suggesting potential horizontal gene transfer. CrAssphage, but not HPyVs, correlated with intI1, sul1, and tetQ (Spearman's rho = 0.50-0.74). Higher numbers of ARGs tended to co-occur in samples with higher crAssphage concentrations, implying sewage contribution to the marine water, with a persisting ARG background. This study provides insight into the ARG pollution status of tropical coastal waters and suggests crAssphage as a proxy for ARG pollution, which could facilitate effective management policies to minimize ARG dissemination in marine environments.

Integrated analyses of fecal indicator bacteria, microbial source tracking markers, and pathogens for Southeast Asian beach water quality assessment.

The degradation of coastal water quality from fecal pollution poses a health risk to visitors at recreational beaches. Fecal indicator bacteria (FIB) are a proxy for fecal pollution; however the accuracy of their representation of fecal pollution health risks at recreational beaches impacted by non-point sources is disputed due to non-human derivation. This study aimed to investigate the relationship between FIB and a range of culturable and molecular-based microbial source tracking (MST) markers and pathogenic bacteria, and physicochemical parameters and rainfall. Forty-two marine water samples were collected from seven sampling stations during six events at two tourist beaches in Thailand. Both beaches were contaminated with fecal pollution as evident from the GenBac3 marker at 88%-100% detection and up to 8.71 log10 copies/100 mL. The human-specific MST marker human polyomaviruses JC and BK (HPyVs) at up to 4.33 log10 copies/100 mL with 92%-94% positive detection indicated that human sewage was likely the main contamination source. CrAssphage showed lower frequencies and concentrations; its correlations with the FIB group (i.e., total coliforms, fecal coliforms, and enterococci) and GenBac3 diminished its use as a human-specific MST marker for coastal water. Human-specific culturable AIM06 and SR14 bacteriophages and general fecal indicator coliphages also showed less sensitivity than the human-specific molecular assays. The applicability of the GenBac3 endpoint PCR assay as a lower-cost prescreening step prior to the GenBac3 qPCR assay was supported by its 100% positive predictive value, but its limited negative predictive values required subsequent qPCR confirmation. Human enteric adenovirus and Vibrio cholerae were not found in any of the samples. The HPyVs related to Vibrio parahaemolyticus, Vibrio vulnificus, and 5-d rainfall records, all of which were more prevalent and concentrated during the wet season. More monitoring is therefore recommended during wet periods. Temporal differences but no spatial differences were observed, suggesting the need for a sentinel site at each beach for routine monitoring. The exceedance of FIB water quality standards did not indicate increased prevalence or concentrations of the HPyVs or Vibrio spp. pathogen group, so the utility of FIB as an indicator of health risks at tropical beaches maybe challenged. Accurate assessment of fecal pollution by incorporating MST markers could lead to developing a more effective water quality monitoring plan to better protect human health risks in tropical recreational beaches.