Predicting Antibiotic Resistance and Assessing the Risk Burden from Antibiotics: A Holistic Modeling Framework in a Tropical Reservoir.

Predicting the hotspots of antimicrobial resistance (AMR) in aquatics is crucial for managing associated risks. We developed an integrated modeling framework toward predicting the spatiotemporal abundance of antibiotics, indicator bacteria, and their corresponding antibiotic-resistant bacteria (ARB), as well as assessing the potential AMR risks to the aquatic ecosystem in a tropical reservoir. Our focus was on two antibiotics, sulfamethoxazole (SMX) and trimethoprim (TMP), and on Escherichia coli (E. coli) and its variant resistant to sulfamethoxazole-trimethoprim (EC_SXT). We validated the predictive model using withheld data, with all Nash-Sutcliffe efficiency (NSE) values above 0.79, absolute relative difference (ARD) less than 25%, and coefficient of determination (R2) greater than 0.800 for the modeled targets. Predictions indicated concentrations of 1-15 ng/L for SMX, 0.5-5 ng/L for TMP, and 0 to 5 (log10 MPN/100 mL) for E. coli and -1.1 to 3.5 (log10 CFU/100 mL) for EC_SXT. Risk assessment suggested that the predicted TMP could pose a higher risk of AMR development than SMX, but SMX could possess a higher ecological risk. The study lays down a hybrid modeling framework for integrating a statistic model with a process-based model to predict AMR in a holistic manner, thus facilitating the development of a better risk management framework.

Virus surrogates throughout a full-scale advanced water reuse system.

Water reuse as an alternative water supply is increasing throughout the world due to water stress and scarcity; however, there are no standard practices for monitoring virus pathogens in such systems. This study aimed to identify suitable surrogates for virus fate, transport, and removal throughout a water reuse scheme. Various microbial targets (11 viruses, two phage, and three bacteria) were monitored using molecular and culture methods across all treatment stages in a wastewater reclamation facility and advanced water treatment facility. Criteria were established for identifying suitable surrogates, which included reliable detection, observable fate and transport, calculable log-reduction values (LRVs), correlations with other targets, and various morphological types. In total, five viruses (PMMoV, AiV, GII NoV, AdV, FRNA GII) met these stringent criteria and were suggested as potential virus surrogates. These surrogates enabled successful comparison of assigned versus actual LRVs throughout a water reuse scheme. Results suggest that virus pathogens are effectively removed throughout water reuse treatment and the suggested surrogates can be utilized for monitoring treatment performance and ensuring public health safety. This study provides a framework that water utilities across the world can reference for establishing virus monitoring practices.

Removal efficiency of antibiotic residues, antibiotic resistant bacteria, and genes across parallel secondary settling tank and membrane bioreactor treatment trains in a water reclamation plant.

Antimicrobial resistance is recognized as a potent threat to human health. Wastewater treatment facilities are viewed as hotspots for the spread of antimicrobial resistance. This study provides comprehensive data on the occurrences of 3 different antibiotic resistant opportunistic pathogens (with resistance to up to 5 antibiotics), 13 antibiotic resistant genes and intI1, and 22 different antimicrobial residues in a large water reclamation plant (176 million gallons per day) that runs a conventional Modified Ludzack-Ettinger (MLE) reactor followed by a secondary settling tank (SST) and membrane bioreactor (MBR) in parallel. All the antibiotic resistant bacteria and most of the antibiotic resistance genes were present in the raw influent, ranging from 2.5 × 102-3.7 × 106 CFU/mL and 1.2× 10-1-6.5 × 1010 GCN/mL, respectively. MBR outperformed the SST system in terms of ARB removal as the ARB targets were largely undetected in MBR effluent, with log removals ranging from 2.7 to 6.8, while SST only had log removals ranging from 0.27 to 4.6. Most of the ARG concentrations were found to have significantly higher in SST effluent than MBR permeate, and MBR had significantly higher removal efficiency for most targets (p < 0.05) except for sul1, sul2, blaOXA48, intI1 and 16S rRNA genes (p > 0.05). As for the antibiotic residues (AR), there was no significant removal from the start to the end of the treatment process, although MBR had higher removal efficiencies for azithromycin, chloramphenicol, erythromycin, erythromycin-H2O, lincomycin, sulfamethoxazole and triclosan, compared to the SST system. In conclusion, MBR outperformed SST in terms of ARB and ARGs removal. However low removal efficiencies of most AR targets were apparent.

