2 Finding of Literature Review

2.1 Introduction

Antimicrobial resistance (AMR) is when microbes (bacteria and fungi) develop the ability to resist the drugs that are designed to combat them, causing the standard treatments to become ineffective (UK Science and Innovation Network, 2018). Once resistant microbes are in the environment, there is potential to spread, colonise, or cause infections in people and animals. If they retain their activity in the environment, they can apply selective pressures on the microbial population and amplify resistant bacteria (UK Science and Innovation Network, 2018).

The co-location of antibiotics and antibiotic resistant genes (ARGs) in waste water treatment plants (WWTPs) can (and does) select for novel combinations of AMR that can be shared between microorganisms by horizontal gene transfer (HGT).

Recent estimates indicate that drug resistant infections are increasing, with approximately 50,000 lives lost each year to antibiotic resistant infections in Europe and the United States (O'Neill, 2019). A European wide study, of 244 hospitals, has shown that AMR bacteria are spreading in hospitals, with deaths caused by carbapenem-resistant K. pneumoniae having increased six-fold since 2007. This literature review is to determine the key drivers, sources, pathways and receptors of AMR within the environment.

2.2 Drivers

There are four main drivers of AMR:

● Antimicrobials and their residues (of which there are four subclasses: antibiotics, antifungals, antivirals and antiparasitics); ● Metals; ● Biocides (e.g. surfactants and disinfectants); and ● Antibiotic resistant genes (Singer, Shaw, Rhodes, & Alwyn, 2016) Although there are many other chemicals, natural and xenobiotic, which are also known to select for AMR, these will not be discussed here. Similarly, the antimicrobial focus will be on impacts of antibiotics.

2.2.1 Antibiotics

Antibiotics are designed to inhibit growth or destroy microorganisms. However, where concentrations are too low to be fatal, this allows for the bacteria to select for resistance (UNEP, 2017). Overuse and improper use of antimicrobials are recognised as key drivers of the emergence and spread of AMR (Food and Agriculture Organization of the United Nations, 2016). Once in the environment, antibiotics can last between a few hours and 300 days (Taso & Cho, 2016). As a result, antibiotic residues that remain within the environment as a result of extensive use of antibiotics and consistent emissions can be considered as a persistent organic contaminant (Ben, et al., 2019).

2.2.2 Metals

The presence of heavy metals has been associated with the reduction of susceptibility of bacterial populations bacteria to antimicrobials and so heavy metals are associated with the emergence and spread of AMR. (Food and Agriculture Organization of the United Nations, 2016)

2.2.3 Biocides

Biocides are commonly used in hospitals, cosmetics, household cleaning products, farmyards and for industrial purposes. These behave in a similar way to antibiotics, in that sub-lethal doses can allow microorganisms to select for antimicrobial resistant genes (Singer, Shaw, Rhodes, & Alwyn, 2016)

2.2.4 Antibiotic resistant genes

When antibiotics and antibiotic resistant genes (ARGs) are in contact, bacteria can select for novel combinations of ARG (Singer, Shaw, Rhodes, & Alwyn, 2016). These bacteria are then able to “share” combinations of AMR through a process known as horizontal gene transfer (UNEP, 2017).

2.3 Sources

There are several sources of these AMR drivers within the environment. These are namely: human and animal waste, aquaculture, crop pesticides, and antimicrobial manufacturing waste.

2.3.1 Human Waste

Human waste can carry AMR pathogens and up to 80% of consumed antibiotics are excreted through urine and faeces (UNEP, 2017). If this waste enters waste water treatment, it will have one of three fates:

○ Biodegradation; ○ Absorption to sewage sludge; or ○ Exit in the sewage sludge unchanged (Singer, Shaw, Rhodes, & Alwyn, 2016). However, in many countries around the world, a high percentage of waste is not treated and is instead discharged directly into the environment. In Dhaka, Bangladesh, 70% of waste water is untreated (UK Science and Innovation Network, 2018). In high-income countries, untreated wastewater may still inadvertently enter the environment directly through combined sewage overflows. Further to this, wastewater treatment plants may not be sufficient where there are high levels of bacteria present, such as waste water from healthcare facilities (UK Science and Innovation Network, 2018). A European study found trace levels of antimicrobials and evidence of resistant bacteria within treated sewage sludge (Wellington, et al., 2013).

2.3.2 Animal Waste

According to The State of the World’s Antibiotics 2015, two-thirds (65,000 tonnes) of all antibiotics produced each year are used in animal husbandry (Gelband, et al., 2015). Antibiotics are increasingly being used nontherapeutically or to encourage growth (Food and Agriculture Organization of the United Nations, 2016). Antibiotics are widely used in non-European countries without veterinary supervision due to them being readily available over the counter at a low cost (Laxminarayan, et al., 2013). The demand and use of antibiotics in animal husbandry is likely to increase in low- and middle- income countries as demand for animal protein increases.

Similar to human waste, a large percent of antibiotics within animals is excreted through faeces and urine (30 to 90% (Berensden, Wegh, Memelink, Zuidema, & Stolker, 2015).

The transmission of antibiotic resistant genes from animals to humans is not well understood. However, it has been demonstrated as probable within 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 (Singer, Shaw, Rhodes, & Alwyn, 2016).

