3 Using NCA as a solution for removal of driving agents of AMR

As stated in section 2.1.1, the key driving agents of AMR are clinical antimicrobials, metals and biocides. Due to limited resources for conducting this review only clinical antimicrobials and metals have been examined in detail within this section of the report.

3.1 Appropriateness of NCA to combatting clinical antimicrobials

3.1.1 Review of economic justifications

Clinical antimicrobials are used within the pharmacological industry, agricultural industry (animal husbandry) and within the aquaculture industry. Clinical drugs present within the water environment come predominantly from agricultural waste and effluent. Animal husbandry accounts for 60-80% of global clinical microbial use, predominantly anti-bacterial. Whilst the use of anti-bacterials as growth promoters has been banned in Europe and regions of North America, it is still used extensively throughout the developing world (Singer et al, 2016).

The degradation of antimicrobials is dependent upon the relation between their physicochemical properties and the microbial activities/aeration of the recipient soil/water in which they are released. Many studies have analysed the rate of degradation under different conditions and, whilst they do vary, the vast majority of antimicrobials have half-lives of <90 days in soils, and many of the most commonly used clinical antimicrobials have half-lives of <40 days (Albero et al 2018). Shorter half-lives are observed in aqueous environments, including soil-aqueous phases, due to reduced surface areas for adsorption, thereby increasing the availability of the antimicrobials for microbial biodegradation (Clarke and Smith , 2011).

Improved education, infection prevention and diagnostic capabilities have drastically reduced the use of clinical microbials within Europe and North America whilst improved techniques and policies have led to a drastic decline in the use (Holmes et al 2016) Despite this, there is an absence of evidence to confidently correlate the quantitative relationship between the frequency of AMR and volume of clinical microbial administration. Studies have shown that emergence of resistance under a constant selective pressure typically occurs on much shorter timescales than the decline in AMR following removal of the selection pressure. Therefore, for a significant reduction in a specific AMR, an equally significant reduction in all factors contributing to maintaining the selection pressure to which the AMR is providing the advantage must occur (Austin et al 1999). Due to the relatively insignificant effect of reduced clinical antimicrobials, many sources argue that other driving agents are responsible for the continued increase in AMR (Holmes et al 2016).

Whilst the use of clinical antimicrobials is still excessive in areas of the developing world, it may also be argued that, with improved education and awareness, the contamination of water and the environment by these compounds may be effectively reduced (Singer et al 2016). Reducing the contamination in this manner, alongside the short half-lives of the majority of clinical antimicrobials calls into question the economic justification for the use of NCA in dealing with this particular driving agent.

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3.1.2 Review of environmental justifications

The NCA concept often has the benefit of improving biodiversity. It has been shown that clinical antimicrobials, specifically anti-bacterials, have greater persistence when incorporated within the guts of small mammals and birds. Pathogenic exogenous micro-organisms (microorganisms introduced to closed biological systems, known as ‘disease-causing’) are the main target of clinical antimicrobials, such pathogens harmful to humans can often be found residing in the guts of many animals without causing harm. Returning to the metaphor of ‘using a spider to catch a fly’, there is therefore a significant risk that colonisation of areas by wildlife where NCA methods have been applied may lead to increased occurrence of exogenous microbial resistance genes (Wellington et al 2013). This becomes an even greater concern when considering bird populations; the vast distances covered both by feeding and migration patterns can result in AMR genes being distributed from one micro-organism reservoir to another very rapidly. This, in addition to the mixing of exogenous microbes in their guts, results in drastic increases in AMR as a result of HGT mechanisms (Smet et al 2010).

3.1.3 Interlinking with Mott MacDonald work

For the reasons discussed in section 3.1; at this present time there does not appear to be substantial justification for the use of NCA in the treatment of clinical antimicrobials rather than improved waste water treatment, reduced untreated waste discharges and reduced use of clinical antimicrobials. It is recommended that further research be done into the specific techniques and methods that would be incorporated into the NCA, so that the risk of unintended negative consequences may be accurately compared/mitigated against the benefit gained in using NCA over other methods.

Further research is also required into the degradation pathways of clinical antimicrobials within the environment so that a proper assessment of the daughter products and their interaction and role in generating AMR may be analysed.