Food waste compost and digestate as novel fertilizers: Impacts on antibiotic resistome and potential risks in a soil-vegetable system.

As a novel agricultural practice, the reuse of food waste compost and digestate as fertilizers leads to a circular economy, but inevitably introduces bio-contaminants such as antibiotic resistance genes (ARGs) into the agroecosystem. Moreover, heavy metal and antibiotic contamination in farmland soil may exert selective pressures on the evolution of ARGs, posing threats to human health. This study investigated the fate, influencing mechanisms and potential risks of ARGs in a soil-vegetable system under different food waste fertilization and remediation treatments and soil contamination conditions. Application of food waste fertilizers significantly promoted the pakchoi growth, but resulted in the spread of ARGs from fertilizers to pakchoi. A total of 56, 80, 84, 41, and 73 ARGs, mobile genetic elements (MGEs) and metal resistance genes (MRGs) were detected in the rhizosphere soil (RS), bulk soil (BS), control soil (CS), root endophytes (RE), and leaf endophytes (LE), respectively. Notably, 7 genes were shared in the above five subgroups, indicating a specific soil-root-endophytes transmission pathway. 36 genes were uniquely detected in the LE, which may originate from airborne ARGs. The combined application of biochar and fertilizers reduced the occurrence of ARGs and MGEs to some extent, showing the remediation effect of biochar. The average abundance of ARGs in the RS, BS and CS was 3.15 × 10-2, 1.31 × 10-2 and 2.35 × 10-1, respectively. Rhizosphere effects may reduce the abundance of ARGs in soil. The distribution pattern of ARGs was influenced by the types of soil, endophyte and contaminant. MGEs is the key driver shaping ARGs dynamics. Soil properties and pakchoi growth status may affect the bacterial composition, and consequently regulate ARGs fate, while endophytic ARGs were more impacted by biotic factors. Moreover, the average daily doses of ARGs from pakchoi consumption is 107-109 copies/d/kg, and its potential health risks should be emphasized.

Assessing the additional health burden of antibiotic resistant Enterobacteriaceae in surface waters through an integrated QMRA and DALY approach.

Antibiotic resistant Enterobacteriaceae pose a significant threat to public health. However, limited studies have evaluated the health risks associated with exposure to antibiotic-resistant bacteria (ARB), especially in natural environments. While quantitative microbial risk assessment (QMRA) assesses microbial risks in terms of the probability of infection, it does not account for the severity of health outcomes. In this study, a QMRA-DALY model was developed to integrate QMRA with health burden (disability-adjusted life years (DALY)) from infections caused by ARB. The model considers uncertainties in probability of infection and health burden assessment using Monte Carlo simulations. The study collected antimicrobial resistance (AMR) surveillance data from surface waters with different land uses. Results revealed water bodies with agricultural land use to be the main AMR hotspots, with the highest additional health burden observed in infections caused by meropenem-resistant E. coli (∆DALY = 0.0105 DALY/event) compared to antibiotic-susceptible E. coli. The estimated ∆DALY for antibiotic-resistant K. pneumoniae was lower than for antibiotic-resistant E. coli (highest ∆DALY = 0.00048 DALY/event). The study highlights the need for better evaluation of AMR associated health burden, and effective measures to mitigate the risks associated with antibiotic-resistant bacteria in natural environments.

Low concentration quaternary ammonium compounds promoted antibiotic resistance gene transfer via plasmid conjugation.

