Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage.

Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use metagenomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.

Antimicrobial activity of pharmaceutical cocktails in sewage treatment plant effluent - An experimental and predictive approach to mixture risk assessment.

Municipal wastewater contains multi-component mixtures of active pharmaceutical ingredients (APIs). This could shape microbial communities in sewage treatment plants (STPs) and the effluent-receiving ecosystems. In this paper we assess the risk of antimicrobial effects in STPs and the aquatic environment for a mixture of 18 APIs that was previously detected in the effluent of a European municipal STP. Effects on microbial consortia (collected from a separate STP) were determined using respirometry, enumeration of culturable microorganisms and community-level physiological profiling. The mixture toxicity against selected bacteria was assessed using assays with Pseudomonas putida and Vibrio fischeri. Additional data on the toxicity to environmental bacteria were compiled from literature in order to assess the individual and expected joint bacterial toxicity of the pharmaceuticals in the mixture. The reported effluent concentration of the mixture was 15.4 nmol/l and the lowest experimentally obtained effect concentrations (EC10) were 242 nmol/l for microbial consortia in STPs, 225 nmol/l for P. putida and 73 nmol/l for V. fischeri. The lowest published effect concentrations (EC50) of the individual antibiotics in the mixture range between 15 and 150 nmol/l, whereas 0.9-190 µmol/l was the range of bacterial EC50 values found for the non-antibiotic mixture components. Pharmaceutical cocktails could shape microbial communities at concentrations relevant to STPs and the effluent receiving aquatic environment. The risk of antimicrobial mixture effects was completely dominated by the presence of antibiotics, whereas other pharmaceutical classes contributed only negligibly to the mixture toxicity. The joint bacterial toxicity can be accurately predicted from the individual toxicity of the mixture components, provided that standardized data on representative bacterial strains becomes available for all relevant compounds. These findings argue for a more sophisticated bacterial toxicity assessment of environmentally relevant pharmaceuticals, especially for those with a mode of action that is known to specifically affect prokaryotic microorganisms.

Development of a mediated whole cell-based electrochemical biosensor for joint toxicity assessment of multi-pollutants using a mixed microbial consortium.

Since most risk assessment for toxicants is based on individual single-species test, the deduction of such results to ecosystem evaluation is afflicted with uncertainties. Herein, we successfully developed a p-benzoquinone mediated whole-cell electrochemical biosensor for multi-pollutants toxicological analysis by co-immobilizing mixed strains of microorganism, including Escherichia coli (gram-negative bacteria), Bacillus subtilis (gram-positive bacteria) and Saccharomyces cerevisiae (fungus). The individual and combined toxicities of heavy metal ions (Cu(2+), Cd(2+)), phenol (3,5-dichlorophenol) and pesticides (Ametryn, Acephate) were examined. The experimental results showed that the order of toxicity for individual toxicant was ranked as Cu(2+) > 3,5-dichlorophenol (DCP) > Ametryn > Cd(2+) > Acephate. Then the toxic unit (TU) model was applied to determine the nature of toxicological interaction of the toxicants which can be classified as concentration additive (IC50mix = 1TU), synergistic (IC50mix < 1TU) and antagonistic (IC50mix > 1TU) responses. The binary combination of Cu(2+) + Cd(2+), Cu(2+) + DCP, Cu(2+) + Acephate, DCP + Acephate, Acephate + Ametryn were analyzed and the three kind of joint toxicity effects (i.e. additive, synergistic and antagonistic) mentioned above were observed according to the dose-response relationship. The results indicate that the whole-cell electrochemical biosensor based on mixed microbial consortium is more reasonable to reflect the joint biotoxicity of multi-pollutants existing in real wastewater, and combined effects of toxicants is extremely necessary to be taken into account in ecological risk assessment. Thus, present study has provided a promising approach to the quality assessment of wastewater and a reliable way for early risk warning of acute biotoxicity.

