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.

Human Health Risk Assessment (HHRA) for environmental development and transfer of antibiotic resistance.

BACKGROUND: Only recently has the environment been clearly implicated in the risk of antibiotic resistance to clinical outcome, but to date there have been few documented approaches to formally assess these risks. OBJECTIVE: We examined possible approaches and sought to identify research needs to enable human health risk assessments (HHRA) that focus on the role of the environment in the failure of antibiotic treatment caused by antibiotic-resistant pathogens. METHODS: The authors participated in a workshop held 4-8 March 2012 in Québec, Canada, to define the scope and objectives of an environmental assessment of antibiotic-resistance risks to human health. We focused on key elements of environmental-resistance-development "hot spots," exposure assessment (unrelated to food), and dose response to characterize risks that may improve antibiotic-resistance management options. DISCUSSION: Various novel aspects to traditional risk assessments were identified to enable an assessment of environmental antibiotic resistance. These include a) accounting for an added selective pressure on the environmental resistome that, over time, allows for development of antibiotic-resistant bacteria (ARB); b) identifying and describing rates of horizontal gene transfer (HGT) in the relevant environmental "hot spot" compartments; and c) modifying traditional dose-response approaches to address doses of ARB for various health outcomes and pathways. CONCLUSIONS: We propose that environmental aspects of antibiotic-resistance development be included in the processes of any HHRA addressing ARB. Because of limited available data, a multicriteria decision analysis approach would be a useful way to undertake an HHRA of environmental antibiotic resistance that informs risk managers.

Assessment of the effects of oil sands naphthenic acids on the growth and morphology of Chlamydomonas reinhardtii using microscopic and spectromicroscopic techniques.

Naphthenic acid fraction components (NAFCs) are thought to be a primary agent of toxicity in oil sands process waters (OSPWs) produced by industrial activity in Canada's Athabasca oil sands. They are a complex, poorly characterized mixture of compounds whose mechanisms of toxicity are not well understood. In this work, it was discovered that the unicellular green algae Chlamydomonas reinhardtii are much more tolerant of NAFCs than predicted based on comparison to Chlamydomonas spp. isolated from the OSPW tailings ponds, with exponential growth occurring at 100 mg L(-1) NAFC. Two cell wall mutants of C. reinhardtii exhibited greater tolerance to NAFC exposure. NAFC exposure induced changes in growth form and morphology were most pronounced in wild-type cells. Confocal scanning laser microscopy and Fourier-transform infrared spectromicroscopy indicated changes in cell wall surface proteins and their confirmation after exposure to NAFCs. Such alterations of cell wall proteins are consistent with the effects of surfactants on green algae, and indicate a possible role for classic naphthenic acids in the NAFC mixture to cause surfactant-mediated toxicity. The much greater tolerance to NAFCs under laboratory conditions indicates the likelihood that NAFCs do not act alone as agents of toxicity in algae such as C. reinhardtii, rather they seem to act in combination with other environmental factors to potentiate toxicity.

Molecular and microscopic assessment of the effects of caffeine, acetaminophen, diclofenac, and their mixtures on river biofilm communities.

The authors examined effects of three common contaminants, caffeine (CF), acetaminophen (AC), and diclofenac (DF), as well as their mixtures on the development, functioning, and biodiversity of river biofilm communities. Biofilms were cultivated in rotating annular reactors. Treatments included AC, CF, DF, AC + CF, AC + DF, CF + DF, AC + CF + DF at 5 µg/L, and their molar equivalent as carbon and nutrients. Incubations using ¹4C-labeled AC, DF, and CF indicated that 90% of the CF, 80% of the AC, and less than 2% of the DF were converted to CO2. Digital imaging revealed a variety of effects on algal, cyanobacterial, and bacterial biomass. Algal biomass was unaffected by AC or CF in combination with DF but significantly reduced by all other treatments. Cyanobacterial biomass was influenced only by the AC + DF application. All treatments other than AC resulted in a significant decrease in bacterial biomass. Diclofenac or DF + CF and DF + AC resulted in increases in micrometazoan grazing. The denaturing gradient gel electrophoresis of Eubacterial community DNA, evaluated by principal component analysis and analysis of similarity, indicated that relative to the control, all treatments had effects on microbial community structure (r = 0.47, p < 0.001). However, the AC + CF + DF treatment was not significantly different from its molar equivalent carbon and nutrient additions. The Archaeal community differed significantly in its response to these exposures based on community analyses, confirming a need to integrate these organisms into ecotoxicological studies.

Advanced imaging techniques for assessment of structure, composition and function in biofilm systems.

