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.

Cryptosporidium and Giardia in Wastewater and Surface Water Environments.

and spp. are significant contributors to the global waterborne disease burden. Waterways used as sources of drinking water and for recreational activity can become contaminated through the introduction of fecal materials derived from humans and animals. Multiple studies have reported the occurence or concentrations of these pathogens in the environment. However, this information has not been comprehensively reviewed. Quantitative microbial risk assessment (QMRA) for and can be beneficial, but it often relies on the concentrations in environmental sources reported from the literature. A thorough literature review was conducted to develop an inventory of reported and concentrations in wastewater and surface water available in the literature. This information can be used to develop QMRA inputs. and (oo)cyst concentrations in untreated wastewater were up to 60,000 oocysts L and 100,000 cysts L, respectively. The maximum reported concentrations for and in surface water were 8400 oocysts L and 1000 cysts L, respectively. A summary of the factors for interpretation of concentration information including common quantification methods, survival and persistence, biofilm interactions, genotyping, and treatment removal is provided in this review. This information can help in identifying assumptions implicit in various QMRA parameters, thus providing the context and rationale to guide model formulation and application. Additionally, it can provide valuable information for water quality practitioners striving to meet the recreational water quality or treatment criteria. The goal is for the information provided in the current review to aid in developing source water protection and monitoring strategies that will minimize public health risks.

Finite element analysis of FGM dental crowns using phase-field approach.

Functionally graded materials (FGMs) - categorized in advanced composite materials - are specially designed to reduce the stresses and failure due to material mismatches. Advances in manufacturing techniques have brought FGMs into use in a variety of applications. However, the numerical analysis is still challenging due to the difficulties in simulations of non-homogeneous material domains of complex parts. Presenting a numerical procedure that both facilitates the implementation of material non-homogeneity in geometrically complex mediums, and increases the accuracy of the calculations using a phase-field approach, this study investigates the usage of FGMs in dental prostheses. For this purpose, a porcelain fused to metal (PFM) mandibular first molar FGM crown is simulated and analyzed under the maximum masticatory bite force, and eventually the results are compared to a PFM crown prepared conventionally.

Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen.

Synthetic biology aims to systematically design and construct novel biological systems that address energy, environment, and health issues. Herein, we describe the development of a synthetic genetic system, which comprises quorum sensing, killing, and lysing devices, that enables Escherichia coli to sense and kill a pathogenic Pseudomonas aeruginosa strain through the production and release of pyocin. The sensing, killing, and lysing devices were characterized to elucidate their detection, antimicrobial and pyocin release functionalities, which subsequently aided in the construction of the final system and the verification of its designed behavior. We demonstrated that our engineered E. coli sensed and killed planktonic P. aeruginosa, evidenced by 99% reduction in the viable cells. Moreover, we showed that our engineered E. coli inhibited the formation of P. aeruginosa biofilm by close to 90%, leading to much sparser and thinner biofilm matrices. These results suggest that E. coli carrying our synthetic genetic system may provide a novel synthetic biology-driven antimicrobial strategy that could potentially be applied to fighting P. aeruginosa and other infectious pathogens.

Temporal characterization of biocycles of mycelium-bound composites made from bamboo and Pleurotus ostreatus for indoor usage.

Mycelium-bound composites (MBCs) are materials obtained by growing fungi on a ligno-cellulosic substrate which have various applications in packaging, furniture, and construction industries. MBCs are particularly interesting as they are sustainable materials that can integrate into a circular economy model. Indeed, they can be subsequently grown, used, degraded, and re-grown. Integrating in a meaningful biocycle for our society therefore demands that MBCs fulfil antagonistic qualities which are to be at the same time durable and biodegradable. In this study, we conduct experiments using MBCs made from the fungus species Pleurotus ostreatus grown on bamboo microfibers substrate. By measuring the variations of the mechanical properties with time, we provide an experimental demonstration of a biocycle for such composites for in-door applications. We found that the biocycle can be as short as 5 months and that the use of sustainable coatings is critical to increase the durability of the composites while maintaining biodegradability. Although there are many scenarios of biocycles possible, this study shows a tangible proof-of-concept example and paves the way for optimization of the duration of each phase in the biocycle depending on the intended application and resource availability.

Wood-Veneer-Reinforced Mycelium Composites for Sustainable Building Components.

The demand for building materials has been constantly increasing, which leads to excessive energy consumption for their provision. The looming environmental consequences have triggered the search for sustainable alternatives. Mycelium, as a rapidly renewable, low-carbon natural material that can withstand compressive forces and has inherent acoustic and fire-resistance properties, could be a potential solution to this problem. However, due to its low tensile, flexural and shear strength, mycelium is not currently widely used commercially in the construction industry. Therefore, this research focuses on improving the structural performance of mycelium composites for interior use through custom robotic additive manufacturing processes that integrate continuous wood fibers into the mycelial matrix as reinforcement. This creates a novel, 100% bio-based, wood-veneer-reinforced mycelium composite. As base materials, Ganoderma lucidum and hemp hurds for mycelium growth and maple veneer for reinforcement were pre-selected for this study. Compression, pull-out, and three-point bending tests comparing the unreinforced samples to the veneer-reinforced samples were performed, revealing improvements on the bending resistance of the reinforced samples. Additionally, the tensile strength of the reinforcement joints was examined and proved to be stronger than the material itself. The paper presents preliminary experiment results showing the effect of veneer reinforcements on increasing bending resistance, discusses the potential benefits of combining wood veneer and mycelium's distinct material properties, and highlights methods for the design and production of architectural components.

