Corrected and Republished from: "Isolation and Characterization of the First Freshwater Cyanophage Infecting Pseudanabaena".

Cyanobacteria are the major primary producers in both freshwater and marine environments. However, the majority of freshwater cyanophages remain unknown due to the limited number of cyanophage isolates. In this study, we present a novel lytic freshwater cyanophage, PA-SR01, which was isolated from the Singapore Serangoon Reservoir. To our knowledge, this is the first isolate of a cyanophage that has been found to infect the cyanobacterium Pseudanabaena. PA-SR01 has a narrow host range, a short latent period, and is chloroform sensitive. PA-SR01 is a member of Siphoviridae with a long noncontractile tail. It is a double-stranded DNA virus with a 137,012-bp genome. Functional annotation for the predicted open reading frames (ORFs) of the PA-SR01 genome identified genes with putative functions related to DNA metabolism, structural proteins, lysis, host-derived metabolic genes, and DNA packaging. Out of 166 predicted ORFs, only 17 ORFs have homology with genes with known function. Phylogenetic analysis of the major capsid protein and terminase large subunit further suggests that phage PA-SR01 is evolutionary distinct from known cyanophages. Metagenomics sequence recruitment onto the PA-SR01 genome indicates that PA-SR01 represents a new evolutionary lineage of phage which shares considerable genetic similarities with phage sequences in aquatic environments and could play key ecological roles. IMPORTANCE This study presents the isolation of the very first freshwater cyanophage, PA-SR01, that infects Pseudanabaena, and fills an important knowledge gap on freshwater cyanophages as well as cyanophages infecting Pseudanabaena.

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.

Picocyanobacterial-bacterial interactions sustain cyanobacterial blooms in nutrient-limited aquatic environments.

Cyanobacterial blooms (CBs) and concomitant water quality issues in oligotrophic/mesotrophic waters have been recently reported, challenging the conventional understanding that CBs are primarily caused by eutrophication. To elucidate the underlying mechanism of CBs in nutrition-deficient waters, the changes in Chlorophyll a (Chl-a), cyanobacterial-bacterial community composition, and certain microbial function in Qingcaosha Reservoir, the global largest tidal estuary storage reservoir, were analyzed systematically and comprehensively after its pilot run (2011-2019) in this study. Although the water quality was improved and stabilized, more frequent occurrences of bloom level of Chl-a (>20 µg L-1) in warm seasons were observed during recent years. The meteorological changes (CO2, sunshine duration, radiation, precipitation, evaporation, and relative humidity), water quality variations (pH, total organic carbon content, dissolved oxygen, and turbidity), accumulated sediments as an endogenous source, as well as unique estuarine conditions collectively facilitated picocyanobacterial-bacterial coexistence and community functional changes in this reservoir. A stable and tight co-occurrence pattern was established between dominant cyanobacteria (Synechococcus, Cyanobium, Planktothrix, Chroococcidiopsis, and Prochlorothrix) and certain heterotrophic bacteria (Proteobacteria, Actinobacteria, and Bacteroidetes), which contributed to the remineralization of organic matter for cyanobacteria utilization. The relative abundance of chemoorganoheterotrophs and bacteria related to nitrogen transformation (Paracoccus, Rhodoplanes, Nitrosomonas, and Zoogloea) increased, promoting the emergence of CBs in nutrient-limited conditions through enhanced nutrient recycling. In environments with limited nutrients, the interaction between photosynthetic autotrophic microorganisms and heterotrophic bacteria appears to be non-competitive. Instead, they adopt complementary roles within their ecological niche over long-term succession, mutually benefiting from this association. This long-term study confirmed that enhanced nutrient cycling, facilitated by cyanobacterial-bacterial symbiosis following long-term succession, could promote CBs in oligotrophic aquatic environments devoid of external nutrient inputs. This study advances understanding of the mechanisms that trigger and sustain CBs under nutritional constraints, contributing to developing more effective mitigation strategies, ensuring water safety, and maintaining ecological balance.

Screening of the PA14NR Transposon Mutant Library Identifies Genes Involved in Resistance to Bacteriophage Infection in Pseudomomas aeruginosa.

