Detection of antibacterial-like activity on a silica surface: fluoroquinolones and their environmental metabolites

Background, scope, and aims

Antibacterial fluoroquinolones (FQs) are third-generation antibiotics that are commonly used as therapeutic treatments of respiratory and urinary tract infections. They are used far less in intensively farmed animal production systems, though their use may be permitted in the veterinary treatments of flocks or in medicated feeds. When used, only a fraction of ingested parent FQ actually reaches the in vivo target site of infection, while the remainder is excreted as the parent FQ and its metabolized products. In many species?? metabolism, enrofloxacin (EF) is converted into ciprofloxacin (CF) while both FQs are classified as parent FQs in human treatments. It is therefore likely that both FQs and their metabolic products will contribute to a common pool of metabolites in biological wastes. Wastes from intensive farming practices are either directly applied to agricultural land without treatment or may be temporarily stored prior to disposal. However, human waste is treated in sewage treatment plants (STPs) where it is converted into biosolids. In the storage or treatment process of STPs, FQs and their in vivo metabolites are further converted into other environmental metabolites (FQEMs) by ex vivo physicochemical processes that act and interact to produce complex mixtures of FQEMs, some of which have antibacterial-like activities. Biosolids are then often applied to agricultural land as a fertilizer amendment where FQs and FQEMs can be further converted into additional FQEMs by soil processes. It is therefore likely that FQ-contaminated biowaste-treated soils will contain complex mixtures of FQEMs, some of which may have antibacterial-like activities that may be expressed on bacteria endemic to the receiving agricultural soil environment. Concern has arisen in the scientific and in the general community that repeated use of FQ-contaminated biowaste as fertilizer amendments of nutrient-impoverished agricultural land may create a selective environment in which FQ-resistant bacteria might grow. The likelihood of this happening will depend, to some extent, on whether bioactive FQEMs are first synthesized from the parent FQs by the action and interaction of in vivo and ex vivo processes producing bioactive FQEMs in biowastes and biosolids. The postulated creation of a selective environment will also depend, in part, on whether such bioactive FQEMs are biologically available to bacteria, which may, in turn, be influenced by soil type, amendment regime, and the persistence of the bioactive FQEMs. Additionally, soil bacteria and soil processes may be affected in different ways or extents by bioactive FQEMs that could possibly act additively or synergistically at ecological targets in these non-target bacteria. This is an important consideration, since, while parent FQs have well-defined ecological targets (DNA gyrase and topoisomerase IV) and modes of bactericidal action, the FQEMs and their possible modes of action on the many different species of soil bacteria is less well studied. It is therefore understandable that there is a lack of conclusive evidence directly attributing biosolid usage to any increase in FQ-resistant bacteria detected in biowaste-amended agricultural soil. However, a lack of evidence may simply imply that a causal relationship between biosolid usage programs and any detection of low levels of FQ-resistant bacteria in soils has yet to be established, rather than an assumption of no relationship whatsoever. Based on results presented in this paper, the precautionary principle should be applied in the usage of FQ-contaminated biosolids as fertilizer amendments of agricultural land. The aim of this research was to test whether any bioactive FQEMs of EF could be synthesized by aerobic fermentation processes using Mycobacterium gilvum (American Tissue Culture Collection) and a mixed culture of microorganisms derived from an agricultural soil. High-performance thin-layer chromatography (HPTLC) and bioautography were tested as screening techniques in the detection and analysis of bioactive FQEMs.

Materials and methods

FQEMs derived from M. gilvum and mixed (soil) culture aerobic ferments were fractionated using preparative HPTLC. A standard strain of Escherichia coli was then used as the reporter organism in a bioautography assay in the detection of bioactive-FQEMs on a mid-section of the HPTLC plate. Plate sections were reassembled, and a photograph was taken under low-intensity ultraviolet (UV) light to reveal regions that contained analytes that had UV chromophores and antibacterial-like activities.