Ecotoxicological assessment of antibiotics: A call for improved consideration of microorganisms.

Antibiotics play a pivotal role in the management of infectious disease in humans, companion animals, livestock, and aquaculture operations at a global scale. Antibiotics are produced, consumed, and released into the environment at an unprecedented scale causing concern that the presence of antibiotic residues may adversely impact aquatic and terrestrial ecosystems. Here we critically review the ecotoxicological assessment of antibiotics as related to environmental risk assessment (ERA). We initially discuss the need for more specific protection goals based on the ecosystem service concept, and suggest that the ERA of antibiotics, through the application of a mode of toxic action approach, should make more use of ecotoxicological endpoints targeting microorganisms (especially bacteria) and microbial communities. Key ecosystem services provided by microorganisms and associated ecosystem service-providing units (e.g. taxa or functional groups) are identified. Approaches currently available for elucidating ecotoxicological effects on microorganisms are reviewed in detail and we conclude that microbial community-based tests should be used to complement single-species tests to offer more targeted protection of key ecosystem services. Specifically, we propose that ecotoxicological tests should not only assess microbial community function, but also microbial diversity ('species' richness) and antibiotic susceptibility. Promising areas for future basic and applied research of relevance to ERA are highlighted throughout the text. In this regard, the most fundamental knowledge gaps probably relate to our rudimentary understanding of the ecological roles of antibiotics in nature and possible adverse effects of environmental pollution with subinhibitory levels of antibiotics.

Qualitative toxicity assessment of silver nanoparticles on the fresh water bacterial isolates and consortium at low level of exposure concentration.

Silver nanoparticles (AgNPs) pose a high risk of exposure to the natural environment owing to their extensive usage in various consumer products. In the present study we attempted to understand the harmful effect of AgNPs at environmentally relevant low concentration levels (≤1ppm) towards two different freshwater bacterial isolates and their consortium. The standard plate count assay suggested that the AgNPs were toxic towards the fresh water bacterial isolates as well as the consortium, though toxicity was significantly reduced for the cells in the consortium. The oxidative stress assessment and membrane permeability studies corroborated with the toxicity data. The detailed electron microscopic studies suggested the cell degrading potential of the AgNPs, and the FT-IR studies confirmed the involvement of the surface groups in the toxic effects. No significant ion leaching from the AgNPs was observed at the applied concentration levels signifying the dominant role of the particle size, and size distribution in bacterial toxicity. The reduced toxicity for the cells in the consortium than the individual isolates has major significance in further studies on the ecotoxicity of the AgNPs.

Effects of increasing organic loading rate on performance and microbial community shift of an up-flow anaerobic sludge blanket reactor treating diluted pharmaceutical wastewater.

The performance of an up-flow anaerobic sludge blanket (UASB) reactor was investigated in the treatment of diluted pharmaceutical fermentation wastewater for a continuous operation of 140 days. The dynamics and compositions of the microbial community were monitored using polymerase chain reaction (PCR)-restriction fragment length polymorphism (PCR-RFLP) analysis. Increase of the organic loading rate (OLR) from 2.7 kg COD/m(3) d to 7.2 COD/m(3) d led to an increase in the COD removal efficiency from 83% to 91%. The dominant bacteria shifted from Proteobacteria (23.8%), Chloroflexi (14.5%) and Firmicutes (4.0%) to Firmicutes (48.4%), Bacteroidetes (9.5%) and Proteobacteria (5.4%). For archeaon, the dominant groups changed from Thermoplasmata (24.4%), Thermoprotei (18.0%) and Methanobacteria (30.8%) to Thermoplasmata (70.4%) and Methanomicrobia (16.8%). Firmicutes, Bacteroidetes, Thermoplasmata and Methanobacteria could outcompete other species and dominated in the reactor under higher OLR. The results indicated that, to some extent, microbial community shift could reflect the performance of the reactor and a significant community shift corresponded to a considerable process event.