3.2 Appropriateness of using NCA to combat metals-driven AMR

3.2.1 Role of heavy metals in generating AMR

Heavy metals within water and soil matrices impact microbial communities and represent an important vector in the maintenance and proliferation of AMR. Heavy metals such as Zn, Cu, Mn, Ni, Cr and Fe provide essential nutrients for micro-organism growth with some resistance enzymes also requiring metal co-factors to function e g. Metallo-beta-lactamases requires zinc for sufficient resistance activity. However, in high enough concentrations they may also act as antimicrobials, whereby the metal ions become displaced from their designated (nutrient) binding sites and instead block essential functional sites. This results in diminished enzyme functions and damage to DNA (Rodgers. K et al, 2018).

The accumulation of other heavy metals with a non-biological role such as Pb and Cd may cause oxidative stress, lipid peroxidation and mutagenesis. The toxicity of heavy metals and the misconception that clinical antimicrobials are the sole cause of AMR results in many metals, particularly Cd and Pb, being used as an alternative to clinical antimicrobials (Seiler and Berendonk, 2012). .

The toxicity of heavy metals in the environment is strongly dependent on conditions affecting the valence state, and therefore bioavailability, of metal ions. Such conditions include pH, https://mottmac-my.sharepoint.com/personal/bryony_osbourn_mottmac_com/Documents/AMR/Natural concentrations of organic matter, and the redox potential of the soil, sediment or water in which they are located (Singer.A et al 2016).

To further complicate matters, a small change in conditions will result in many metals exhibiting contrary behaviours to that predicted. For example; under low-pH conditions the solubility of Pb, Cd and Zn is increased and therefore often considered effective to use within aquaculture If, however, high contents of organic matter are present within the sediment, as if often the case in closed water systems, the sediment will act as sorption sinks for these metals. This will therefore prevent them being transported within the water column where they may be used and/or treated but instead results in the accumulation of high concentration of heavy metals within the aquatic sediment. The use of heavy metals without proper understanding of their fate within the environment has resulted in anthropogenically increased concentrations existing far above their natural environmental concentrations (Seiler and Berendonk 2012).

Due to co-selection, resistance to heavy metals often has the synergistic effect of providing resistance to other antimicrobials; particularly those of a clinical nature. The key mechanisms for metal resistance include (Rodgers. K et al, 2018):

● Sequestration of toxic metals: metal binding minimises the concentration of free toxic ions in the cytoplasm. Such binding may be via sorption, protein-metal associations, or release of organic chelators.

● Detoxification through reduction of intracellular ions: An example of this is the use of mercury reductase which reduces Hg2+ to the less toxic Hg0, this will then diffuse out of the cell at a faster rate due to its low evaporation point.

● Extrusion of toxic ions by efflux mechanisms: cation/anion antiporters mediate resistance to metal ions by extrusion of metals from the cytoplasm through the inner and outer membrane to the surrounding environment. Such antiporters are commonly known as “multi-drug efflux pumps”.

The resistance mechanism listed above couple with clinical AMR due to either cross-resistance (e.g. multi-drug efflux pumps) or co-resistance. Co-resistance occurs due to the genes providing metal-resistance being frequently located next to clinical AMR on same plasmid, resulting in non-intentional HGT of clinical AMR genes in response to increased metal toxicity (Seiler and Berendonk, 2012).

Heavy metal toxicity is therefore considered to be the dominant driving force in the development and dissemination of AMR within water and the environment. Given that heavy metals do not degrade within the environment, not only do selective pressures derived from metals persist longer than pharmaceutical and clinical antimicrobial pressures, but due to industrial and urban pollution, the scale of the selection pressure is far more extensive than any other driving agent (Holmes et al, 2015).

3.2.2 Legacy of heavy metal pollution – where can NCA be used?

3.2.2.1 Agriculture

Due to high nutrient density; biosolids (also referred to as ‘sludge’) from waste water treatment plants, aquaculture and animal waste are recycled for use as fertiliser for the agricultural industry.

Many developed countries have existing legislation concerning the application of sludge to reduce the concentration of leached metals on the receiving soil and adjacent water body (Singer et al 2012). Whilst such legislation exists for biosolids, many countries have little https://mottmac-my.sharepoint.com/personal/bryony_osbourn_mottmac_com/Documents/AMR/Natural concerning the use of manure which, in additional to the nutritional benefit, is often applied in excess to improve soil cohesion and reduce erosion (Ramos et al 2006).

It is not economically feasible to use NCA to reduce the concentration of heavy metals within biosolids and manure due the economic value derived from the high nutrient density. Alternatively, there is justification for the use of NCA in intercepting runoff from fields, thus reducing the concentration of metals entering surface water bodies or infiltrating to the aquifer.