Results and discussion

Many fractionated FQEMs displayed antibacterial-like activity while bound to silica gel HPTLC plates. These results also provide evidence that sufficient quantities of biologically active FQEMs were biologically available from a silica gel surface to prevent the adherent growth of E. coli. Six to seven FQEMs derived from EF using aerobic fermentation processes had antibacterial-like activities, while two FQEMs were also detectable using UV light. Furthermore, similar banding patterns of antibacterial-like activity were observed in both the monoculture (M. gilvum) and mixed culture bioautography assays, indicating that similar processes operated in both aerobic fermentations, either producing similar biologically active FQEMs or biologically active FQEMs that had similar physicochemical properties in both ferments. The simplest explanation for these findings is that the tested agricultural soil also contained mycobacteria that metabolized EF in a similar way to the purchased standard monoculture M. gilvum. Additionally, the marked contrast between the bioautography results and the UV results indicated that the presence of UV chromophores is not a prerequisite for the detection of antibacterial-like activity.

Conclusions

A reliance on spectrophotometric techniques in the detection of bioactive FQEMs in the environment may underestimate component antibacterial-like activity and, possibly, total antibacterial-like activity expressed by EF and its FQEMs. The described bioautography method provides a screening technique with which antibacterial-like activities derived from EF and possibly other FQs can be detected directly on silica gel HPTLC plates.

Recommendations

It is recommended that both bioassay and instrumental analytical techniques be used in any measurement of hazard and risk relating to antibacterial-like activities in the environment that are derived from fluoroquinolone antibiotics and their environmental metabolites.     

Soil and biodiversity

It is shown that the soil diversity-biodiversity system in terrestrial ecosystems operates in spatiotemporal unity, which manifests itself at different hierarchical levels of their structural-functional organization: successional-evolutionary, zonal geographic, landscape, biogeocenotic, soil-type, horizon-layer, geochemical, and the levels of elementary soil processes and soil fertility. Arguments confirming the functional relationship between organisms and soils are considered. Effective biodiversity conservation is possible on the basis of an ecosystems approach involving simultaneous conservation of soil diversity.     

Bromination of selected pharmaceuticals in water matrices

The bromination of five selected pharmaceuticals (metoprolol, naproxen, amoxicillin, phenacetin, and hydrochlorothiazide) was studied with these compounds individually dissolved in ultra-pure water. The apparent rate constants for the bromination reaction were determined as a function of the pH, obtaining the sequence amoxicillin > naproxen ? hydrochlorothiazide ≈ phenacetin ≈ metoprolol. A kinetic mechanism specifying the dissociation reactions and the species formed for each compound according to its pK_a value and the pH allowed the intrinsic rate constants to be determined for each elementary reaction. There was fairly good agreement between the experimental and calculated values of the apparent rate constants, confirming the goodness of the proposed reaction mechanism.In a second stage, the bromination of the selected pharmaceuticals simultaneously dissolved in three water matrices (a groundwater, a surface water from a public reservoir, and a secondary effluent from a WWTP) was investigated. The pharmaceutical elimination trend agreed with the previously determined rate constants. The influence of the main operating conditions (pH, initial bromine dose, and characteristics of the water matrix) on the degradation of the pharmaceuticals was established. An elimination concentration profile for each pharmaceutical in the water matrices was proposed based on the use of the previously evaluated apparent rate constants, and the theoretical results agreed satisfactorily with experiment. Finally, chlorination experiments performed in the presence of bromide showed that low bromide concentrations slightly accelerate the oxidation of the selected pharmaceuticals during chlorine disinfection.     

N deposition as a threat to the World's protected areas under the Convention on Biological Diversity

This paper combines the world’s protected areas (PAs) under the Convention on Biological Diversity (CBD), common classification systems of ecosystem conservation status, and current knowledge on ecosystem responses to nitrogen (N) deposition to determine areas most at risk. The results show that 40% (approx. 11% of total area) of PAs currently receive >10 kg N/ha/yr with projections for 2030 indicating that this situation is not expected to change. Furthermore, 950 PAs are projected to receive >30 kg N/ha/yr by 2030 (approx. twice the 2000 number), of which 62 (approx. 11,300 km²) are also Biodiversity Hotspots and G200 ecoregions; with forest and grassland ecosystems in Asia particularly at risk. Many of these sites are known to be sensitive to N deposition effects, both in terms of biodiversity changes and ecosystem services they provide. Urgent assessment of high risk areas identified in this study is recommended to inform the conservation efforts of the CBD.     