2.3.3 Aquaculture

Antimicrobials are used worldwide within aquaculture, but the quantities and types are not known (UK Science and Innovation Network, 2018). It is estimated that up to 75% of antibiotics used in aquaculture may be lost to the environment (UNEP, 2017). Nguyen Dang Giang et al (2015) 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.

2.3.4 Crop pesticides

Antimicrobials are widely used as pesticides for crop disease management. These are similar to antimicrobials used in human medicine. Using these has the potential to select for resistant microbes in the environment (UK Science and Innovation Network, 2018).

2.3.5 Antimicrobial manufacturing waste

Manufacturers of antimicrobials may release antimicrobials and their residues into the environment, where there are no effective control measures in place. There are no international standards for wastewater limits for antimicrobials (UK Science and Innovation Network, 2018). The Dangerous Substances Directive 76/464/EEC lists substances that are so toxic, persistent, or bioaccumulative that efforts are required to prevent their release into the environment. However, antimicrobials are not listed here and so are not routinely tested for (Wellington, et al., 2013).

2.4 Pathways

Selected pathways through which AMR can transfer within the environment are discussed here.

2.4.1 Municipal Waste water

As discussed in 2.3.1., municipal waste water can act as a pathway for AMR and antibiotic residues. This will ultimately discharge into surface watercourses, coastal waters and potentially groundwater.

2.4.2 Manure and sewage sludge applied to land

As discussed in 2.3.1 and 2.3.2, manure and sewage sludge can contain AMR bacteria and antibiotic residues that will facilitate AMR. When applied to land this can contaminate soils, groundwater and surface waters through runoff. A recent study demonstrated that land spreading of composed sludge on a field will likely lead to the spread of antimicrobial resistance genes in the soil and wider environment (Su, et al., 2015).

2.4.3 Aquaculture contaminated waters

The unregulated use of antimicrobials in aquaculture, a fast-expanding sector in low- and middle-income countries (Food and Agriculture Organization of the United Nations, 2016), poses a serious risk of AMR developing and spreading at a local and global level. The extent and persistence of antimicrobial residues in aquaculture is currently unknown (Food and Agriculture Organization of the United Nations, 2016). It is becoming common for shrimps to be transported around the world in frozen blocks. This water may contain antimicrobial residues and AMR bacteria which can then contaminate kitchens, foods and consumers (Food and Agriculture Organization of the United Nations, 2016).

2.4.4 Crop spraying

Spraying of antimicrobials onto crops could potentially cause AMR and selection of resistant microbes. The extent of this pathway and how it interacts within the environment is not well studied.

2.4.5 Airborne particles/ bioaerosols

There is potential for airborne particles of antimicrobial residues to result from land spreading of contaminated manure or sewage sludge. Several studies have recorded antibiotics downwind of feedlots at concentrations similar to that found in rivers downstream of sewage outlets (Singer, Shaw, Rhodes, & Alwyn, 2016).

Receptors

Concerns regarding AMR are in regard to human health and so, humans are the ultimate receptor. However, there are other receptors within the environment that will facilitate or encourage AMR before ultimately reaching the human receptor:

2.4.6 Groundwater

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 (Singer, Shaw, Rhodes, & Alwyn, 2016). Groundwater may then be used for irrigiation, drinking water and act as a pathway to surface waters and coastal waters.

2.4.7 Surface waters

These include rivers, lakes and coastal waters. Studies have found detectable levels of resistant bacteria in surface waters (UK Science and Innovation Network, 2018). Furthermore, it has been suggested that sediments within surface and coastal waters could act as reservoirs of resistance genes and bacteria (Food and Agriculture Organization of the United Nations, 2016). Contamination of coastal waters is particularly concerning where there are shellfish beds; these are filter feeders and so there are greater chances that these will act as reservoirs for AMR or antibiotic residues. Surface water may then be used for irrigiation, drinking water and act as a pathway to groundwater and coastal waters.

2.4.8 Food Products

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 (Food and Agriculture Organization of the United Nations, 2016).

2.4.9 Ecosystems

Ecosystems (marine and terrestrial) and the flora and fauna within them are considered to be receptors and many are protected by environmental law. Literature reviews have documented ecologically relevant effects on a range of target organisms, such as pollution-induced autoimmunity, from exposure to sub-lethal concentrations of pollutants, including antibiotics. Bactericidal antibiotics can stimulate bacteria to produce reactive oxygen species (ROS), which are highly deleterious molecules that can interfere with the normal functions of oxygen-respiring organisms (Kohanski et al., 2010). ROS are mutagens, which can result in a DNA damage and repair cascade by low levels of antibiotics leading to an increase in mutation rates, which can result in the emergence of multidrug resistance (Kohanski et al., 2010).

In the case of plants, there is evidence to suggest that seed germination, root elongation and overall plant health can be sensitive to sub-inhibitory concentrations of antibiotics and metals in the soil (Pan and Chu, 2016). Exposure to sub-lethal concentrations of pollutants such as antibiotics, biocides, and metals can

induce pollution-induced parasitization, which in turn has been shown to increase susceptibility to the toxic effects of the pollutant (Khan and Thulin,1991).

Sediments, although considered an environmental reservoir for AMR, are not considered as a receptor separately for the purposes of this study.