During the pandemic of COVID-19, the amounts of quaternary ammonium compounds (QACs) used to inactivate the virus in public facilities, hospitals and households increased, which raised concerns about the evolution and transmission of antimicrobial resistance (AMR). Although QACs may play an important role in the propagation of antibiotic resistance gene (ARGs), the potential contribution and mechanism remains unclear. Here, the results showed that benzyl dodecyl dimethyl ammonium chloride (DDBAC) and didecyl dimethyl ammonium chloride (DDAC) significantly promoted plasmid RP4-mediated ARGs transfer within and across genera at environmental relevant concentrations (0.0004-0.4 mg/L). Low concentrations of QACs did not contribute to the permeability of the cell plasma membrane, but significantly increased the permeability of the cell outer membrane due to the decrease in content of lipopolysaccharides. QACs altered the composition and content of extracellular polymeric substances (EPS) and were positively correlated with the conjugation frequency. Furthermore, transcriptional expression levels of genes encode for mating pairing formation (trbB), DNA replication and translocation (trfA), and global regulators (korA, korB, trbA) are regulated by QACs. And we demonstrate for the first time that QACs decreased the concentration of extracellular AI-2 signals, which was verified to be involved in regulating conjugative transfer genes (trbB, trfA). Collectively, our findings underscore the risk of increased disinfectant concentrations of QACs on the ARGs transfer and provide new mechanisms of plasmid conjugation.

Quaternary ammonium compounds of emerging concern: Classification, occurrence, fate, toxicity and antimicrobial resistance.

Amplified hygiene and precautionary measures are of utmost importance to control the spread of COVID-19 and future infection; however, these changes in practice are projected to trigger a rise in the purchase, utilisation and hence, discharge of many disinfectants into the environment. While alcohol-based, hydrogen peroxide-based, and chlorine-based compounds have been used widely, quaternary ammonium compounds (QACs) based disinfectants are of significant concern due to their overuse during this pandemic. This review presents the classification of disinfectants and their mechanism of action, focusing on QACs. Most importantly, the occurrence, fate, toxicity and antimicrobial resistance due to QACs are covered in this paper. Here we collated evidence from multiple studies and found rising trends of concern, including an increase in the mass load of QACs at a wastewater treatment plant (WWTP) by 331% compared to before the COVID-19 pandemic, as well as an increases in the concentration of 62% in residential dust, resulting in high concentrations of QACs in human blood and breast milk and suggesting that these could be potential sources of persistent QACs in infants. In addition to increased toxicity to human and aquatic life, increased use of QACs and accelerated use of antibiotics and antimicrobials during the COVID-19 pandemic could multiply the threat to antimicrobial resistance.

Population-based variations of a core resistome revealed by urban sewage metagenome surveillance.

Sewage-based surveillance is widely employed to understand the occurrence and distribution of antimicrobial resistance (AMR) in urban community. However, there are limited studies which investigated the sewage of different sources within community. The present study used metagenomics to decipher the AMR profiles in five sources: local residence's source, animal source, migrant workers' source, clinical source , and urban wastewater treatment plant influent. A core resistome of ARGs was found across all samples, accounting for 81.4%-93.3% of the abundance of total resistome with only 17.3% diversity, irrespective of the sewage sources. Clinically relevant ARGs were identified in the core resistome across all wastewater sources. This included genes conferring resistance to beta-lactams as biomarkers of hospital sewage. The pet center wastewater showed a high abundance of genes encoding resistance to tetracycline, which is a commonly used veterinary antibiotic. The resistome profile of sewage from the migrant workers' dormitories showed a slight variation to that of the local residential population, suggesting possible differences in the human gut resistome of the foreign/migrant population, with biomarkers of genes encoding resistance to fosfomycin, fosmidomycin, kasugamycin, MLS, and polymyxin. The co-localization of ARGs and plasmid, MGEs and integrative and conjugative elements (ICEs) could explain variations in the core resistome, presumably a result of high antibiotic selection pressure. Further analysis showed a specific host-associated resistance pattern, in which core hosts mediated the core resistome profile. The core BMRGs were also co-localized with MGEs/ICEs and carried by core potential bacterial hosts. Local healthy population carried the lowest ARG load (copy number discharged by each person per day) but contributed the highest ARG burden (copy number discharged by the population). This study elucidates population-based variations of a core resistome, and further provides important insights into source tracking and management of AMR in urban environments.

Prevalence and characterization of antibiotic resistant bacteria in raw community sewage from diverse urban communities.

