A. Summaries of reviewed papers

A.1 Initiatives for Addressing Antimicrobial Resistance in the Environment: Current Situation and Challenges (UK Science and Innovation Network, 2018)

A.1.1 Human and Animal Contamination

● AMR is when microbes (i.e. bacteria and fungi) develop the ability to defeat the drugs designed to combat them- causing a threat to public health. Pathogenic antimicrobialresistant microbes can cause infections in humans that are difficult, and sometimes impossible, to treat. The report highlights data identifying the potential for the environment (waterways and soils) to be a source of pathogenic antimicrobial-resistant microbes that could affect human health. ● Contamination of the environment can occur from human and animal waste, pharmaceutical manufacturing waste, and use of antimicrobial pesticides for crops. However, the scale and risk of this is not fully understood. ● Scientific evidence shows that antimicrobials and resistance do spread in the environment and people exposed to resistance pathogens like Methicillin-resistant Staphylococcus aureus (MRSA) in environmental waters are at increased risk of infection from this exposure. ● Basic sanitation, including access to facilities for disposing of human waste safely, is critically important for preventing many diseases. ● Human waste: waste can carry antimicrobial-resistant pathogens, there is an increased risk of infections where this waste is discharged directly into the environment without treatment.

Wastewater treatment plants are essential for reducing bacteria; although this might not be sufficient where there are high levels of bacteria such as in healthcare facilities. Resistant microbes can persist and grow within healthcare plumbing systems. Studies have found detectable levels of resistant bacteria in surface waters (rivers, coastal waters). Human waste may enter the environment inadvertently through direct release into water bodies by overflow of combined sewers. In many countries around the world, a high percentage of human sewage is not treated appropriately. In Dhaka, Bangladesh, 70% is discharged directly into the environment. A European study found trace levels of antimicrobials and evidence of resistant bacteria in treated sewage sludge. ● Animal waste and agriculture: animal and human waste can be used as manure. If not treated properly, this could contain antimicrobial-resistant pathogens. Runoff from livestock could contaminate surface and groundwaters with resistant bacteria. Antimicrobials are used worldwide in aquaculture but the quantities and types used are not known. Smith et al. estimated that 1mg of antimicrobial agents was used per kg of production in Norway, yet

Chile used more than 600mg per kg of salmon produced. Based on surface water samples for antimicrobial residues, it is estimated that approximately 5,800 tonnes of enrofloxacin, 1,800 tonnes of sulphadiazine, 12,300 tonnes of sulphamethoxazole and 6,400 tonnes of trimethoprim are discharged into the Mekong Delta every year from terrestrial livestock discharge and shrimp and fish culture systems in the region. Several studies have found evidence suggesting that a farm-to-environment-to-human route of transmission may occur.

However, the public health impact of AMR from agriculture exposures are not well understood. ● Once resistant microbes are in the environment, there is potential to spread, colonize, or cause infections in other people or animals. If these antimicrobials retain their activity in the

environment, they can apply selective pressure on the microbial population and amplify resistant bacteria. ● Preventing Exposure: Treating recreational waters or segregating them from other contaminated environmental surface waters. For potable water, finishing treatment plants and maintaining water supply systems is required to enhance the probability of AMR free water. Sewage needs to be kept from fisheries and bivalve beds. Water for irrigation needs to be kept uncontaminated. However, there is a lack of available technologies for low- and middle-income countries.

A.1.2 Antimicrobial Manufacturing Waste

● Release of active pharmaceutical ingredients (APIs) into the environment may occur when antimicrobials are manufactured without effective control measures in place. The manufacturing process can result in a high amount of antimicrobials in the surrounding environment (e.g. soil, water) which may lead to selecting for antibiotic-resistant bacteria. ● Significance of how manufacturing waste might contaminate the environment is unclear. ● Manufacturers do not voluntarily disclose APIs released into the environment in their discharge water. ● There are no international standards for wastewater limits for antimicrobials. ● Localised discharges from manufacturing plans might lead to more antimicrobial contamination than the excretion of drugs that people use for therapy (human waste).

A.1.3 Antimicrobials Used as Crop Pesticides

● Antimicrobials are widely used as pesticides for crop disease management. In some cases, these antimicrobials are the same, or closely related to, antimicrobials used in human medicine. Using antimicrobials as crop pesticides has the potential to select for resistant microbes present in the environment. This is of particular concern if the microbe can cause human infection or confers transferable resistance mechanisms to antimicrobials commonly used to treat human infections. Of particular concern are cases where antibiotic use on crops increases or when the environment exposed to the pesticide is contaminated with multi-drug resistant microbes.

