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An integrated life cycle assessment (LCA) and quantitative microbial risk assessment (QMRA) were conducted to assess microalgae-mediated wastewater disinfection (M-WWD). M-WWD was achieved by replacing ultraviolet disinfection with a microalgal open raceway pond in an existing sewage treatment plant (STP) in India. Regarding impacts on human health, both M-WWD and STP yielded comparable life cycle impacts, around 0.01 disability-adjusted life years (DALYs) per person per year. However, QMRA impacts for M-WWD (0.053 DALYs per person per year) were slightly lower than that for STP while considering exposure to E. coli O157:H7 and adenovirus. Additionally, a comparative LCA resolved the dilemma about the appropriate utilization of microalgal biomass. Among biodiesel, biocrude, and biogas production, the lowest impacts of 0.015 DALYs per person per year were obtained for biocrude for 1 m3 water treated by M-WWD. Electricity consumption in microalgae cultivation was a major environmental hotspot. Overall, M-WWD, followed by production of microalgal biocrude, emerged as a sustainable alternative from environmental and public health perspectives. These findings set the foundation for pilot-scale M-WWD system development, testing, and economic evaluation. Such comprehensive investigations, encompassing LCA, QMRA, and resource recovery scenarios, offer crucial insights for stakeholders and decision-makers in wastewater treatment and environmental management.

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An integrated approach was employed in the present study to combine life cycle assessment (LCA) with quantitative microbial risk assessment (QMRA) to assess an existing sewage treatment plant (STP) at Roorkee, India. The midpoint LCA modeling revealed that high electricity consumption (≈ 576 kWh.day-1) contributed to the maximum environmental burdens. The LCA endpoint result of 0.01 disability-adjusted life years per person per year (DALYs pppy) was obtained in terms of the impacts on human health. Further, a QMRA model was developed based on representative sewage pathogens, including E. coli O157:H7, Giardia sp., adenovirus, norovirus, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The public health risk associated with intake of pathogen-laden aerosols during treated water reuse in sprinkler irrigation was determined. A cumulative health risk of 0.07 DALYs pppy was obtained, where QMRA risks contributed 86 % of the total health impacts. The annual probability of illness per person was highest for adenovirus and norovirus, followed by SARS-CoV-2, E. coli O157:H7 and Giardia sp. Overall, the study provides a methodological framework for an integrated LCA-QMRA assessment which can be applied across any treatment process to identify the hotspots contributing maximum environmental burdens and microbial health risks. Furthermore, the integrated LCA-QMRA approach could support stakeholders in the water industry to select the most suitable wastewater treatment system and establish regulations regarding the safe reuse of treated water.

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Concerns over fecal contamination in stormwater canals have promoted the need for pollution control strategies, including the use of microbial source tracking, to identify fecal contamination in the Greater New Orleans Area. Surface water samples were collected over a 12-month period at five canal locations within Jefferson Parish, Louisiana. Quantitative polymerase chain reaction and the IDEXX method were used to assess the concentrations of coliforms, Escherichia coli (E. coli) and human fecal 183 bacteroides (HF183) in stormwater samples. A 100% positive detection rate of total coliforms and E. coli was observed across all tested sites. Despite the closeness of the five sites, when averaged across all sampling time points, Kruskal-Wallis tests indicated that E. coli was present at significantly different concentrations in these locations (χ2(5) = 19.8, p = 0.0005). HF183 was detected in 62% of the water samples collected during the stormwater sampling. Without further testing for HF183 markers, the conclusion from this study would have been that fecal contamination from an unknown source was always present at varying levels during the study period. Analysis of HF183 markers therefore adds another layer of conclusions to the results deductible from E. coli concentrations. A 100% E. coli detection rate, high E. coli concentrations coupled with low rates of HF183 detection particularly at the Esplanade, Poplar Street, and Bonnabel Boat Launch sites, the sites closest to the lake outlet, throughout the study period, indicate that fecal contamination at these stormwater canal sites comes primarily from non-human sources. However, the Metairie Road and Napoleon Avenue sites, which have the highest HF183 detection rates, on top of chronic pollution by other non-human sources, are also influenced by human fecal pollution, possibly because of human development and faulty infrastructure. This study highlights the advantages of the use of microbial source-tracking methods to complement traditional indicator bacteria.