Community sewage is a useful medium for antimicrobial resistance (AMR) surveillance of populations residing in urban dwellings as it provides the resolution of tracking sources of AMR from different waste streams within a community. Antibiotic resistance profiles of 4 opportunistic pathogens were compared from 3 sewage sources: raw community sewage from 2 housing estates, a foreign workers dormitory and a pet centre. Sewage was collected from receiving manholes and a culture-based method was used to quantify opportunistic pathogens Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Enterococcus spp. K. pneumoniae was the most prevalent opportunistic pathogen detected while P. aeruginosa was the least abundant across the sample. A total of 892 bacterial isolates were selected for antibiotic susceptibility testing using the VITEKⓇ 2 Compact System (bioMérieux, Singapore). E. coli isolated from the pet centre exhibited higher resistance towards all 16 antibiotics tested, and P. aeruginosa showed at least 75% resistance towards the classes of antibiotics, except for carbapenems, aminoglycosides and quinolones (below 30%). For K. pneumoniae, resistance towards the classes of antibiotics was below 10%, except for penicillin and nitrofurans. Enterococcus spp. had high resistance towards gentamicin and clindamycin (both almost 100%), while resistance towards vancomycin was below 18% for all 4 locations. Multidrug resistant (MDR) P. aeruginosa and Enterococcus spp. showed the highest prevalence with percentages above 96%. MDR E. coli and MDR K. pneumoniae was lower, ranging between 9% to 54% and 8% to 23% respectively. Highest prevalence of MDR E. coli and K. pneumoniae were detected in the pet centre, with a percentage of 54% and 23% respectively. These results show that wastewater based surveillance is not only a useful method of understanding emerging trends of AMR across the community but also provides the resolution of capturing AMR data from key sewage sources based on population demographics. This information could provide a basis for devising better control measures to mitigate the spread of MDR.

A new modelling framework for assessing the relative burden of antimicrobial resistance in aquatic environments.

The infections caused by antibiotic resistant bacteria (ARB) can lead to higher medical costs, prolonged hospital stays, and increased mortality compared to bacteria that are susceptible to antibiotics. Challenges exist in quantifying the potential risk/burden associated with antimicrobial resistance (AMR) as there is a lack of dose-response models available for pathogens which are resistant to antibiotics, in addition to the fact that very little is known regarding the health risks posed by antibiotic resistant genes (ARG). In this paper, we proposed a new modelling framework to evaluate the relative burden of AMR in natural aquatic environments. With this framework, an AMR burden score for each sample was calculated based on burden coefficients assigned for each ARB and ARG, as well as weighted burdens for the separate ARBs and ARGs components. The method developed in this study was applied to assess the relative burden of AMR in local aquatic environments with different land uses at different seasons. The collected filed data were used to verify the applicability of the proposed relative burden assessment method. Through the established method, the spatial and temporal hotspots of AMR were identified, which could provide useful information to agencies for better control and management of AMR emergence in natural aquatic environments.

Emerging contaminants in wastewater, stormwater runoff, and surface water: Application as chemical markers for diffuse sources.

Diffuse sources of pollution such as sewer leakages, sewer overflows, illicit discharges and stormwater runoff affect the urban surface water quality but often remain unknown. Therefore, the development of chemical markers for identifying and characterizing the origin of diffuse sources of pollution in urban surface waters is a requisite for protecting and managing urban water resources. In this study, the occurrence of 31 emerging contaminants (ECs) in untreated wastewater, treated wastewater, urban stormwater runoff, agricultural stormwater runoff, and freshwater bodies was investigated. Artificial sweeteners (ASs), pharmaceuticals and personal care products (PPCPs) were more frequently detected in the collected water samples. In raw wastewater, 21 target ECs were detected 100% in the collected samples with median concentrations ranging from 49.6 to 77,721 ng/L, while in freshwater bodies, only 13 compounds were found with detection frequency >50%. The median concentration of the majority of detected ECs in freshwater samples was below 100 ng/L. The suitability of ECs as chemical markers of diffuse sources in an urban watershed was assessed using a suite of criteria, including the detection frequency (DF), detection ratio (DR) (i.e. the ratio between median concentration and method quantification limit of a compound) and attenuation rates (i.e., biodegradation, sorption and abiotic degradation) in wastewater treatment processes. In addition, we propose a new key criterion, the concentration ratio (CR) of labile to conservative compounds, to evaluate the applicability of suitable chemical markers for source tracking. Using this new set of criteria (i.e. CR, DF, DR and attenuation rates), our analysis showed that among the investigated ECs, only acesulfame (ACE), acetaminophen (ACT), cyclamate (CYC), saccharin (SAC) were suitable as chemical markers of diffuse sources in surface waters. For caffeine (CF), N,N-diethyl-meta-toluamide (DEET), crotamiton (CTMT), triclocarban (TCC) and triclosan (TCS), their median concentration ratio to sucralose (SUC) in water bodies was consistently higher than that in raw wastewater, suggesting that these compounds might be unsuitable as chemical markers of sewage leakage in surface waters for this study area.

