Library
This page organizes various scientific literature and background research regarding antibiotic resistant bacteria (ARBs), the pour-plate method versus the novel Idexx method, how antibiotic pollution occurs, Turbidity and TSS applictaion, as well as, remote sensing.
UCLA IoES Practicum
The Institute of the Environment and Sustainability’s practicum is an annual project that offers seniors the ability to produce tangible scientific work. As part of UCLA’s Environmental Science Major, this specific practicum is a project that will be recurring and focused on researching the effects of antibiotic pollution leading to antibiotic resitant bacteria, as well as, other parameters monitoring California's coastal water quality using remote sensing and in situ data.
Antibiotic Resistant Bacteria
The persistence of Antibiotic-Resistant Genes (ARGs) as they relate to Antibiotic-Resistant Bacteria (ARBs) in the environment is not only misunderstood but very difficult to trace within the environment because of lack of standardization in detection techniques and difficulty quantifying the amount of exposure to ARGs that cause harm tohuman health. The major sources of ARB are a result of overprescription of antibiotics, the use of antimicrobial products in hospitals, and the mass use of broad-spectrum antibiotics in livestock management. The key focus of our research will be understanding how the use of broad-spectrum antibiotics used to both promote growth and prevent disease in livestock creates an issue of ARG persistence in the environment. The effects of the use of antibiotics in agriculture and overprescription has led to 700,000 deaths annually and creates a major global health crisis without much knowledge by the public (Li 1). Being able to identify the locations of these major point sources of ARG and ARB proliferation in agriculture will help us understand how they can persist in runoff and eventually help eliminate the concentration of ARGs in our coastal ecosystems.
Antibiotics encompass a wide range of chemical compounds that can be produced naturally, semi-synthetically, and synthetically (Manyi-Loh et al., 2018). In humans and animals, antibiotics are used to fight bacterial infections by either killing bacteria or stopping bacterial growth (CDC, 2020). However, like us, bacteria are constantly evolving and passing on new genetic material that makes them increasingly resistant to the antibiotics designed to inhibit them. In 1942, the first antibiotic-resistant bacteria (ARB), Staphylococcus aureus, was discovered, and the race between effective antibiotics and bacterial resistance has not stopped since (CDC, 2020). The creation of and selection for ARB is caused by the consistent, frequent, and, in some cases, improper use of antibiotics (Le et al., 2018). Every time an antibiotic is used, there is an opportunity for bacteria to become resistant (Wu et al., 2019). When there is exposure to an antibiotic, microbial communities share resistant genes across genera to positively select for new resistant strains, even at heightened metabolic cost (Griffin et al., 2020). Bacteria can become resistant to antibiotics by either intracellular mutation or by acquiring mobilized antibiotic-resistant genes (ARG) from other bacteria (Wu et al., 2019). ARGs are found in plasmids, which serve as mobile carriers for genetic information. Horizontal transfer of plasmids allows bacteria to adapt to antibiotics and acquire these ARGs at an alarmingly quick rate in the human body (Stalder et al., 2019). This process can alter the human microbiome and cause health disturbances. The human microbiome is a complex ecosystem consisting of bacteria, viruses, archaea, or eukaryotes that co-evolve together from various selective pressures like antibiotics, diet, and lifestyle (Baron et al., 2018). Bacteria naturally compete against each other and acquire ARGs as a survival mechanism (Rolain et al., 2016).
How do Antibiotics Enter the Environment?
Antibiotics can enter the environment from a variety of places including industries, people, and farming. The manufacturing of antibiotics creates wastewater from the factories which is discharged into the environment via streams, lagoons, and holding ponds. Even if this water is treated to try to remove the pollutants, the concentration of antibiotics can be magnitudes higher than in waters where accidental exposure occurs (Larsson, 2014). This is because wastewater treatment plants are not designed to treat specialized micropollutants such as antibiotics (Pruden, 2006). As a result, both discharge locations from antibiotic manufacturing as well as wastewater treatment plants are locations where antibiotics may enter the environment. People are also a source of antibiotics entering the environment. An increasing number of people are demanding antibiotic treatments for illnesses, even if a bacterial infection is not the cause of infection. Each year, 30% of outpatient, oral antibiotic prescriptions in the U.S, totaling 34 million prescriptions, are written unnecessarily (Fleming-Dutra, 2016). A common issue is when people do not complete the full course of their antibiotics treatments and dispose of the antibiotics improperly (Larsson, 2014). Additionally, up to 95% of antibiotics can be excreted from humans in an unaltered state (Pruden, 2006). Combined with wastewater treatment plants’ limited abilities to filter out antibiotics, this means an increasing number of antibiotics are being released into the environment unaltered, allowing for bacteria to develop resistance to them. Over 20.5 million lbs of antibiotics were sold in the U.S in 2018, and 65% were used for animal farming (Wallinga, 2020). Similarly to people, 95% of antibiotics can be excreted from animals in an unaltered state (Pruden, 2006). This means that manures from animal farming can carry high levels of antibiotics if the animals had received antibiotics. When manures are used for crop farming, these antibiotics are transferred to the crops. This means antibiotics are being transferred from animals to crops and from crops to people via the food system (Finley, 2013).Along each stage, bacteria are exposed to antibiotics and able to build resistance. Once in the environment, antibiotics, like many other pollutants, can move. For example, discharge sites have been shown to contaminate groundwater in some cases, leading to contamination in drinking water as well (Larsson, 2014). Tracking the movement of antibiotics in the environment can give insight into where ARB may be located.