Multidrug-resistant P. aeruginosa infections pose a serious public health threat due to the rise in antimicrobial resistance. Phage therapy has emerged as a promising alternative. However, P. aeruginosa has evolved various mechanisms to thwart phage attacks, making it crucial to decipher these resistance mechanisms to develop effective therapeutic strategies. In this study, we conducted a forward-genetic screen of the P. aeruginosa PA14 non-redundant transposon library (PA14NR) to identify dominant-negative mutants displaying phage-resistant phenotypes. Our screening process revealed 78 mutants capable of thriving in the presence of phages, with 23 of them carrying insertions in genes associated with membrane composition. Six mutants exhibited total resistance to phage infection. Transposon insertions were found in genes known to be linked to phage-resistance such as galU and a glycosyl transferase gene, as well as novel genes such as mexB, lasB, and two hypothetical proteins. Functional experiments demonstrated that these genes played pivotal roles in phage adsorption and biofilm formation, indicating that altering the bacterial membrane composition commonly leads to phage resistance in P. aeruginosa. Importantly, these mutants displayed phenotypic trade-offs, as their resistance to phages inversely affected antibiotic resistance and hindered biofilm formation, shedding light on the complex interplay between phage susceptibility and bacterial fitness. This study highlights the potential of transposon mutant libraries and forward-genetic screens in identifying key genes involved in phage-host interactions and resistance mechanisms. These findings support the development of innovative strategies for combating antibiotic-resistant pathogens.

Coexistence of Synechococcus and Microcystis Blooms in a Tropical Urban Reservoir and Their Links with Microbiomes.

Bacteria play a crucial role in driving ecological processes in aquatic ecosystems. Studies have shown that bacteria-cyanobacteria interactions contributed significantly to phytoplankton dynamics. However, information on the contribution of bacterial communities to blooms remains scarce. Here, we tracked changes in the bacterial community during the development of a cyanobacterial bloom in an equatorial estuarine reservoir. Two forms of blooms were observed simultaneously corresponding to the lotic and lentic characteristics of the sampling sites where significant spatial variabilities in physicochemical water quality, cyanobacterial biomass, secondary metabolites, and cyanobacterial/bacterial compositions were detected. Microcystis dominated the upstream sites during peak periods and were succeeded by Synechococcus when the bloom subsided. For the main body of the reservoir, a mixed bloom featuring coccoid and filamentous cyanobacteria (Microcystis, Synechococcus, Planktothricoides, Nodosilinea, Raphidiopsis, and Prochlorothrix) was observed. Concentrations of the picocyanobacteria Synechococcus remained high throughout the study, and their positive correlations with cylindrospermopsin and anatoxin-a suggested that they could produce cyanotoxins, which pose more damaging impacts than previously supposed. Succession of different cyanobacteria (Synechococcus and Microcystis) following changes in nutrient composition and ionic strength was demonstrated. The microbiomes associated with blooms were unique to the dominant cyanobacteria. Generic and specialized bloom biomarkers for the Microcystis and downstream mixed blooms were also identified. Microscillaceae, Chthoniobacteraceae, and Roseomonas were the major heterotrophic bacteria associated with Microcystis bloom, whereas Phycisphaeraceae and Methylacidiphilaceae were the most prominent groups for the Synechococcus bloom. Collectively, bacterial community can be greatly deviated by the geological condition, monsoon season, cyanobacterial density, and dominant cyanobacteria.

Responses of cyanobacterium Microcystis aeruginosa under single and repeated ofloxacin exposure.

Antibiotics are omnipresent and pseudo-persistent in the environment. Yet, their potential ecological risks under repeated exposure, which is more environmentally relevant, are understudied. Therefore, this study used ofloxacin (OFL) as the probe chemical to investigate the toxic effects of different exposure scenarios-single dose of high concentration (4.0 µg/L) and multiple additions of low concentrations-towards the cyanobacterium Microcystis aeruginosa. Flow cytometry was employed to measure a collection of biomarkers, including endpoints related with biomass, single cell properties and physiological status. Results showed that the single dose of the highest OFL level inhibited cellular growth, chl-a content and cell size of M. aeruginosa. In contrast, OFL induced stronger chl-a autofluorescence and higher doses tended to have more remarkable effects. Repeated low OFL doses can more significantly increase the metabolic activity of M. aeruginosa than a single high dose. Viability and cytoplasmic membrane were not affected by OFL exposure. Oxidative stress was observed for the different exposure scenarios, with fluctuating responses. This study demonstrated the different physiological responses of M. aeruginosa under different OFL exposure scenarios, providing novel insights into the toxicity of antibiotics under repeated exposure.