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With a focus on five sites in an impaired, densely populated area in the New Orleans area, we investigated the temporal and spatial variability of standard FIB and a marker of human-associated pollution (Bacteroides HF183). With all sites combined, only a weak positive correlation (r = 0.345; p = 0.001) was observed between E. coli and HF183. Also, specific conductivity (r = - 0.374; p < 0.0001) and dissolved oxygen (r = - 0.390; p < 0.0001) were observed to show a weak moderate correlation with E. coli. These correlations increased to moderately negative when HF183 was correlated with specific conductivity (r = - 0.448; p < 0.0001) and dissolved oxygen (r = - 0.455; p < 0.0001). E. coli contamination was generally highest at the sites in the canal that are situated in the most densely populated part of the watershed while HF183 was frequently detected across all sites. E. coli concentrations were significantly higher (p < 0.05) when HF183 was present. HF183 was detected at significantly higher concentrations in samples that exceeded the EPA water quality standard (WQS) than those that did not (p < 0.05). Dissolved oxygen and specific conductivity were significantly lower when E. coli WQS was exceeded or when HF183 was present (p < 0.05). Rainfall impacted E. coli concentrations and HF183 differently at the study sites. While HF183 and E. coli concentrations levels were significantly higher (p < 0.05) if the days prior to sampling had been wet, the frequency of detection of HF183 was unimpacted, as comparable detection rates were recorded during wet and dry weather conditions. Without testing for HF183, it would have been assumed, based on testing for E. coli alone, that human fecal pollution was only associated with densely populated areas and rainfall events. E. coli alone may not be an effective indicator of sewage pollution at the study sites across all weather conditions and may need to be complemented with HF183 enumeration to optimize human fecal pollution identification and management at the watershed level.

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The current global Severe Acute Respiratory Syndrome- Coronavirus-2 (SARS-CoV-2) epidemic has heightened calls for studies to evaluate respiratory exposure for wastewater treatment workers. In this global first study, we assess occupational health risks to wastewater treatment plant (WWTP) operators from inhalation of aerosolized SARS-CoV-2 using a Quantitative Microbiological Risk Assessment (QMRA) framework. The following considerations were used to develop the QMRA and assess the illness risks to workers: a) the proportion of the population who are infected and thus responsible for shedding SARS-CoV-2 into raw wastewater; b) the concentration of SARS-CoV-2 in raw and treated wastewater; c) the volume of aerosolized water inhaled by a WWTP operator during work; d) humidity and temperature-dependent viability of coronaviruses in aerosolized waste water; e) estimation of the amount, frequency, and duration of exposure; and f) exposure doses. The variables were then fed into an exponential dose response model to estimate the risks in three scenarios representing low-grade, moderate and aggressive outbreaks. These scenarios were designed on the assumption of 0.03%, 0.3% and 3% of the wastewater-generating population being infected with SARS-CoV-2. In terms of averaged-out illness risk profiles, the individual illness risks for low grade, moderate and aggressive outbreak scenarios respectively are 0.036, 0.32 and 3.21 illness cases per 1000 exposed WWTP operators. Our study suggests that the risk of accidental occupational exposure to SARS-CoV-2 in raw wastewater, via inhalation at the WWTP environment, is negligible, particularly when less than 0.3% of the population served by the plant are actively infected.

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Biofilm production, gel and esp gene carriage was enumerated among forty six vancomycin resistant enterococci (VRE) and vancomycin susceptible enterococci (VSE) beach isolates. A higher proportion (61.54%) of biofilm producers was observed among beach sand as compared to beach water enterococci isolates (30%) indicating that enterococci within the sand column may be more dependent on biofilm production for survival than their beach water counterparts. Correlation analysis revealed strongly negative correlation (r=-0.535, p=0.015) between vancomycin resistance and biofilm formation. Given the observation of high prevalence of biofilm production among beach sand and the concomitant absence of esp gene carriage in any of the isolate, esp gene carriage may not be necessary for the production of biofilms among beach sand isolates. On the whole beach sand and water isolates demonstrated clearly different prevalence levels of vancomycin resistance, biofilm formation, esp and gel gene carriage. Application of these differences may be found useful in beach microbial source tracking studies. Tested starved cells still produced biofilm albeit at lower efficiencies. Non-dividing enterococci in beach sand can survive extended periods of environmental hardship and can resume growth or biofilm production in appropriate conditions thus making them infectious agents with potential health risk to recreational beach users.