Mechanical properties of dense mycelium-bound composites under accelerated tropical weathering conditions.

Mycelium, as the root of fungi, is composed of filamentous strands of fine hyphae that bind discrete substrate particles into a block material. With advanced processing, dense mycelium-bound composites (DMCs) resembling commercial particleboards can be formed. However, their mechanical properties and performance under the working conditions of particleboards are unknown. Here, we show how weathering conditions affect the DMC stress and elastic modulus. DMC was made using Ganoderma lucidum mycelium grown on a substrate of sawdust and empty fruit bunch. The DMC was then subjected to weathering under tropical conditions over 35 days and tested under flexural, tensile, and compressive loading with reference to international standards. After exposure to specified weathering conditions, the maximum stress in flexure, tension, and compression decreased substantially. The addition of a protective coating improved the resistance of DMC to weathering conditions; however, the difference between coated and uncoated samples was only found to be statistically significant in tensile strength.

Effect of common foods as supplements for the mycelium growth of Ganoderma lucidum and Pleurotus ostreatus on solid substrates.

The transition from a linear to a circular economy is urgently needed to mitigate environmental impacts and loss of biodiversity. Among the many potential solutions, the development of entirely natural-based materials derived from waste is promising. One such material is mycelium-bound composites obtained from the growth of fungi onto solid lignocellulosic substrates, which find applications such as insulating foams, textiles, packaging, etc. During growth, the fungus degrades and digests the substrate to create a web-like stiff network called mycelium. The development of the mycelium is influenced by several factors, including the substrate composition. As food waste accounts for nearly 44% of total municipal solid waste, incorporating food in the substrate composition could be a means to increase the nutrients absorbed by the fungus. In this paper, we study the effects of the addition of food supplements on the growth of two fungal species, Ganoderma lucidum and Pleurotus ostreatus. The substrates, the food supplements, and the mycelia are characterized using Fourier-transform infrared spectroscopy, scanning electron microscopy, and optical microscopy. Our results show that addition of barley as a supplement significantly boosts the growth of G. lucidum and P. ostreatus. Using a common food as a nutritious enrichment for the development of mycelium is a simple and straightforward strategy to create waste-based mycelium-bound biocomposites for a large range of applications, on-site, therefore promoting a circular economy.

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.

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 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.

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.

Precipitation influences pathogenic bacteria and antibiotic resistance gene abundance in storm drain outfalls in coastal sub-tropical waters.

Stormwater contamination can threaten the health of aquatic ecosystems and human exposed to runoff via nutrient and pathogen influxes. In this study, the concentrations of 11 bacterial pathogens and 47 antibiotic resistance genes (ARGs) were determined by using high-throughput microfluidic qPCR (MFQPCR) in several storm drain outfalls (SDOs) during dry and wet weather in Tampa Bay, Florida, USA. Data generated in this study were also compared with the levels of fecal indicator bacteria (FIB) and sewage-associated molecular markers (i.e., Bacteroides HF183 and crAssphage markers) in same SDOs collected in a recent study (Ahmed et al., 2018). Concentration of FIB, sewage-associated markers, bacterial pathogens and many ARGs in water samples were relatively high and SDOs may be potentially hotspots for microbial contamination in Tampa Bay. Mean concentrations of culturable E. coli and Enterococcus spp. were tenfold higher in wet compared to dry weather. The majority of microbiological contaminants followed this trend. E. coli eaeA, encoding the virulence factor intimin, was correlated with levels of 20 ARGs, and was more frequently detected in wet weather than dry weather samples. The blaKPC gene associated with carbapenem resistant Enterobacteriaceae and the beta-lactam resistant gene (blaNPS) were only detected in wet weather samples. Frequency of integron genes Intl2 and Intl3 detection increased by 42% in wet weather samples. Culturable E. coli and Enterococcus spp. significantly correlated with 19 of 47 (40%) ARG tested. Sewage-associated markers crAssphage and HF183 significantly correlated (p < 0.05) with the following ARGs: intl1, sul1, tet(M), ampC, mexB, and tet(W). The presence of sewage-associated marker genes along with ARGs associated with sewage suggested that aging sewage infrastructure contributed to contaminant loading in the Bay. Further research should focus on collecting spatial and temporal data on the microbiological contaminants especially viruses in SDOs.

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.

A predicted S-type pyocin shows a bactericidal activity against clinical Pseudomonas aeruginosa isolates through membrane damage.

The nucleic acid sequence at the positions 1067817-1066321 of Pseudomonas aeruginosa PAO1 genome was predicted to encode a novel S-type pyocin, designated S5, based on the genome sequence. However, its antimicrobial spectrum, activity and mechanism have not been investigated. Herein, we report that pyocin S5 has an antimicrobial activity against seven clinical P. aeruginosa isolates (DWW3, InA, InB, In3, In4, In7, and In8). Among them, DWW3 is most sensitive with a minimum inhibitory concentration of 12.6 microg/ml and a killing percentage of 95.7 at 225 microg/ml. Further, we demonstrated that the antimicrobial mechanism of pyocin S5 is membrane damage, evidenced by the leakage of intracellular materials, the increase of membrane permeability, and cell surface disruption.