Heavy metals in a typical city-river-reservoir system of East China: Multi-phase distribution, microbial response and ecological risk.

The rapid construction of artificial reservoirs in metropolises has promoted the emergence of city-river-reservoir systems worldwide. This study investigated the environmental behaviors and risks of heavy metals in the aquatic environment of a typical system composed of main watersheds in Suzhou and Jinze Reservoir in Shanghai. Results shown that Mn, Zn and Cu were the dominant metals detected in multiple phases. Cd, Mn and Zn were mainly presented in exchangeable fraction and exhibited high bioavailability. Great proportion and high mobility of metals were found in suspended particulate matter (SPM), suggesting that SPM can greatly affect metal multi-phase distribution process. Spatially, city system (CiS) exhibited more serious metal pollution and higher ecological risk than river system (RiS) and reservoir system (ReS) owing to the diverse emission sources. CiS and ReS were regarded as critical pollution source and sink, respectively, while RiS was a vital transportation aisle. Microbial community in sediments exhibited evident spatial variation and obviously modified by exchangeable metals and nutrients. In particular, Bacteroidetes and Firmicutes presented significant positive correlations with most exchangeable metals. Risk assessment implied that As, Sb and Ni in water may pose potential carcinogenic risk to human health. Nevertheless, ReS was in a fairly safe state. Hg was the main risk contributor in SPM, while Cu, Zn, Ni and Sb showed moderate risk in sediments. Overall, Hg, Sb and CiS were screened out as priority metals and system, respectively. More attention should be paid to these priority issues to promote the sustainable development of the watershed.

Isolation and Characterization of the First Freshwater Cyanophage Infecting Pseudanabaena.

Cyanobacteria are the major primary producers in both freshwater and marine environments. However, the majority of freshwater cyanophages remain unknown due to the limited number of cyanophage isolates. In this study, we present a novel lytic freshwater cyanophage, PA-SR01, which was isolated from the Singapore Serangoon Reservoir. To our knowledge, this is the first isolate of a cyanophage that has been found to infect the cyanobacterium Pseudanabaena PA-SR01 has a narrow host range, a short latent period, and is chloroform sensitive. Distinct from the majority of cyanophage isolates, PA-SR01 has a tailless morphology. It is a double-stranded DNA virus with a 137,012-bp genome. Functional annotation for the predicted open reading frames (ORFs) of the PA-SR01 genome identified genes with putative functions related to DNA metabolism, structural proteins, lysis, host-derived metabolic genes, and DNA packaging. Out of 166 predicted ORFs, only 17 ORFs have homology with genes with known function. Phylogenetic analysis of the major capsid protein and terminase large subunit further suggests that phage PA-SR01 is evolutionary distinct from known cyanophages. Metagenomics sequence recruitment onto the PA-SR01 genome indicates that PA-SR01 represents a new evolutionary lineage of phage which shares considerable genetic similarities with phage sequences in aquatic environments and could play key ecological roles.IMPORTANCE This study presents the isolation of the very first freshwater cyanophage, PA-SR01, that infects Pseudanabaena, and fills an important knowledge gap on freshwater cyanophages as well as cyanophages infecting Pseudanabaena.

Developing Surrogate Markers for Predicting Antibiotic Resistance "Hot Spots" in Rivers Where Limited Data Are Available.

Pinpointing environmental antibiotic resistance (AR) hot spots in low-and middle-income countries (LMICs) is hindered by a lack of available and comparable AR monitoring data relevant to such settings. Addressing this problem, we performed a comprehensive spatial and seasonal assessment of water quality and AR conditions in a Malaysian river catchment to identify potential "simple" surrogates that mirror elevated AR. We screened for resistant coliforms, 22 antibiotics, 287 AR genes and integrons, and routine water quality parameters, covering absolute concentrations and mass loadings. To understand relationships, we introduced standardized "effect sizes" (Cohen's D) for AR monitoring to improve comparability of field studies. Overall, water quality generally declined and environmental AR levels increased as one moved down the catchment without major seasonal variations, except total antibiotic concentrations that were higher in the dry season (Cohen's D > 0.8, P < 0.05). Among simple surrogates, dissolved oxygen (DO) most strongly correlated (inversely) with total AR gene concentrations (Spearman's ρ 0.81, P < 0.05). We suspect this results from minimally treated sewage inputs, which also contain AR bacteria and genes, depleting DO in the most impacted reaches. Thus, although DO is not a measure of AR, lower DO levels reflect wastewater inputs, flagging possible AR hot spots. DO measurement is inexpensive, already monitored in many catchments, and exists in many numerical water quality models (e.g., oxygen sag curves). Therefore, we propose combining DO data and prospective modeling to guide local interventions, especially in LMIC rivers with limited data.