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Partial gene sequences of phenylalanyl-tRNA synthase alpha subunit (pheS) and RNA polymerase alpha subunit (rpoA) were evaluated for species delineation and detection of recombination among enterococci populations recovered from a bathing beach impacted by low tide river flow. At inter-species level, a maximum similarity of 86.5% and 94.8% was observed among the enterococci pheS and rpoA sequence, respectively. A superimposed plot of delimited pairwise similarity values obtained for 266 pair-wise observations revealed that while there was a harmony between species identity obtained from both genes, pheS was more discriminatory than rpoA. The difference was more pronounced for inter-species comparison. A number of putative recombination events between indigenous and non-indigenous strains was detected based on a library of aligned sequences. Virulence genes cyl, esp, gelE and asa were detected in 7, 22, 100 and 63%, respectively among river isolates but at lower proportion of 0, 20, 67 and 42%, respectively among beach water isolates. Random amplified polymorphic DNA profiling presented evidence suggesting low tide river as a source of fecal enterococci entering the recreation beach water. Multilocus sequence typing analysis of a number of Enterococcus faecalis isolates presented four sequence types, ST59, 117, 181 and 474. The presence of genetically diverse fecal enterococci with associated virulence traits and a background of recombination events in surface recreational water could present a potential public health risk.

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Median enterococci counts of beach water samples gradually increased at statistically significant levels (χ2: 26.53, df: 4; p<0.0001) with increasing proximity to river influx. The difference in proportion of antibiotic resistant enterococci in beach water and river water samples was statistically significant (p<0.05) for the tested antibiotics with river isolates generally presenting higher resistance frequencies. Virulence genes cyl, esp, gelE and asa were detected at varying frequencies (7.32%, 21.95%, 100% and 63.41% respectively) among river isolates. On the other hand, the prevalence of these genes was lower (0%, 20%, 67.27% and 41.82% respectively) among beach water isolates. Multi-Locus-Sequence-Typing analysis of Enterococcus faecalis presented four sequence types (ST) one of which shared six out of seven tested loci with ST6, a member of the clonal complex of multi-drug resistant strains associated with hospital outbreaks.

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We report the first study on the occurrence of high-level aminoglycoside-resistant (HLAR) Enterococci in coastal bathing waters and beach sand in Malaysia. None of the encountered isolates were resistant to high levels of gentamicin (500 µg/mL). However, high-level resistance to kanamycin (2,000 µg/mL) was observed in 14.2 % of tested isolates, the highest proportions observed being among beach sand isolates. High-level resistance to kanamycin was higher among Enterococcus faecalis and Enterococcus faecium than Enterococcus spp. Chi-square analysis showed no significant association between responses to tested antibiotics and the species allocation or source of isolation of all tested Enterococci. The species classification of encountered Enterococci resistance to vancomycin was highest among Enterococcus spp. (5.89 %) followed by E. faecium (4.785) and least among E. faecalis. A total of 160 isolates were also tested for virulence characteristics. On the whole, caseinase production was profoundly highest (15.01 %) while the least prevalent virulence characteristic observed among tested beach Enterococci was haemolysis of rabbit blood (3.65 %). A strong association was observed between the source of isolation and responses for each of caseinase (C = 0.47, V = 0.53) and slime (C = 0.50, V = 0.58) assays. Analysis of obtained spearman's coefficient showed a strong correlation between caseinase and each of the slime production (p = 0.04), gelatinase (p = 0.0035) and haemolytic activity on horse blood (p = 0.004), respectively. Suggestively, these are the main virulent characteristics of the studied beach Enterococci. Our findings suggest that recreational beaches may contribute to the dissemination of Enterococci with HLAR and virulence characteristics.

