Kristian K. Brandt

Management options for reducing the release of antibiotics and antibiotic resistance genes to the environment

Background: There is growing concern worldwide about the role of polluted soil and water environments in the development and dissemination of antibiotic resistance. Objective: Our aim in this study was to identify management options for reducing the spread of antibiotics and antibiotic-resistance determinants via environmental pathways, with the ultimate goal of extending the useful life span of antibiotics. We also examined incentives and disincentives for action. Methods: We focused on management options with respect to limiting agricultural sources; treatment of domestic, hospital, and industrial wastewater; and aquaculture. Discussion: We identified several options, such as nutrient management, runoff control, and infrastructure upgrades. Where appropriate, a cross-section of examples from various regions of the world is provided. The importance of monitoring and validating effectiveness of management strategies is also highlighted. Finally, we describe a case study in Sweden that illustrates the critical role of communication to engage stakeholders and promote action. Conclusions: Environmental releases of antibiotics and antibiotic-resistant bacteria can in many cases be reduced at little or no cost. Some management options are synergistic with existing policies and goals. The anticipated benefit is an extended useful life span for current and future antibiotics. Although risk reductions are often difficult to quantify, the severity of accelerating worldwide morbidity and mortality rates associated with antibiotic resistance strongly indicate the need for action.

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. Citation: Ashbolt NJ, Amézquita A, Backhaus T, Borriello P, Brandt KK, Collignon P, Coors A, Finley R, Gaze WH, Heberer T, Lawrence JR, Larsson DG, McEwen SA, Ryan JJ, Schönfeld J, Silley P, Snape JR, Van den Eede C, Topp E. 2013. Human health risk assessment (HHRA) for environmental development and transfer of antibiotic resistance. Environ Health Perspect 121:993–1001; http://dx.doi.org/10.1289/ehp.1206316

Cu Exposure under Field Conditions Coselects for Antibiotic Resistance as Determined by a Novel Cultivation-Independent Bacterial Community Tolerance Assay

Environmental reservoirs of antibiotic resistance are important to human health, and recent evidence indicates that terrestrial resistance reservoirs have expanded during the antibiotic era. Our aim was to study the impact of Cu pollution as a selective driver for the spread of antibiotic resistance in soil. Bacteria were extracted from a well-characterized soil site solely contaminated with CuSO₄ more than 80 years ago and from a corresponding control soil. Pollution-induced bacterial community tolerance (PICT) to Cu and a panel of antibiotics was determined by a novel cultivation-independent approach based on [³H]bromodeoxyuridine (BrdU) incorporation into DNA and by resistance profiling of soil bacterial isolates on solid media. High Cu exposure selected for Cu-tolerant bacterial communities but also coselected for increased community-level tolerance to tetracycline and vancomycin. Cu-resistant isolates showed significantly higher incidence of resistance to five out of seven tested antibiotics (tetracycline, olaquindox, nalidixic acid, chloramphenicol, and ampicillin) than Cu-sensitive isolates. Our BrdU-PICT data demonstrate for the first time that soil Cu exposure coselects for resistance to clinically important antibiotics (e.g., vancomycin) at the bacterial community-level. Our study further indicates that Cu exposure provides a strong selection pressure for the expansion of the soil bacterial resistome.

Toxic Effects of Linear Alkylbenzene Sulfonate on Metabolic Activity, Growth Rate, and Microcolony Formation of Nitrosomonas and Nitrosospira Strains