A.2 Frontiers 2017 Emerging Issues of Environmental Concern (UNEP, 2017)

● It is the low concentration contamination that is of particular importance- the concentration is too low to be lethal to exposed bacteria, but sufficient to select for resistance. At low antibiotic concentration, the acquisition of resistance may be reliant more on gene transfer from another bacterium, known as horizontal gene transfer. ● Up to 75% of antibiotics used in aquaculture may be lost into the surrounding environment. ● 70% of antibiotics are used by animals. ● Major waste flows including waste water, manures and agricultural runoff contain antibiotic residues and antibiotic-resistant bacteria. ● Up to 80% of consumed antibiotics are excreted through urine and faeces. ●

A.3 Drivers, dynamics and epidemiology of antimicrobial resistance in animal production (Food and Agriculture Organization of the United Nations, 2016)

● Overuse of antimicrobials and improper use in many parts of the world are recognized as key drivers of the emergence and spread of AMR. ● It is projected that two thirds of the future growth of antimicrobial use will be for animal production. Although antimicrobial use in animal food production has been substantially reduced in high-income countries in recent years, data available indicate that livestock antimicrobial use will continue to increase in low- and middle-income countries during the next decades due to the growing demand for animal protein. As a consequence, there is likely to be an increase in resistance to commonly used antimicrobials in these countries and regions. ● It has been observed that enterococci of animal origin can also colonize the human gut (Werner et al., 2013). ● Strong and direct evidence for AMR transmission via food is still limited. However, there is evidence of AMR occurrence not only in animal-derived foodstuffs but also in vegetables.

This raises the issue of global trade and travel in the transboundary dissemination of resistance genes. ● Antimicrobials are commonly used non-therapeutically in livestock production as a kind of

“insurance” in addition to other animal disease risk management procedures. In some non-

European countries, antimicrobials are widely used by farmers without veterinary supervision due to their relatively low cost and ready availability for sale over the counter (Laxminarayan et al., 2013). ● Only a few countries in Europe (e.g. Netherlands, Denmark, Sweden) currently conduct integrated surveillance of antimicrobial use and AMR in humans, animals and food products of animal origin. ● Heavy metals: these may be used in agriculture as part of livestock feed supplements, and in a Chinese study were detected in manure from pig farms (Zhu et al., 2013). Heavy metals have been associated with the emergence and spread of AMR in environmental bacteria due to co-selection. The presence of heavy metals has also been associated with the reduction of susceptibility of bacterial populations and commensal bacteria to antimicrobials. ● The extent and persistence of antimicrobial residues in aquaculture production systems is currently unknown, but they are likely to be greatly diluted in the environment. ● Resistance genes and bacteria resistant to sulphonamides and trimethoprim have also been isolated from the sediment under aquaculture farms in the Baltic Sea and persisted in the environment for at least 6 years. Sediments could act as reservoirs of resistance genes and bacteria in local fish farms and in humans via food distribution. The substances used widely in aquaculture are the same as those licensed for therapy of infectious diseased in humans and livestock. ● Risk factors for the emergence of AMR in agriculture at national and international level: – Legislative framework and governance; – Financial status and stability; – Degree of international imports and exports; – Human resources: population size, education and expertise; – Culture; – Structure and organization of the various agricultural systems in use nationally. ● Aquaculture is a fast-expanding agricultural sector in many LMICs, and the unregulated use of antimicrobials in many of these countries poses serious risks of AMR developing and

spreading at local and global level- the latter through international trade. A significant amount of water from shrimp farms is shipped along with shrimp in frozen blocks, transported from farms directly to international consumers. This water can contain antimicrobial residues and

AMR bacteria. This may then come in contact with kitchen surfaces, other food stuffs, and consumers themselves, enabling the global spread of bacteria and resistance genes. ● Both pathogenic and non-pathogenic resistant bacteria can be transmitted from livestock to humans via food consumption, or via direct contact with animals or their waste in the environment. Any mechanism that helps spread bacteria has the potential to spread resistant bacteria. Resistance may also be conferred by the exchange of genetic elements between bacteria of the same or different strains of species, and such transfer can occur in the environment where resistant bacteria have the opportunity to mix with a susceptible bacterial population, such as in the human or animal gut, in slurry spread on agricultural soil, or in aquatic environments.