Occurrence and distribution of viruses and picoplankton in tropical freshwater bodies determined by flow cytometry.

This study aimed to examine the drivers in shaping the occurrence and distribution of total viruses and picoplankton in tropical freshwater ecosystems. Flow cytometry was used to quantify the concentrations of total viruses, picoheterotrophs, picophytoplankton, and picocyanobacteria. Three land use patterns (urban-, agriculture- and parkland-dominated) were evaluated using ArcGIS. Significant correlations were observed between water-borne microbial targets and water quality parameters (0.175 ≤ |r| ≤ 0.441), nutrients (0.250 ≤ r ≤ 0.570) and land use factors (0.200 ≤ |r| ≤ 0.460). In particular, the concentrations of total viruses and picoheterotrophic cells were higher in catchments whereas the abundances of picophytoplankton and picocyanobacteria were higher in reservoirs. Total viruses and picoplankton had higher concentrations in urban- and agriculture-dominated areas, probably due to anthropogenic inputs and agricultural inputs, respectively. Although surface water is a complex matrix influenced by niche-based (i.e., physicochemical properties, nutrients, land use impact etc.) and neutral-based factors (i.e., ecological drift, dispersal and species), land use patterns could help to elucidate the occurrence and distribution of the total microbial community at the macroscopic level. Meanwhile, inter-correlations among viruses, picoplankton and picoheterotrophs (0.715 ≤ r ≤ 0.990) also substantiates their mutual interactions in influencing the microbial community. Furthermore, the relationships between total microbial cells and bacterial and viral indicators were also investigated. Concentrations of total viruses, picoplankton and picoheterotrophs were positively correlated with bacterial indicators (0.427 ≤ r ≤ 0.590) and viral indicators (0.201 ≤ r ≤ 0.563). These results indicated that faecal and viral contamination could contribute to the numbers of total viruses and bacteria.

Occurrence of microbial indicators, pathogenic bacteria and viruses in tropical surface waters subject to contrasting land use.

Fecal indicator bacteria, such as Escherichia coli (E.coli) and Enterococcus, have been widely used to indicate the presence of pathogens. However, the suitability of fecal indicator bacteria to represent health risks is still being challenged, particularly in tropical aquatic environments. The objective of this study is to understand the occurrence and prevalence of indicators and pathogens in areas with contrasting land use, as well as to identify the major correlations between indicators, pathogens and environmental parameters. The spatial and temporal variation of indicators and pathogens was studied to examine the distribution patterns for areas with different land use, and the impact of seasonal changes on microbial populations. A total of 234 water samples were sampled for two years from reservoirs and their tributaries, and tested for fecal indicator bacteria, coliphages, human specific markers, pathogenic bacteria and viruses. The prevalence of indicators and pathogens in reservoirs were generally low, while relatively high concentrations were observed in tributaries to varying degrees. Of the enteric viruses, norovirus GII was among the most prevalent and had the highest concentration. Although strong correlations were found between indicators, only relatively weak correlations were found between indicators and pathogens. The results in this study showed that none of the bacteria/phage indicators were universal predictors for pathogens. Inclusion of the alternative indicators, Methanobrevibacter smithii, Bacteroides and human polyomaviruses (HPyVs) to monitoring programs could help to determine whether the fecal source was human. The microbial distribution patterns allow the classification of sampling sites to different clusters and thus, help to identify sites which have poor water quality. This approach will be useful for water quality management to pinpoint factors that influence water quality and help to prioritize sites for restoration of water quality.

Draft Genome Sequences of Four Multidrug-Resistant Pseudomonas aeruginosa Isolates from Hospital Wastewater in Singapore.