Multi-phase distribution, spatiotemporal variation and risk assessment of antibiotics in a typical urban-rural watershed.

The widespread consumption and continuous discharge of antibiotics have threatened the ecological health of urban-rural watershed. In this study, multi-phase distribution, spatiotemporal variation and ecological risk of 18 antibiotics in rivers and lakes from Suzhou City were investigated based on urban-rural gradient. The total antibiotic concentration in surface water, suspended particulate matter (SPM) and sediments was 39.28-2578 ng/L, 6.16-171.09 ng/L and 12.67-2249 ng/g, respectively. High detection frequency (>76%) and concentration of antibiotics in multi-phase suggested universal pollution. Quinolones (QNs) and tetracycline (TCs) were the dominant antibiotics detected. The partitioning coefficient (KP) value of SPM-water was 1.43-29.93 times larger than sediment-water, indicating that SPM can greatly affect the fate and distribution of antibiotics. Significant positive correlations between antibiotics and environmental parameters (e.g. TOC, TP and TN) revealed combined contamination and similar pollution sources. Antibiotic pollution exhibited evident spatiotemporal variation. For spatial variation, urban area showed more serious antibiotic pollution and greater ecological risk than rural and suburb areas, especially for sediments. Besides, antibiotic level and risk in rivers were higher than lakes. For seasonal variation, in case of surface water, rural area exhibited higher content in winter, while greater content was detected in autumn and spring in urban and suburb areas, respectively. The highest antibiotic content in SPM and sediments was all measured in winter owing to weak degradation ability. Ecological risk assessment based on risk quotients (RQs) indicated that norfloxacin (NFX), ciprofloxacin (CFX) and anhydroerythromycin (ETM-H2O) in surface water presented medium to high risk throughout the entire year, while sulfadiazine (SDZ) and enrofloxacin (EFX) in sediments showed higher accumulation potential. Thus, these five antibiotics were selected as the priority antibiotics for pollution control. In short, this study improves the understanding of antibiotic fates in the urban-rural watershed and provides scientific basis for the authorities to regulate antibiotic pollution.

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.

Occurrence and Fate of Benzophenone-Type UV Filters in a Tropical Urban Watershed.

The study investigated the occurrence and fate of seven benzophenone-type UV filters (i.e., 2,4-dihydroxybenzophenone (2,4OH-BP), 2,2',4,4'-tetrahydroxybenzophenone (2,2',4,4'OH-BP), 2-hydroxy-4-methoxybenzophenone (2OH-4MeO-BP), 2,2'-Dihydroxy-4,4'-dimethoxybenzophenone (2,2'OH-4,4'MeO-BP), 2,2'-dihydroxy-4-methoxybenzophenone (2,2'OH-4MeO-BP), 4-hydroxybenzophenone (4OH-BP), and 4,4'-dihyroxybenzophenone (4DHB)) in a tropical urban watershed consisting of five major tributaries that discharge into a well-managed basin. Total benzophenone concentrations (∑CBPs) varied from 19-230.8 ng L-1 in overlying bulk water, 48-115 ng L-1 in pore water, 295-5813 ng g-1 dry weight (d.w.) in suspended solids, and 6-37 ng g-1 d.w. in surficial sediments, respectively. The tributaries (∑CBPs: 19-231 ng L-1) were the main source of benzophenone compounds entering the basin (∑CBPs: 20-81 ng L-1). In the water column, the vertical concentration profile in the aqueous phase was uniform while concentrations in the suspended solids decreased with depth. Different distribution profiles were also identified for benzophenones in suspended solids and sediments. A preliminary risk assessment suggested that the seven BPs were unlikely to pose ecotoxicological risks to local aquatic organisms except for 2OH-4MeO-BP in the case of an intermittent release.

Occurrence, distribution and risk assessment of pesticides in a river-reservoir system.