A.4 Epidemic of carbapenem-resistant Klebsiella pneumoniae in Europe is driven by nosocomial spread (David, et al., 2019)

● A European wide study (244 hospitals involved across Europe) has shown that antimicrobial resistant bacteria are spreading in hospitals. ● Klebsiella pneumoniae is resistant to drugs called carbapenems which are antibiotics used only when the infection cannot be treated by anything else. ● Deaths caused by carbapenem-resistant K.pneumoniae have increased six-fold since 2007. ● The research emphasises the importance of infection control and ongoing surveillance of antibiotic-resistant bacteria to ensure that new strains are detected early.

A.5 Antimicrobial resistance in the Gul Cooperation Council region: a proposed framework to assess threats, impacts and mitigation measures associated with AMR in the marine and aquatic environment (Le Quesne, et al., 2018)

● The WHO considers antimicrobial resistance as one of the most pressing global issues which poses a fundamental threat to human health, development and security (WHO, 2016). ● Microbial resistance to antibiotics spans all known classes of natural and synthetic agents (D’Costa et al., 2006) and drug resistant infections are rising with recent estimates suggesting that up to 50,000 lives are lost each year to antibiotic resistant infections in

Europe and the US alone (O’Neill, 2016). ● The journal suggests that genetic diversity and abundance of AMR in non-clinical settings has been underestimated and that the environment plays in integral role in enabling the development of AMR. ● Due to specific demographic and environmental factors, the Gulf Cooperation Council region may be particularly susceptible to the threat of AMR, with marine and aquatic environment potentially playing a specific role in its development and propagation. ● Demographic factors include: – Rapid population growth; – Significant international population movements; – Heavy antibiotic use; and – Insufficient antibiotic stewardship. ● Environmental factors include: – Notable outputs of untreated sewage effluent; – High ambient water temperatures; – Elevated concentrations of heavy metals; and – Poorly regulated use of antimicrobials in veterinary settings.

A.6 Review of Antimicrobial Resistance in the Environment and its Relevance to Environmental Regulators (Singer, Shaw, Rhodes, & Alwyn, 2016)

● Low levels of antibiotics, metals, biocides can directly select (and co-select) for ARGs within the AMR pathways. ● Sub-lethal effects of antibiotics, metals and biocides on organisms, their microbiome and ecosystem services (e.g. rivers, coastal waters and sol) can impact the health, yield, and safety of economically important food products and wider biome. ● There are three well-characterised classes of resistance driving chemicals: – Antimicrobials (four subclasses: antibiotics, antifungals, antivirals, and antiparasitics); – Heavy metals; – Biocides (i.e. disinfectants and surfactants). ● However, there are many other chemicals, natural (e.g. plant derived) and xenobiotic which are also known to select for resistance. ● Drivers of resistance: antibiotics used in humans and in animals. According to The State of the World’s Antibiotics 2015, two thirds of all the antibiotics produced globally each year (65,000 of 100,000 tonnes) are used in animal husbandry. ● Pathways for antibiotics: – Municipal and industrial wastewater: Antibiotics excreted by humans will enter wastewater treatment plants with one of three fates: ○ Biodegradation; ○ Absorption to sewage sludge; or ○ Exit in the sewage sludge unchanged. – Persistence of an antibiotic in a WWTP is a function of: ○ Influent composition; ○ Salinity; ○ Temperature; ○ Nature of WWTP (e.g. trickling bed, activated sludge, membrane bioreactor); ○ Hydraulic retention time. – Greywater, reclaimed and black water – Veterinary and livestock: when animals consume antibiotics, as much as 30 to 90% is released into the manure and urine. Animal excreta has been shown to contaminate the environment with antibiotic resistance bacteria and antibiotics. The transmission of antibiotic resistant bacteria and genes from animals to humans has been demonstrated in the literature (Smith et al. 2013). A recent review of the academic literature that address the issue of antibiotic use in agriculture suggests that only seven studies (five percent) argued that there was no link between antibiotic consumption in animals and resistance in humans, while 100 (72%) found evidence of a link. – Land application of manure and sludge: An estimated 37% of biosolids are land applied in Europe. ● Drivers of resistance- Biocides: Biocides are disinfectants that are commonly used in hospitals, cosmetics, household cleaning products, wipes and furniture preservatives, farmyards for purposes such as wheel and foot washes, and a range of industrial processes, including the control of fouling and scouring of pipes including oil wells. In much the same way that sub-lethal concentrations of antibiotics can select for antimicrobial resistant genes,