ABSTRACT Strong inhibitory effects of the anionic surfactant linear alkylbenzene sulfonate (LAS) on four strains of autotrophic ammonia-oxidizing bacteria (AOB) are reported. TwoNitrosospira strains were considerably more sensitive to LAS than two Nitrosomonas strains were. Interestingly, the two Nitrosospira strains showed a weak capacity to remove LAS from the medium. This could not be attributed to adsorption or any other known physical or chemical process, suggesting that biodegradation of LAS took place. In each strain, the metabolic activity (50% effective concentration [EC50], 6 to 38 mg liter−1) was affected much less by LAS than the growth rate and viability (EC50, 3 to 14 mg liter−1) were. However, at LAS levels that inhibited growth, metabolic activity took place only for 1 to 5 days, after which metabolic activity also ceased. The potential for adaptation to LAS exposure was investigated with Nitrosomonas europaea grown at a sublethal LAS level (10 mg liter−1); compared to control cells, preexposed cells showed severely affected cell functions (cessation of growth, loss of viability, and reduced NH4+ oxidation activity), demonstrating that long-term incubation at sublethal LAS levels was also detrimental. Our data strongly suggest that AOB are more sensitive to LAS than most heterotrophic bacteria are, and we hypothesize that thermodynamic constraints make AOB more susceptible to surfactant-induced stress than heterotrophic bacteria are. We further suggest that AOB may comprise a sensitive indicator group which can be used to determine the impact of LAS on microbial communities.

Selection for Cu-tolerant bacterial communities with altered composition, but unaltered richness, via long-term Cu exposure.

ABSTRACT Toxic metal pollution affects the composition and metal tolerance of soil bacterial communities. However, there is virtually no knowledge concerning the responses of members of specific bacterial taxa (e.g., phyla or classes) to metal toxicity, and contradictory results have been obtained regarding the impact of metals on operational taxonomic unit (OTU) richness. We used tag-coded pyrosequencing of the 16S rRNA gene to elucidate the impacts of copper (Cu) on bacterial community composition and diversity within a well-described Cu gradient (20 to 3,537 μg g−1) stemming from industrial contamination with CuSO4 more than 85 years ago. DNA sequence information was linked to analysis of pollution-induced community tolerance (PICT) to Cu, as determined by the [3H]leucine incorporation technique, and to chemical characterization of the soil. PICT was significantly correlated to bioavailable Cu, as determined by the results seen with a Cu-specific bioluminescent biosensor strain, demonstrating a specific community response to Cu. The relative abundances of members of several phyla or candidate phyla, including the Proteobacteria, Bacteroidetes, Verrumicrobia, Chloroflexi, WS3, and Planctomycetes, decreased with increasing bioavailable Cu, while members of the dominant phylum, the Actinobacteria, showed no response and members of the Acidobacteria showed a marked increase in abundance. Interestingly, changes in the relative abundances of classes frequently deviated from the responses of the phyla to which they belong. Despite the apparent Cu impacts on Cu resistance and community structure, bioavailable Cu levels did not show any correlation to bacterial OTU richness (97% similarity level). Our report highlights several bacterial taxa responding to Cu and thereby provides new guidelines for future studies aiming to explore the bacterial domain for members of metal-responding taxa.

Sulfate Reduction Dynamics and Enumeration of Sulfate-Reducing Bacteria in Hypersaline Sediments of the Great Salt Lake (Utah, USA)

Bacterial sulfate reduction activity (SRA) was measured in surface sediments and slurries from three sites in the Great Salt Lake (Utah, USA) using radiolabeled 35S-sulfate. High rates of sulfate reduction (363 ± 103 and 6,131 ± 835 nmol cm-3 d-1) were measured at two sites in the moderately hypersaline southern arm of the lake, whereas significantly lower rates (32 ± 9 nmol cm-3 d-1) were measured in the extremely hypersaline northern arm. Bacterial sulfate reduction was strongly affected by salinity and showed an optimum around 5-6% NaCl in the southern arm and an optimum of around 12% NaCl in the more hypersaline northern arm of the lake. High densities of sulfate-reducing bacteria (SRB) ranging from 2.2 × 107 to 6.7 × 108 cells cm-3 were determined by a newly developed tracer MPN-technique (T-MPN) employing sediment media and 35S-sulfate. Calculation of specific sulfate reduction rates yielded values comparable to those obtained in pure cultures of SRB. However, when using a conventional MPN technique with synthetic media containing high amounts of Fe(II), the numbers of SRB were underestimated by 1-4 orders of magnitude as compared to the T-MPN method. Our results suggest that high densities of slightly to moderately halophilic and extremely halotolerant SRB are responsible for the high rates of sulfate reduction measured in Great Salt Lake sediments.