Four multidrug-resistant Pseudomonas aeruginosa isolates were cultured from intensive care unit wastewater. All isolates exhibited resistance to carbapenem and extended-spectrum beta-lactam antibiotics. Genome characterization revealed the presence of beta-lactamase resistance genes (bla PAO and bla OXA), and three out of the four isolates carried the bla NDM-1 gene encoding resistance against carbapenems.

Microbial water quality and the detection of multidrug resistant E. coli and antibiotic resistance genes in aquaculture sites of Singapore.

Poor microbial water quality jeopardizes the health and safety of food produced by aquaculture farms. Three fish farms and transect sites in Singapore were assessed for microbial water quality and antimicrobial resistance determinants. Of the 33 multidrug resistant E. coli isolated from surface waters of the Johor Straits, 81.8% were ESBL producers. The relative abundance of sul1, qnrA and intI1 genes were higher in sediments than surface waters. Among the surface water samples, higher concentrations (10-1-101) of beta-lactamases (blaSHV, blaOXA, blaCTX-M) were detected in the transect sites. This study highlights a potential antimicrobial resistance transmission chain from environmental waters, to animal carriers and humans.

Effect of Rainfall on the Microbial Water Quality of a Tropical Urban Catchment.

Wet weather conditions have been associated with increased bacterial and viral counts in surface waters. Moreover, heavy rainfall and flooding were found to be the most common events preceding waterborne disease outbreaks associated with extreme weather conditions. This study aimed to examine the effect of rainfall on the quality of surface waters and to determine its suitability for primary contact recreation during wet weather conditions. A total of 228 catchment water samples were collected during wet and dry periods. Parameters that were found to increase with increasing rainfall were , enterococci, somatic coliphages, and turbidity, whereas total dissolved solids were found to decrease. Positive correlations ( < 0.05) were observed between cumulative rainfall and geometric mean concentrations of , enterococci, somatic coliphages, and turbidity ( = 0.69-0.95), whereas a negative correlation was observed between cumulative rainfall and total dissolved solids ( = -0.58). In addition, a rapid decline in water quality was observed during heavy rainfall that resulted in failure to meet recreational water quality guidelines. In view of public health and safety, primary recreational activities in the water catchment may not be advisable during or immediately after a rainfall event due to poor water quality.

Occurrence of Traditional and Alternative Fecal Indicators in Tropical Urban Environments under Different Land Use Patterns.

This study evaluated the geospatial distribution of fecal indicator bacteria (FIB) (i.e., Escherichia coli, Enterococcus spp.) and the alternative fecal indicator pepper mild mottle virus (PMMoV) in tropical freshwater environments under different land use patterns. Results show that the occurrence and concentration of microbial fecal indicators were higher for urban than for parkland-dominated areas, consistent with land use weightage. Significant positive correlations with traditional FIB indicate that PMMoV is a suitable indicator of fecal contamination in tropical catchments waters (0.549 ≤ rho ≤ 0.612; P < 0.01). PMMoV exhibited a strong significant correlation with land use weightage (rho = 0.728; P < 0.01) compared to traditional FIB (rho = 0.583; P < 0.01). In addition, chemical tracers were also added to evaluate the potential relationships with microbial fecal indicators. The relationships between diverse variables (e.g., environmental parameters, land use coverage, and chemical tracers) and the occurrence of FIB and PMMoV were evaluated. By using stepwise multiple linear regression (MLR), the empirical experimental models substantiate the impact of land use patterns and anthropogenic activities on microbial water quality, and the output results of the empirical models may be able to predict the sources and transportation of human fecal pollution or sewage contamination. In addition, the high correlation between PMMoV data obtained from quantitative real-time PCR (qPCR) and viral metagenomics data supports the possibility of using viral metagenomics to relatively quantify specific microbial indicators for monitoring microbial water quality (0.588 ≤ rho ≤ 0.879; P < 0.05).IMPORTANCE The results of this study may support the hypothesis of using PMMoV as an alternative indicator of human fecal contamination in tropical surface waters from the perspective of land use patterns. The predictive result of the occurrence of human fecal indicators with high accuracy may reflect the source and transportation of human fecal pollution, which are directly related to the risk to human health, and thereafter, steps can be taken to mitigate these risks.