sub-lethal concentrations of biocides have also been shown to select for common mutations that confer clinically relevant antibiotic resistance. ● Pathways for Biocides: Similar to antibiotics, most notably WWTPs. ● Drivers of resistance: Metals. Major urban inputs of heavy metals to WWTPs come from household effluent, drainage water, business effluent (e.g. car washes, dental uses), atmospheric deposition, and traffic related emissions. Metals such as Pb, Cu, Zn, Cd, and As have been used as animal growth promoters and nutritional supplements, pesticides, and fungicides in aquaculture and agriculture. The relationship between metal bioavailability, speciation and resistance gene selection is largely unexplored. Bacteria carrying metal resistance genes have been shown to more frequently carry ARGs as compared to those bacteria without metal resistance genes, and these genes can often be found on plasmids. ● Pathways for metals: similar to that of biocides and antimicrobials. Elevated concentrations of metals will be found in urban areas and areas that have experienced mining. ● Drivers of resistance: antibiotic resistance genes. The co-location of antibiotics and ARGs in

WWTPs can (and does) select for novel combinations of AMR that can be shared between microorganisms by horizontal gene transfer (HGT). The competitive and chemically challenging environment of a sewage works offers favourable conditions for the amplification of existing resistance genes. ● WWTP Discharge: Introduction of pollutants (antibiotics, ARGs, biocides and metals) interacts with the native fauna and flora and begin to change the microbial community structure and genetic make up. These changes in the microbial community have been shown to have significant impacts on the aboveground diversity and functioning of terrestrial ecosystems. Polluting into recreational coastal and bathing waters, through combined sewer overflows, will elevate exposure to humans and by extension all wildlife that inhabits and feeds off/within the impacted waters systems. However, little is known about the chronic effects from chemical exposure or the elevated prevalence of ARGs within a food web. ● Land spreading of manure and biosolids: the dissemination of manure and biosolids onto crops and soils increases the ARG exposure risk to: – Animals (wild); – Crops; – Adjacent surface water bodies; – Groundwater; – Farm workers; – Air as dust particles from land spreading or aeolian erosion. ● A recent study demonstrated that land spreading of composted sludge on a field will likely lead to the spread of ARGs in the soil and wider environment (Su et al., 2015). Persistence in soil varies greatly in literature between a few days and 300 days. Persistence increases at low temperatures, unexposed to light, and high organic conditions. The fate will also be sensitive to pH and soil properties. ● Air transmission: several antibiotics have been recorded downwind of feedlots at concentrations similar to that found in rivers downstream of sewage outlets. ● Food plants: a significant number of papers exist in the literature demonstrating the sensitivity and uptake of antibiotics from irrigation, manure or sludge amended soils by crops into the plant biomass. The degree to which this occurs and the risk that it poses to the environment remain poorly studied. ● Aquaculture and shellfish beds: the accumulation and chronic exposure of river, estuarine and coastal environments to antibiotics, biocides and metals can persist and spread AMR

into and from the sediment. The use and misuse of antibiotics in aquaculture has led to an increase in antibiotic resistance in fish pathogens. The implications for the spread of AMR throughout the food web from fish-eating organisms has not been well studied and is a significant knowledge gap. ● Groundwater quality: antibiotics in manure or sludge amended agricultural soils will enter groundwater as a result of rainfall, irrigation and other human activities. Very little has been reported regarding the impact of antibiotic residues in groundwater on the generation of AMR in pathogens.

A.7 Bacterial diversity and antibiotic resistance in water habitats: searching the link with the human microbiome (Vaz-Moreira, Nunes, & Manaia, 2014)

● Only a few bacteria found in waters were, so far, identified in the human-associated microbiome. It is still uncertain in which cases the same species and strain can live in water and colonize humans. In such case, those bacteria may be involved in the direct or indirect transfer of properties, including antibiotic resistance. ● Water habitats host an impressive bacterial diversity. However, only a few lineages are known to harbour antibiotic resistance genes of already recognised clinical relevance.