Desulfobacter halotolerans sp. nov., a halotolerant acetate-oxidizing sulfate-reducing bacterium isolated from sediments of Great Salt Lake, Utah

Summary Dissimilatory acetate-oxidizing sulfate-reducing bacteria were enriched from hypersaline sediments of the Great Salt Lake (Utah, USA) in a synthetic medium. Three mesophilic rod-shaped strains were isolated from deep-agar dilution series, and one strain (GSL-Ac1) was chosen for further studies. Strain GSL-Ac1 showed a high degree of salt tolerance (up to 13% NaCl and 4.5% MgCl 2 · 6H 2 O), but displayed optimum growth with only 1–2% NaCl in the medium. 16S rDNA sequencing placed GSL-Acl in the genus Desulfobacter . On the basis of DNA-DNA hybridization studies and physiological differences, we describe strain GSL-Acl as a new species, Desulfobacter halotolerans sp. nov. Strain GSL-Acl showed the highest NaCl-tolerance reported for any member of the genus Desulfobacter .

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.

Delftia lacustris sp. nov., a peptidoglycan-degrading bacterium from fresh water, and emended description of Delftia tsuruhatensis as a peptidoglycan-degrading bacterium.

Extracellular peptidoglycan is commonly found in natural environments, yet little is known about its biodegradation in nature. We here describe a novel peptidoglycan-degrading bacterium, designated strain 332T, isolated from mesotrophic lake water in Denmark. The strain was a Gram-negative-staining, motile rod. It had chitinase and lysozyme activities, which are relevant to peptidoglycan degradation, and was capable of utilizing several mono- and disaccharides, amino acids and organic acids. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain 332T belonged to the genus Delftia. Fatty acids of the strain included C8:0 and C10:0, which are characteristic of the genus Delftia. The DNA G+C content of the strain was 65.3 mol%. A DNA-DNA hybridization value of 66.2% was found between strain 332T and Delftia tsuruhatensis DSM 17581T. Based on differences in physiological and biochemical characteristics, the strain is considered to represent a novel species, for which the name Delftia lacustris sp. nov. is proposed. The type strain is 332T (=DSM 21246T=LMG 24775T). An emended description of Delftia tsuruhatensis is also presented.

Desulfocella halophila gen. nov., sp. nov., a halophilic, fatty-acid-oxidizing, sulfate- reducing bacterium isolated from sediments of the Great Salt Lake

A new halophilic sulfate-reducing bacterium, strain GSL-But2T, was isolated from surface sediment of the Southern arm of the Great Salt Lake, UT, USA. The organism grew with a number of straight-chain fatty acids (C4-C16), 2-methylbutyrate, L-alanine and pyruvate as electron donors. Butyrate was oxidized incompletely to acetate. Sulfate, but not sulfite or thiosulfate, served as an electron acceptor. Growth was observed between 2 and 19% (w/v) NaCl with an optimum at 4-5% (w/v) NaCl. The optimal temperature and pH for growth were around 34 degrees C and pH 6.5-7.3, respectively. The generation time under optimal conditions in defined medium was around 28 h, compared to 20 h in complex medium containing yeast extract. The G+C content was 35.0 mol%. 16S rRNA gene sequence analysis revealed that strain GSL-But2T belongs to the family Desulfobacteriaceae within the delta-subclass of the Proteobacteria and suggested that strain GSL-But2T represents a member of a new genus. The name Desulfocella halophila gen. nov., sp. nov. is proposed for this organism. The type strain of D. halophila is strain GSL-But2T (= DSM 11763T = ATCC 700426T).