Geospatial distribution of viromes in tropical freshwater ecosystems.

This study seeks to understand the general distribution of virome abundance and diversity in tropical freshwater ecosystems in Singapore and the geospatial distribution of the virome under different landuse patterns. Correlations between diversity, environmental parameters and land use patterns were analyzed and significant correlations were highlighted. Overall, the majority (65.5%) of the annotated virome belonged to bacteriophages. The percentage of Caudovirales was higher in reservoirs whereas the percentages of Dicistroviridae, Microviridae and Circoviridae were higher in tributaries. Reservoirs showed a higher Shannon-index virome diversity compared to upstream tributaries. Land use (urbanized, agriculture and parkland areas) influenced the characteristics of the virome distribution pattern. Dicistroviridae and Microviridae were enriched in urbanized tributaries while Mimiviridae, Phycodnaviridae, Siphoviridae and Podoviridae were enriched in parkland reservoirs. Several sequences closely related to the emerging zoonotic virus, cyclovirus, and the human-related virus (human picobirnavirus), were also detected. In addition, the relative abundance of PMMoV (pepper mild mottle virus) sequences was significantly correlated with RT-qPCR measurements (0.588 < r < 0.879, p < 0.05). This study shows that spatial factors (e.g., reservoirs/tributaries, land use) are the main drivers of the viral community structure in tropical freshwater ecosystems.

Occurrence of Vibrio species, beta-lactam resistant Vibrio species, and indicator bacteria in ballast and port waters of a tropical harbor.

Ballast water discharges are potential sources for the spread of invasive and pathogenic aquatic organisms. Ballast waters from six ships docked in the Port of Singapore were tested to determine if indictor organisms fell within proposed standards for ballast water discharge according to regulation D-2 of the Ballast Water Management Convention (BWMC) guidelines. Vibrio species were cultured on media supplemented with beta-lactam antibiotics to determine the presence of antibiotic resistant Vibrio species in the ballast waters of these vessels. Indicator organisms were quantified using culture media Colilert-18 and Enterolert in ballast waters of six ships docked in a tropical harbor, with uptake from different geographical locations. Of the six ships, one had ballast water originating from the Persian Gulf, another from the East China Sea, and four from the South China Sea. Two of the six ships which carried ballast waters from the East China Sea and the South China Sea did not meet the D-2 stipulated requirements of the Ballast Water Management Convention for indicator organisms with Enterococci values more than three times higher than the acceptable limit of <100CFU/100mL. Using the most-probable-number-PCR (MPN-PCR) method for Vibrio species detection, non-toxigenic species of V. cholerae (2 MPN/100mL), Vibrio vulnificus (>110 MPN/100mL), and Vibrio parahaemolyticus (2 to >110 MPN/100mL) were detected in at least one of six ballast water samples. Using thiosulfate-citrate-bile salts-sucrose agar (TCBS) supplemented with beta-lactam antibiotics (meropenem, ceftazidime), 11 different Vibrio species, exhibiting resistance to beta-lactam antibiotics were isolated; with Vibrio campbellii (44%) and Vibrio alginolyticus (15%) the most detected antibiotic resistant Vibrio species. A practical approach of prioritized screening of high-risk vessels should be conducted to ensure that the water quality meets D-2 standards prior to discharge.

Development of a chemiluminescent DNA fibre optic genosensor to Hepatitis A Virus (HAV).

Hepatitis A virus (HAV) infection has caused substantial morbidity and economic losses to human society, presenting a major public health problem in many parts of the world. Despite the capability for low-concentration detection, current PCR-based techniques are limited by the requirement of specialized lab equipment, trained personnel and a relatively large time-commitment. The need for a prompt in-field quantitative identification of HAV in real samples has led us to develop a chemiluminescent fibre optic genosensor system. In this study, a two-probe sandwich-type hybridization process was implemented on the tip of a fibre optic with an area of 0.12mm2. After optimization of the probes and the working conditions, we showed that the biosensor was able to work for both cDNA and RNA with a relatively large signal/noise ratio and a good sensitivity. An excellent specificity was also confirmed by screening with a broad range of other pathogen samples. The nucleic acid probes method was validated by optimized PCR and qPCR, and may thus be used when field testing would be required.