A.8 The role of the natural environment in the emergence of antibiotic resistance in gram-negative bacteria (Wellington, et al., 2013)

● During the past 10 years multidrug-resistant Gram-negative Enterobacteriaceae have become a substantial challenge to infection control. It has been suggested by clinicians that the effectiveness of antibiotics is in such rapid decline that, depending on the pathogen concerned, their future utility can be measured in decades or even years. ● Unless the rise in antibiotic resistance can be reversed, we can expect to see a substantial rise in incurable infection and fatality in both developed and developing regions. ● The Dangerous Substances Directive 76/464/EEC lists 129 substances that are regarded as so toxic, persistent, or bioaccumulative that efforts to control their release and prevent pollution should be given the highest priority. However, because antibiotics are not listed and are therefore not routinely tested for, their high prevalence in the environment has received little attention. Many antibiotics are not inherently biodegradable, and some synthetic antibiotics can persist in soils for long periods of time at high concentrations. Some substances (e.g. tetracyclines and fluoroquinolones) also persist in the environment for months to years. ● Wildlife as reservoirs of antibiotic resistant genes: There is good evidence that proximity to human populations, rather than direct antibiotic use on land, is sufficient to substantially affect the gut flora of local wildlife. A study comparing levels of resistance in E. coli recovered from animals with varying amounts of contact with people, from wild Atlantic salmon to dogs, support this notion. However, this is different for wild birds, where ecological factors such as migratory behaviour and high population densities increase the likelihood of the presence of clinically relevant resistance genes carried by birds even in areas of low anthropogenic effect. ● Social issues driving antibiotic resistance: Social interventions are essential to reduce antibiotic misuse within the health-care industry and the home. Campaigns aiming to raise awareness and improve antibiotic prescriptions have tended to focus on high-income countries. Various social factors can impede large-scale reductions in antibiotic prescriptions, such as an increasing capacity to afford health care, rising health care expectations, the number of vulnerable individuals who experience repeated infections, and poor professional attitudes. A rapid increase in internet access has resulted in a corresponding increase in the unregulated purchasing of antibiotics, accompanied by lowquality patient care and increased risk of environmental contaminations through unregulated disposal. Public use or misuse of antibiotics is caused by several social factors, including increased incidence of self-medication, ethnic origin, country of residence, income and education level. ● The potential threat posed by the continued evolution of ARGs seems sufficiently grave and imminent that reliance upon stakeholder behavioural change should be considered a highrisk strategy. ● The absence of full environmental fate and effect data on antibiotics inhibits an effective assessment of the potential risk through environmental pathways. ● There is now sufficient evidence to support the hypothesis that one of the most important emerging public health threats is that of large-scale dissemination of multi-resistant pathogens in the hospital environment, the community, and the wider environment. Rapid demographic, environmental, and agricultural changes are all contributing to a global antibiotic resistance crisis, which, if not stopped, will emerge as one of the major causes of death in the coming decades.

A.9 Human health risk assessment of antibiotic resistance associated with antibiotic residues in the environment: a review (Ben, et al., 2019)

● The extensive use of antibiotics leading to the rapid spread of antibiotic resistance poses high health risks to humans, but to date there is still lack of a quantitative model to properly assess the risks. ● Concerns over the health risk of antibiotic residues in the environment are mainly: – The potential hazard of ingested antibiotic residues in the environment altering the human microbiome and promoting emergence and selection for bacteria resistance inhabiting the human body; and – The potential hazard of creating a selection pressure on environmental microbiome and leading to reservoirs of antibiotic resistance in the environment. ● Although the half-life of most antibiotics is not long (hours to hundred days), antibiotic residues that remain in the environment can be considered as a “persistent” organic contaminant due to frequent and extensive use of antibiotics and uninterrupted emissions. ● Antibiotic resistant genes are becoming recognised as an emerging environmental pollutant. ● The paper examines and summarised the available data and information on the four core elements of antibiotic resistance associated with antibiotic residues in the environment: – Hazard identification; – Exposure assessment; – Dose-response assessment; and – Risk characterisation. ● Hazard identification: antibiotic residues in the environment could accelerate the emergency and evolution of antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARG) in the environment. The risks refer to the transmission of environmental ARB and ARGs to humans. ● Exposure assessment: exposure through surface water, wastewater, drinking water, potentially air and dust, soil, food products (meat, eggs, milk, vegetables and grains, fish and shrimp). ● Dose-response: the relationship between the antibiotic concentration and the probability of emergence of antibiotic resistance. ● Risk characterisation: how dangerous are the adverse effects of human exposure to antibiotic resistance associated with environmental antibiotic residues?