The role of land drains in water pollution conveyance is largely ignored within reviews of AMR, yet many studies have found extremely high concentrations of not just metals but of faecal micro-organisms within ditches following rainfall events. A similar trend of high contaminants can also be observed in roadside ditches away from agricultural land, such as within forested areas, due to the leaching of forest soils and wildlife excrement (Falbo et al 2016).

Increased manure spreading may be used during high rainfall due to the benefit of reduced soil detachment, therefore protecting the crops during the wet season. Regular spreading of manure can reduce soil detachment by up to 70% due to increased cohesion but may increase total runoff volume into drainage ditches by up to 30% during each rainfall event (Ramos et al 2006)

Due to a high organic content, a high degree of metal-sorption occurs within soil drains allowing metals to accumulate within ditches for significant periods of time. The presence of a high concentration of metals and micro-organisms within roadside ditches and land drains makes them ideal locations for HGT to occur, generating a pool of AMR genes ready to be washed into nearby water bodies or leached to shallow aquifers following storm events. The dissemination of AMR by HGT is accentuated by the fact that land drains are designed to intersect with one another, resulting in the large-scale mixing of different micro-organism reservoirs and habitats (Schipper et al, 2008).

For these reasons, drainage ditches present ideal targets where NCA may be beneficial in intercepting not only heavy metals, but also clinical antimicrobials and biocides within runoff from the surrounding environment.

Interlinking with Mott MacDonald Work: The use of NCA to remediate drainage ditches and prevent the occurrence and spread of AMR within water catchments would closely tie in with work being done using natural flood alleviation methods to combat flooding.

The potentially significant degree of crossover in this area, and that NCA methods for AMR have potential for effective integrated within NCA to combat flooding, are identified as key findings of this review and are recommended to be explored further.

3.2.2.2 Legacy of industrial pollution

Aquatic sediments are a considerable reservoir of AMR resistant micro-organisms, which are exposed to a vast range of continually changing conditions due to annual cycles in water chemistry within river systems. Naturally stressful conditions render any micro-organisms present more susceptible to stresses imposed by anthropogenic pollution (Rodgers et al, 2018).

Anthropogenic pollution in watercourses can lead to rapidly alternating conditions which may result in a switching of absorption and desorption reactions between metals and aquatic sediments. Sediments may also retain metals and nutrients through sorption on to the surface of mineral and organic particles for significant amounts of time, until destabilisation due to changes in water chemistry results in a mass release (Wellington et al, 2013).

Unlike other pollutants, metals do not degrade. Indigenous micro-organisms persist within aquatic sediments over extremely long timescales and interact with the legacy metal https://mottmac-my.sharepoint.com/personal/bryony_osbourn_mottmac_com/Documents/AMR/Natural contamination, promoting HGT. This process may form layers of enhanced AMR reservoirs in the subsurface.

Additionally, micro-organism community characteristics are related to their responses to physico-chemical properties, pH, nutritional quality of the sediments and source of carbon, irrespective of the pollutant conditions. This can create extreme spatial differences of species composition and community structures, therefore, drastically enhancing the chances of coresistance due to a much more diverse range of AMR genes within the microbiome (Rodgers.K et al, 2018).

Interlinking with Mott MacDonald Work

There is a potentially significant opportunity to target and integrate AMR with regards to industrial waste entering the water courses or remediation of polluted legacy sediments/soils

By making AMR a key factor when assessing the suitability of projects such as discharge permit locations, planning applications, abstractions from or in proximity to watercourses or contaminated land, the dissemination of AMR could be drastically reduced.

Simple measures such as modifying common practice within preliminary risk assessments and desk studies to prevent active involvement in the spread of AMR would not only increase the awareness of staff involved, but also facilitate clients to address these issues.

The potential implication of these findings is that AMR may become a crucial factor when considering suitable remediation strategies within Mott MacDonald’s contaminated land sector. As an additional point to this, it is recommended that assessment of the positive and negative impacts of currently used remediations strategies for common industrial contamination be undertaken with a view to determining the potential role of remediation in the spread of AMR.

3.3 Precautionary considerations regarding use of NCA as an action plan for AMR

3.3.1 Natural production of antimicrobials

Many micro-organisms and organisms, including plants and animals, naturally produce antimicrobial substances; such antimicrobial substances are intended to often at concentrations too low to be effective on entire micro-organism population

There is a significant body of evidence indicating that sub-lethal doses are significantly more likely to increase AMR due to prolonged exposure (Andersson and Hughes. 2014). Many saprophytic organisms are also known to produce a class of beta-lactam antibiotics known as carbapenems; this antibiotic is also heavily administrated within the clinical industry due to its ability to actively inhibit both aerobic and anaerobic gram positive and gram-negative organisms (Holmes et al 2016).