Scientific imaging represents an important and accepted research tool for the analysis and understanding of complex natural systems. Apart from traditional microscopic techniques such as light and electron microscopy, new advanced techniques have been established including laser scanning microscopy (LSM), magnetic resonance imaging (MRI) and scanning transmission X-ray microscopy (STXM). These new techniques allow in situ analysis of the structure, composition, processes and dynamics of microbial communities. The three techniques open up quantitative analytical imaging possibilities that were, until a few years ago, impossible. The microscopic techniques represent powerful tools for examination of mixed environmental microbial communities usually encountered in the form of aggregates and films. As a consequence, LSM, MRI and STXM are being used in order to study complex microbial biofilm systems. This mini review provides a short outline of the more recent applications with the intention to stimulate new research and imaging approaches in microbiology.

Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms.

A major limitation for the use of two-proton laser scanning microscopy (2P-LSM) in biofilm and other studies is the lack of a thorough understanding of the excitation-emission responses of potential fluorochromes. In order to use 2P-LSM, the utility of various fluorochromes and probes specific for a range of biofilm constituents must be evaluated. The fluorochromes tested in this study included classical nucleic acid-specific stains, such as acridine orange (AO) and 4",6"-diamidino-2-phenylindole (DAPI), as well as recently developed stains. In addition, stains specific for biofilm extracellular polymeric substances (EPS matrix components) were tested. Two-photon excitation with a Ti/Sapphire laser was carried out at wavelengths from 760 to 900 nm in 10-nm steps. It was found that autofluorescence of phototrophic organisms (cyanobacteria and green algae) resulted in strong signals for the entire excitation range. In addition, the coenzyme F(420)-related autofluorescence of methanogenic bacteria could be used to obtain images of dense aggregates (excitation wavelength, 780 nm). The intensities of the emission signals for the nucleic acid-specific fluorochromes varied. For example, the intensities were similar for excitation wavelengths ranging from 780 to 900 nm for AO but were higher for a narrower range, 780 to 810 nm, for DAPI. In selective excitation, fading, multiple staining, and combined single-photon-two-photon studies, the recently developed nucleic acid-specific fluorochromes proved to be more suitable regardless of whether they are intended for living or fixed samples. Probes specific for proteins and glycoconjugates allowed two-photon imaging of polymeric biofilm constituents. Selective excitation-emission was observed for Calcofluor White M2R (780 to 800 nm) and SyproOrange (880 to 900 nm). In addition, fluor-conjugated concanavalin A lectins were examined and provided acceptable two-photon emission signals at wavelengths ranging from 780 to 800 nm. Finally, CellTracker, a fluorochrome suitable for long-term labeling of microbial eucaryote cells, was found to give strong emission at wavelengths ranging from 770 to 810 nm. If fluorochromes have the same two-photon excitation cross section, they are suitable for multiple staining and multichannel recording. Generally, if an appropriate excitation wavelength and fluorochrome were used, it was possible to obtain more highly resolved images for thick biofilm samples with two-photon laser microscopy than with conventional single-photon laser microscopy. Due to its potential for higher resolution in light-scattering tissue-like material, such as biofilms, and extremely localized excitation, 2P-LSM is a valuable addition to conventional confocal laser scanning microscopy with single-photon excitation. However, further development of the method and basic research are necessary to take full advantage of nonlinear excitation in studies of interfacial microbial ecology.

Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems.

An assessment of lectin-binding analysis for the characterization of extracellular glycoconjugates as part of the extracellular polymeric substances in environmental microbial communities was performed using fully hydrated river biofilms. The applicability of the method was evaluated for single, dual and triple staining with a panel of fluor-conjugated lectins. It was shown that lectin-binding analysis was able to stain glycoconjugates within biofilm communities. Lectin staining also demonstrated spatial heterogeneity within the biofilm matrix. Furthermore, the application of two or even three lectins was possible if suitable combinations were selected. The lectin-binding analysis can be combined with general nucleic acid stains to collect both nucleic acid and glycoconjugate signals. The effects of incubation time, lectin concentration, fluor labelling, carbohydrate inhibition, order of addition and lectin interactions were studied. An incubation time of 20 min was found to be sufficient for completion of lectin binding. It was not possible to ascertain saturating concentration for individual lectins, therefore a standard concentration was used for the assay. Carbohydrate inhibition tests indicated that fluorescein isothiocyanate (FITC)-conjugated lectins had more specific binding characteristics than tetramethyl rhodamine isothiocyanate (TRITC)- or cyanine dye (CY5)-labelled lectins. The order of addition and the nature of the fluor conjugate were also found to influence the binding pattern of the lectins. Therefore the selection of a panel of lectins for investigating the EPS matrix must be based on a full evaluation of their behaviour in the biofilm system to be studied. Despite this necessity, lectin-binding analysis represents a valuable tool to examine the glycoconjugate distribution in fully hydrated biofilms. Thereby, chemical heterogeneities within extracellular biofilm locations can be identified in order to examine the role (e.g. sorption properties, microenvironments, cell-extracellular polymeric substance interactions) of the extracellular polymeric substances in environmental biofilm systems.