Economic process to produce biohydrogen and volatile fatty acids by a mixed culture using vinasse from sugarcane ethanol industry as nutrient source.

This work evaluates the potential of vinasse (a waste obtained at the bottom of sugarcane ethanol distillation columns) as nutrient source for biohydrogen and volatile fatty acids production by means of anaerobic consortia. Two different media were proposed, using sugarcane juice or molasses as carbon source. The consortium LPBAH1 was selected for fermentation of vinasse supplemented with sugarcane juice, resulting in a higher H2 yield of 7.14 molH2 molsucrose(-1) and hydrogen content in biogas of approx. 31%, while consortium LPBAH2 resulted in 3.66 molH2/molsucrose and 32.7% hydrogen content in biogas. The proposed process showed a rational and economical use for vinasse, a mandatory byproduct of the renewable Brazilian energy matrix.

Biosafety assessment of GFP transplastomic tobacco to rhizosphere microbial community.

Green fluorescent protein (GFP) is one of the most widely studied and exploited proteins in biochemistry, and has many applications as a marker, especially in plant transformation system. Although a number of studies have been conducted to assess the toxify of this protein to specific organisms, little is known about GFP on rhizosphere microbial community, which is regarded as good indicator for environmental risk assessment. Chloroplast genetic engineering has shown superiority over traditional nuclear genetic engineering, and has been used in many aspects of plant genetic engineering. High levels of chloroplast-based protein accumulation make this technology as an ideal strategy to evaluate biosafety of transgenes. In the present study, the effects of field-released GFP transplastomic tobacco (Nicotiana tabacum) on rhizosphere microbes over a whole growth cycle were investigated by using both culture-dependent and culture-independent methods. Compared to wild-type control, transplastomic tobacco had no significant influence on the microbial population at the seedling, vegetative, flowering and senescing stages. However, developmental stages had more influence than ecotypes (GFP-transformed and wild-type). This was confirmed by colony forming unit, Biolog Eco(TM) and PCR-DGGE analysis. Thus, these results suggest chloroplast transformation with a GFP reporter gene has no significant influence on rhizosphere microbial community, and will be potential platform for plant biotechnology in future.

Molecular assessment of the sensitivity of sulfate-reducing microbial communities remediating mine drainage to aerobic stress.

Sulfate-reducing permeable reactive zones (SR-PRZs) are microbially-driven anaerobic systems designed for the removal of heavy metals and sulfate in mine drainage. Environmental perturbations, such as oxygen exposure, may adversely affect system stability and long-term performance. The objective of this study was to examine the effect of two successive aerobic stress events on the performance and microbial community composition of duplicate laboratory-scale lignocellulosic SR-PRZs operated using the following microbial community management strategies: biostimulation with ethanol or carboxymethylcellulose; bioaugmentation with sulfate-reducing or cellulose-degrading enrichments; inoculation with dairy manure only; and no inoculation. A functional gene-based approach employing terminal restriction fragment length polymorphism and quantitative polymerase chain reaction targeting genes of sulfate-reducing (dsrA), cellulose-degrading (cel5, cel48), fermentative (hydA), and methanogenic (mcrA) microbes was applied. In terms of performance (i.e., sulfate removal), biostimulation with ethanol was the only strategy that clearly had an effect (positive) following exposure to oxygen. In terms of microbial community composition, significant shifts were observed over the course of the experiment. Results suggest that exposure to oxygen more strongly influenced microbial community shifts than the different microbial community management strategies. Sensitivity to oxygen exposure varied among different populations and was particularly pronounced for fermentative bacteria. Although the community structure remained altered after exposure, system performance recovered, indicating that SR-PRZ microbial communities were functionally redundant. Results suggest that pre-exposure to oxygen might be a more effective strategy to improve the resilience of SR-PRZ microbial communities relative to bioaugmentation or biostimulation.