Road deicing salt irreversibly disrupts osmoregulation of salamander egg clutches

It has been postulated that road deicing salts are sufficiently diluted by spring rains to ameliorate any physiological impacts to amphibians breeding in wetlands near roads. We tested this conjecture by exposing clutches of the spotted salamander (Ambystoma maculatum) to three chloride concentrations (1 mg/L, 145 mg/L, 945 mg/L) for nine days, then transferred clutches to control water for nine days, and measured change in mass at three-day intervals. We measured mass change because water uptake by clutches reduces risks to embryos associated with freezing, predation, and disease. Clutches in controls sequestered water asymptotically. Those in the moderate concentrations lost 18% mass initially and regained 14% after transfer to control water. Clutches in high concentration lost 33% mass and then lost an additional 8% after transfer. Our results suggest that spring rains do not ameliorate the effects of deicing salts in wetlands with extremely high chloride concentrations.     

Integrating Media Studies of Climate Change into Transdisciplinary Research: Which Direction Should We Be Heading?

Research in the area of media coverage on climate change communication represents one of the most prolific areas of inquiry within communication and mass communication studies. This body of literature, which ranges from empirical to critical studies, continues to expand. Much research has focused on representations of climate change causes, effects, and human actions, while some has assessed the impacts of these representations. What is broadly missing from this literature, however, is a discussion of how we might integrate media analysis into transdisciplinary collaborative research aimed at creating solutions to the social, environmental, and economic issues intertwined with climate change. Given the magnitude of problems the society and science are currently grasping with, it behooves us to understand how media studies can contribute most effectively to characterizing and solving problems. We maintain that the move toward integrating media studies into transdisciplinary collaborative research marks an essential transition for environmental communication in general, but climate change communication in particular, given the urgency and magnitude of creating meaningful adaptation and mitigation strategies to address this pressing, complex challenge. Drawing on our work as part of a large transdisciplinary sustainability science team, we provide a case study for understanding what collaborations are key to moving media studies into a transdisciplinary context and the key opportunities and barriers that come along with that move. We argue that media studies must increasingly engage directly in collaboration with other researchers, stakeholders, and communities to serve on-the-ground decision-making and enhance society's ability to take action.     

A Systematic Approach to the Evaluation of RCRA Disposal Facilities Under Future Climate‐Induced Events

The ability of near‐surface disposal facility cover designs to meet percolation performance criteria can be influenced by naturally occurring climatic mechanisms as well as anthropogenic forcing. This study was conducted to determine the effect of climate‐induced events on percolation based, probabilistic distributions derived from historical climate data. Water balance predictions were evaluated using the HELP model, employing several variations of degradation in a traditional RCRA disposal facility cover design over a 100‐year simulation period. Results demonstrated that changes in precipitation and temperature can influence performance. The analysis also revealed that when both precipitation and temperature are increased, warmer temperatures tend to offset some of the impact from greater precipitation. ©2015 Wiley Periodicals, Inc.     

Understanding the Causes of Spills From the Supply and Handling of Chemicals at Resource Construction Sites: A Case Study

An intermediate bulk container (IBC) was punctured during its handling, releasing a refined oil product onto land at a large construction site in an environmentally sensitive region of Australia. Understanding and controlling the risks from fuel, oil, and chemical spills on the current project was of critical importance as part of the project's overall approval, and ongoing compliance depended on the project committing to minimizing all chemical and petroleum hydrocarbon spills on the site. The telehandler (forklift) did not pierce the plastic of the IBC directly (as was expected to be the case) but rather one of the tynes caught on the underside of the metal base plate (pallet belly plate), despite numerous controls being in place at the time of spill (to limit the risks of damaging the IBC), revealing a previously unreported mechanism for a fluid spill from handling of petroleum hydrocarbons and related chemicals. The investigation team used a root cause analysis (RCA) technique, based on the fishbone (Ishikawa) diagram, which was undertaken with 12 expert contributors (from the project) to identify the underlying cause: The inspection process was inadequate. This study is a companion to the article published in Winter 2014 in Remediation (Guerin, 2014) covering multiple causes of spills from plant and equipment commonly used on construction and remediation projects. ©2015 Wiley Periodicals, Inc.