The water environment from river to reservoir has been considered as a hybrid river-reservoir system due to pronounced environmental properties. This study investigated the distribution and potential environmental behavior of pesticides in river-reservoir system, examining 31 target pesticides in water phase from a key drinking water source (Dongjiang River). The concentrations of ∑8OCPs, ∑16OPPs and ∑7SPs with the corresponding occurrence were in the range of 107.57-340.35 ng/L (moderate level), 232.65-1197.95 ng/L (moderate level) and 125.23-245.09 ng/L (low level), respectively. Ecological risk assessments indicated that most of the pesticides posed a high level of risk to the aquatic organisms. Moreover, seasonal agricultural application, rainfall and temperature could influence the levels and compositions of ∑8OCPs, ∑16OPPs and ∑7SPs in the river system, while seasonal hydrological processes could only influence their compositions in the reservoir system. In the wet season, the levels of ∑8OCPs and ∑7SPs decreased from the river system to reservoir system probably via biogeochemical processes, while the level of ∑16OPPs decreased along the environmental gradient probably via dilution process. Additionally, longer hydraulic retention time of the reservoir system could contribute to the biogeochemical processes for OPPs and SPs exported from the river system, but it might fail to facilitate this process for OCPs. Taken together, this study highlighted that the distributions and environmental behavior of pesticides in river-reservoir system varied seasonally from river system to reservoir system.

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.

Relationship of Microbiota and Cyanobacterial Secondary Metabolites in Planktothricoides-Dominated Bloom.

The identification of phytoplankton species and microbial biodiversity is necessary to assess water ecosystem health and the quality of water resources. We investigated the short-term (2 days) vertical and diel variations in bacterial community structure and microbially derived secondary metabolites during a cyanobacterial bloom that emerged in a highly urbanized tropical reservoir. The waterbody was largely dominated by the cyanobacteria Planktothricoides spp., together with the Synechococcus, Pseudanabaena, Prochlorothrix, and Limnothrix. Spatial differences (i.e., water depth) rather than temporal differences (i.e., day versus night) better-explained the short-term variability in water quality parameters and bacterial community composition. Difference in bacterial structure suggested a resource-driven distribution pattern for the community. We found that the freshwater bacterial community associated with cyanobacterial blooms is largely conserved at the phylum level, with Proteobacteria (ß-proteobateria), Bacteroidetes, and Actinobacteria as the main taxa despite the cyanobacterial species present and geographical (Asia, Europe, Australia, and North America) or climatic distinctions. Through multivariate statistical analyses of the bacterial community, environmental parameters, and secondary metabolite concentrations, we observed positive relationships between the occurrences of cyanobacterial groups and off-flavor compounds (2-methyisoborneol and ß-ionone), suggesting a cyanobacterial origin. This study demonstrates the potential of 16S rRNA gene amplicon sequencing as a supporting tool in algal bloom monitoring or water-resource management.

Occurrences and Characterization of Antibiotic-Resistant Bacteria and Genetic Determinants of Hospital Wastewater in a Tropical Country.

Wastewater discharged from clinical isolation and general wards at two hospitals in Singapore was examined to determine the emerging trends of antibiotic resistance (AR). We quantified the concentrations of 12 antibiotic compounds by analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS), antibiotic-resistant bacteria (ARB), the class 1 integrase gene (intI1), and 16 antibiotic resistance genes (ARGs) that confer resistance to 10 different clinically relevant antibiotics. A subset of 119 antibiotic-resistant isolates were phylogenetically classified and tested for the presence of ARGs encoding resistance to ß-lactam antibiotics (blaNDM, blaKPC, blaSHV, blaCTX-M), amikacin [aac(6')-Ib], co-trimoxazole (sul1, sul2, dfrA), ciprofloxacin (qnrA, qnrB), and the intI1 gene. Among these resistant isolates, 80.7% were detected with intI1 and 66.4% were found to carry at least 1 of the tested ARGs. Among 3 sampled locations, the clinical isolation ward had the highest concentrations of ARB and the highest levels of ARGs linked to resistance to ß-lactam (blaKPC), co-trimoxazole (sul1, sul2, dfrA), amikacin [aac(6')-Ib], ciprofloxacin (qnrA), and intI1 We found strong positive correlations (P < 0.05) between concentrations of bacteria resistant to meropenem, ceftazidime, amikacin, co-trimoxazole, and ciprofloxacin and abundances of blaKPC, aac(6')-Ib, sul1, sul2, dfrA, qnrA, and intI1 genes.

Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis.

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously. Microcystis and Cylindrospermopsis are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for Cylindrospermopsis species and the c-phycocyanin beta subunit-like gene for Microcystis species. Duplex assays were developed for two molecular techniques-qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.

Nano-scale and micron-scale plastics amplify the bioaccumulation of benzophenone-3 and ciprofloxacin, as well as their co-exposure effect on disturbing the antioxidant defense system in mussels, Perna viridis.

Plastics ranging from nano-scale to micron-scale are frequently ingested by many marine animals. These particles exhibit biotoxicity and additionally perform as vectors that convey and amass adsorbed chemicals within organisms. Meanwhile, the frequency of detection of the benzophenone-3 and ciprofloxacin can be adsorbed on plastic particles, then accumulated in bivalves, causing biotoxicity. To understand their unknown accumulative kinetics in vivo affected by different plastic sizes and toxic effect from co-exposure, several scenarios were set up in which the mode organism were exposed to 0.6 mg/L of polystyrene carrying benzophenone-3 and ciprofloxacin in three sizes (300 nm, 38 µm, and 0.6 mm). The live Asian green mussels were chosen as mode organism for exposure experiments, in which they were exposed to environments with plastics of different sizes laden with benzophenone-3 and ciprofloxacin, then depurated for 7 days. The bioaccumulation and depuration kinetics of benzophenone-3 and ciprofloxacin were measured using HPLC-MS/MS after one week of exposure and depuration. Meanwhile, their toxic effect were investigated by measuring the changes in six biomarkers (condition index, reactive oxygen species, catalase, glutathione, lipid peroxidation, cytochrome P450 and DNA damage). The bioconcentration factors in mussels under different exposure conditions were 41.48-111.75 for benzophenone-3 and 6.45 to 12.35 for ciprofloxacin. The results suggested that microplastics and nanoplastics can act as carriers to increase bioaccumulation and toxicity of adsorbates in mussels in a size-dependent manner. Overproduction of reactive oxygen species caused by microplastics and nanoplastics led to increased DNA damage, lipid peroxidation, and changes in antioxidant enzymes and non-enzymatic antioxidants during exposure. Marked disruption of antioxidant defenses and genotoxic effects in mussels during depuration indicated impaired recovery. Compared to micron-scale plastic with sizes over a hundred micrometers that had little effect on bivalve bioaccumulation and toxicity, nano-scale plastic greatly enhanced the biotoxicity effect.

Microcystis genotypes in a tropical freshwater lake: Discovery of novel MIB-producing Microcystis with potentially unique synthesis pathway.

Harmful algal blooms (HABs) are a threat to freshwater systems over the world due to the production of hepatotoxins like microcystin (MC), and nuisance taste and odour (T&O) compounds like 2-methylisoborneol (MIB). While MCs are known to cause detrimental effects to both water quality and human health, MIB is only reported to cause aesthetical problems. In this study, we investigated a tropical, urban lake that was experiencing persistent MC and MIB events. Although it was dominated by Microcystis blooms, analysis revealed that the toxigenic Microcystis were not the only species driving the MC concentrations. Additionally, there was also a lack of causative species for the MIB events. Through isolation, we have identified three toxigenic Microcystis found to produce four different variants of MCs, and two novel non-toxigenic Microcystis that were capable of producing MIB. The ability to produce MIB had never been previously reported for this species. Compared to other major producers such as Planktothricoides sp. and Streptomyces sp., the MIB synthase genes of our Microcystis sp. strains were partial, illustrating the possibility of unique synthesis pathways. The Microcystis sp. strains were found to produce about 2.77-5.22 fg MIB cell-1, with a majority of the contents (70-80 %) existing in the extracellular phase. Correlation analysis of field study indicated that phosphorus limitation may have an indirect effect on non-toxigenic Microcystis abundance and proportion by influencing the toxigenic genotype, suggesting that current measures to control HABs may favour the proliferation of the non-toxigenic Microcystis. The potential for Microcystis sp. to produce MIB through unique synthesis pathway, coupled with the potential dominance of non-toxigenic genotypes in Microcystis blooms, signals the possibility that non-toxigenic Microcystis should be monitored as well.