How do ARBs and ARGs Move Through the Environment?
ARB populations develop via the natural selection process described above. Therefore, when a group of bacteria is exposed to antibiotics, a population of ARB may be established in that area of exposure. This is especially true for prolonged antibiotic exposure. Unlike other pollutants, ARB are often enriched after treatment because the treatment will only kill the bacteria without resistance, leaving the “strongest” bacteria to survive and reproduce (Pruden, 2006). Tracking antibiotic movement in the environment, several potential ‘hot spots’ for the enrichment and transmission of resistant bacteria” can be identified (Finley, 2013, p. 705). These locations would be ones in which bacteria can spread, and are exposed to antibiotics to build resistance. It includes locations talked about above such as industrial discharge sites, farms, and communities (European Union, 2014).
While ARB are an important piece of the puzzle, the real interest for human health is the pathway of ARGs through the environment. This is because while ARGs often follow the pathway of ARB and the pathway of antibiotics in the environment, they often persist longer in the pathway (Pruden, 2006). ARGs can move through the environment in 3 ways: vertical gene transfer, horizontal gene transfer, and dissemination. Vertical gene transfer refers to the spread of genes from parent to offspring. Bacteria often reproduce by a parent bacteria duplicating itself. Therefore, if the parent cell has ARGs, the daughter cell does too. Horizontal gene transfer is how bacteria cells share DNA with other bacteria cells. There are three types of horizontal gene transfer: conjugation, transduction, and transformation. Conjugation refers to the process of a donor bacteria cell forming a bridge with a receiver bacteria cell. DNA can then be transferred through the bridge from the donor to the receiver bacteria (Burmeister, 2015). If the DNA transferred contained ARGs, the receiver cell can reproduce the ARGs. Transduction refers to the process of a phage (virus) infecting a bacteria cell (Burmeister, 2015). Phages will insert their DNA into a bacteria cell, using the bacterial organs to make copies of the viral DNA. Once copies of the viral DNA have been made, they are packaged into other viruses. However, sometimes host bacteria DNA is packaged with viral DNA. Therefore, when the viruses are released from the bacteria host to infect other bacteria, the host bacteria DNA is also spread. If the host bacteria contained ARGs, these ARGs can be spread through viral infections. Transformation refers to the uptake of DNA from the environment (Burmeister, 2015). This process is especially important in tracking antibiotic resistance because “even if cells carrying ARGs have been killed, DNA released to the environment has been observed to persist” (Pruden, 2006, p.7445). Researchers have found an increasing number of ARGs in soils since the 1940s, which protected them from being broken down (Wright, 2010). This is important because even if a population of ARB is eliminated, the resistant gene could surface again at a later time, although unlikely to be in a pathogen (Palme, 2018). It also means hydrology serves as a major pathway forARG transfer. Dissemination refers to the spread of ARGs, often through the spread of ARB. ARB hitchhike “ from humans to animals, and vice versa, often through various environmental pathways, including foodstuffs, animal wastes, and water sources” (Finley, 2013, p.708). As the bacteria move around, so do the genes they carry. Therefore, stopping the spread of ARB is an important step in slowing the spread of ARGs. However, even after the bacteria dies,their DNA and the ARGs they carry can persist through horizontal gene transfer. ARGs in water treatment plant filters have led to ARGs in drinking water through dissemination (Larsson, 2014).
Antibiotics encompass a wide range of chemical compounds that can be produced naturally, semi-synthetically, and synthetically (Manyi-Loh et al., 2018). They are used to stop bacterial growth (“bacteriostatic”) or kill bacterial growth (“bactericidal”) with minimal damage to the host receiving them (Milic et al., 2013; Martínez, 2012; Gillings, 2013). They are categorized as either bacteriostatic or bactericidal, as well as by their scope in the pathogens they target as either narrow or broad-spectrum. These drugs are not only used in clinical settings, but they are also regularly used in livestock farming to treat and prevent animal diseases, promote growth, and improve feed-to-weight ratios (You & Silbergeld, 2014; Hong et al., 2013; Hao et al., 2014). Increasing consumer demand for animal protein worldwide is instigating more intensive farming methods (Manyi-Log et al., 2018). Antibiotics given to livestock is resulting in consequent antibiotic residues in animal-derived products and waste streams. Globally, the main classes of antibiotics widely used in agriculture include tetracyclines, aminoglycosides, β-lactams, lincosamides, macrolides, pleuromutilins, and sulphonamides (Finley et al., 2013; De Briyne et al., 2014; Baynes et al., 2016). The overall quantity of antibiotics used in agriculture is estimated to fall between 63,000 and 240,000 tons. In fact, about 80% of antibiotics sold in the U.S. are used for animal production alone (Ventola, 2015). Contrastingly, Sweden is an example of a country that has banned antibiotics for animal growth purposes. According to Swedish agricultural data, there were no recorded loss of production after they implemented this ban (Cogliani et al, 2011). This example shows that such policies have the potential to be effective since reduction of antibiotic use was not followed by pervasive animal disease and successfully reduced antibiotic resistance buildup (Wierup, 2001).