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Bacteriocin-producing Pseudomonas putida strain FStm2 isolated from shark showed broad range of antibacterial activity against all pathogens tested except Bacillus subtilis ATCC11774, MRSA N32064, Proteus mirabilis ATCC12453, Enterococcus faecalis ATCC14506, Salmonella typhimurium ATCC51312, Salmonella mutan ATCC25175, and Aeromonas hydrophila Wbf314. Of the three growth media tested in this study, TSB was observed to support the bacteriocin activity the most. While the highest bacteriocin activity was observed for media supplemented with 1 % NaCl, there was an observed reduction in bacteriocin activity with increasing salt concentration. Although the least bacteriocin activity was observed for marine broth, addition of increasing amounts of tryptone, glucose, or yeast extract increased bacteriocin activity. This was, however, contrary to the effect observed when MgSO4 and MnSO4 were added as supplements. In the presence of α-amylase, lipase, DNase, and RNase, a positive effect on bacteriocin production was observed. Proteinase K strongly inhibited bacteriocin production. Furthermore, the bacteriocins produced were heat stable within the temperature range of 30-70 °C. Bacteriocin activity also was not affected within a wide pH range of 3-9. Exposure to detergents did not inhibit the activity of the bacteriocin at the concentrations tested. Instead, a positive effect on the relative activity of produced bacteriocin was observed as sodium dodecyl sulfate (SDS), EDTA, and Tween 20 at 1 % concentration all improved bacteriocin activity when the cell-free supernatant was tested against Serratia marcescens ATCC 13880. The bacteriocin was purified by ammonium sulfate precipitation and gel filtration on a Superdex-200 column. SDS-PAGE analysis of the partially purified bacteriocin revealed an apparent molecular weight of ~32 kDa.

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We report the first study on the occurrence of antibiotic-resistant enterococci in coastal bathing waters in Malaysia. One hundred and sixty-five enterococci isolates recovered from two popular recreational beaches in Malaysia were speciated and screened for antibiotic resistance to a total of eight antibiotics. Prevalence of Enterococcus faecalis and Enterococcus faecium was highest in both beaches. E. faecalis/E. faecium ratio was 0.384:1 and 0.375:1, respectively, for isolates from Port Dickson (PD) and Bagan Lalang (BL). Analysis of Fisher's exact test showed that association of prevalence of E. faecalis and E. faecium with considered locations was not statistically significant (p < 0.05). Chi-square test revealed significant differences (χ(2) = 82.630, df = 20, p < 0.001) in the frequency of occurrence of enterococci isolates from the considered sites. Resistance was highest to nalidixic acid (94.84 %) and least for chloramphenicol (8.38 %). One-way ANOVA using Tukey-Kramer multiple comparison test showed that resistance to ampicillin was higher in PD beach isolates than BL isolates and the difference was extremely statistically significant (p < 0.0001). Frequency of occurrence of multiple antibiotic resistance (MAR) isolates were higher for PD beach water (64.29 %) as compared to BL beach water (13.51 %), while MAR indices ranged between 0.198 and 0.48. The results suggest that samples from Port Dickson may contain MAR bacteria and that this could be due to high-risk faecal contamination from sewage discharge pipes that drain into the sea water.

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Despite the growing demand of tourism in Malaysia, there are no resolute efforts to develop beaches as tourist destinations. With no incentives to monitor public beaches or to use them in a sustainable manner, they might eventually degenerate in quality as a result of influx of pollutants. This calls for concerted action plans with a view to promoting their sustainable use. The success of such plans is inevitably anchored on the availability of robust quality monitoring schemes. Although significant efforts have been channelled to collation and public disclosure of bacteriological quality data of rivers, beach water monitoring appears left out. This partly explains the dearth of published information related to beach water quality data. As part of an on-going nation-wide surveillance study on the bacteriological quality of recreational beaches, this paper draws on a situation analysis with a view to proffering recommendations that could be adapted for ensuring better beach water quality in Malaysia.

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