The use of natural capital as an action plan for AMR may naturally increase the resistance to the antimicrobial being secreted naturally, thus increasing AMR within that soil layer. Returning to the ‘using a spider to catch a fly’ metaphor, the knock-on effect of introducing or increasing non-native antimicrobials to a system must be considered. For example; if a NCA is used in which an increase in biodiversity occurs, part of which includes an increase in saprophytic organisms, then naturally an increased resistance to carbapenems may occur within that biodome. Any wildlife, particularly birds and small mammals, interacting within this biodome will come in contact with micro-organisms in possession of the carbapenems resistant genes within https://mottmac-my.sharepoint.com/personal/bryony_osbourn_mottmac_com/Documents/AMR/Natural their plasmids; through this SPR example the resistance to carbapenems will spread from one reservoir to another (Wellington et al 2013). The ecological effect of this resistance to the antibiotic producing organism must also be considered; if the produced compound no longer works then the producing organism, in this case the saprophyte, may be outcompeted by other organisms.

Symbiotic relationships may also be inadvertently impacted; Actinomycetes are a diverse family of gram-negative bacteria that have evolved to live in symbiosis with many plants, fungi, insects and sponges. Such organisms profit from the natural products produced by actinomycetes such as nutrients (e.g. nitrogen) and protection against pathogenic microbes due to the secretion of antibiotics (Van der Meij et al 2017). Increased AMR to antibiotics such as those produced by actinomycetes, perhaps by cross- or co-resistance, could have a devastating ecological impact on the host organisms in symbiosis. Disruption to symbiotic relationships may also have impacts on crop production as reduced soil persistence of bacteria such as actinomycetes may drastically reduce the nutrient density of compounds such as nitrogen within agricultural soils (Rosenblueth et al 2018). The potential impacts that increased AMR within the environment may have on symbiotic relationships important to agriculture is something of great importance, particularly concerning what role NCA may have to play, yet this requires much more extensive research in order to be discussed further at this time.

The above is simply an example of how without proper understanding, NCA may counteract the exact thing it is being used to solve later down the line. Ecological and co-evolutionary processes occur at different spatial and time scales. Whilst single, isolated populations are typically dominated by demographic and genetic stochasticity, colonisation and extinction dynamic factors have a larger influence on metapopulations (Smith et al 2003). In order for NCA to be an effective action plan for AMR the controls on both isolated populations and metapopulations must be considered and correctly understood so as to avoid a cascade of effects at a later stage.

3.3.2 Creation of reservoirs of AMR

The risk of creating reservoirs of AMR has been discussed in detail with regards to clinical antimicrobials in section 3.1.2. In addition to what is discussed in section 3.1.2 with regards to clinical antimicrobials, consideration must be given to metals and biocides to ensure that the concentration of these driving agents is not simply reduced from the water environment by increasing/transferring them to the terrestrial environment or biosphere. Examples of ways in this may occur include:

• Bioaccumulation of heavy metals and clinical antimicrobials in plants and animals,

• Alteration of the water conditions causing heavy metals to precipitate out of water courses only to accumulate in soils and leach to aquifers,

• The use of activated soil filters to remove biocides from infiltrating groundwater but result in concentrated layers of legacy sediments occurring within the soil zone (Bester et al 2011).

3.3.3 Interlinking with Mott MacDonald Work

Various sectors within Mott MacDonald could be targeted to increase awareness that the role the natural environment could have in promoting AMR.

One way in which this could be done is by considering what the introduction of non-native plant species to a habitat could have on AMR, and the impact of exposing a microbial population to new and different naturally produced sub-lethal doses of antimicrobials.

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Examples of where this could be applied within current projects within Mott MacDonald include:

• Ecology: include the broad class of antimicrobials occurring naturally within a habitat as part of ecological surveys.

• Landscape Architecture: based on the ecological surveys of the area, consider the negative impact that the mixing of antimicrobials may have when choosing the plant species. Already invasive traits and characteristics must be considered when choosing non-native ornamental species. Therefore, with increased awareness, the class of antimicrobial being introduced could also be considered.

• Flood management: Increased awareness of the AMR role that various natural flood management strategies may have. For example, the planting of non-native trees to form riparian zones https://mottmac-my.sharepoint.com/personal/bryony_osbourn_mottmac_com/Documents/AMR/Natural

• Contaminated land: when using NCA as a remediation tactic, consider the mixing and gene pool implications that artificially creating a habitat where it would not naturally occur may have. For example, artificially created wetlands as a solution for acid mine drainage.