Fecal indicator bacteria and ESBL - E. coli
When conducting surveillance of antibiotic resistance in the environment, fecal indicator bacteria (FIB) are often focused on because of their pathogenic potential and ability to acquire resistance easily (Reinthaler et al., 2003; Collignon, 2009). FIB are bacteria that live in the gut of warm-blooded animals and are introduced into the environment through fecal matter (NOAA GLERL, n.d.). While FIB are generally harmless, they indicate the possible presence of other pathogenic bacteria, viruses, and protozoans found in fecal matter (US EPA, n.d.c). Since testing for the presence of many different pathogens is expensive and difficult, water systems are typically tested for a few subgroups of bacteria instead. The most commonly tested FIB include total coliforms, fecal coliforms, Escherichia coli (E. coli), fecal streptococci, and enterococci. Total coliforms are a widespread group of bacteria that can occur in human feces, but also other places outside the human body. For drinking water, total coliforms are still a standard indicator test because their presence indicates contamination of a water supply by an outside source. Fecal coliforms are a subset of total coliform bacteria, which are more fecal-specific in origin. For recreational waters, this group was used as the primary bacteria indicator until relatively recently when the EPA began recommending E. coli and enterococci as better indicators of health risk from a water system. E. coli is a species of fecal coliform bacteria that is considered the best indicator bacteria to use for recreational water quality monitoring because of its specificity, ease of detection, and how well studied it has been historically (McLain et al., 2016; Wuijts et al., 2017; Vikesland et al., 2017). In the past, ratios of fecal streptococci to fecal coliforms were monitored to determine whether contamination was of human or nonhuman origin. This is no longer recommended as a reliable bacterial source tracking test. Lastly, enterococci are a subgroup of fecal streptococci and are distinguished by their ability to survive in salt water conditions. Because of this, the EPA now recommends enterococci be the indicator of health risk in salt water used for recreation. Since these groups of bacteria commonly reside in the intestines of warm-blooded animals, they are subjected to frequent exposure to antibiotics consumed by their host which creates high selection pressure (Khadori, 2012). As an example, E. coli can be used to measure antibiotic resistance by looking at extended-spectrum β-lactamase producing E. coli (ESBL-E. coli). ESBLs are plasmid-mediated enzymes produced by E. coli that inhibit β-lactam antibiotics (e.g. penicillins, cephalosporins, carbapenems) (Fuentes et al., 2019; Shanthi & Sekar, 2010). These enzymes also show co-resistance to many other classes of antibiotics. Infections caused by ESBLs can range from UTIs to life-threatening sepsis (Rawat & Nair, 2010). Antibiotic-resistant infections result in longer hospitalization times and require intake of stronger and more expensive medicines, resulting in higher medical costs and patient complications. With the spread of antibiotic resistance accelerating across the globe, monitoring bacteria like ESBL-E. coli is critical.
Indicators of Water Quality
In addition to monitoring bacterial content and antibiotic resistance in water systems, water quality measurements also encompass a large variety of chemical, physical, and biological properties. For aquatic ecosystems, there are several widely accepted indicators that are used by most monitoring agencies. These key indicators can include (but are not limited to) temperature, pH, conductivity, dissolved oxygen content, turbidity, total suspended solids (TSS), light absorbance, and nutrient levels.
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The pH of a liquid is the measure of free hydrogen and hydroxyl ions in the water and determines the solubility of chemical constituents (USGS Water Science School, 2019). Since the pH controls the availability of chemicals used as nutrients by living things, there is a pH range for organisms where they exhibit optimum growth. Since the Industrial Revolution, the buildup of CO2 in the atmosphere has contributed to ocean acidification (NOAA, n.d.). As a result, the average pH of the ocean has fallen from approximately 8.2 to 8.1, roughly representing a 30% increase in acidity (NOAA, n.d.).
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Conductivity is a measure of the ability of a liquid or material to carry an electrical current. Salts and other inorganic compounds carry electrical currents, and knowing the conductivity of a sample provides insight on its chemical makeup (US EPA, 2013a).
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The dissolved oxygen percentage in water measures how much oxygen is available for use by aquatic life and is directly linked to the presence of inorganic compounds and temperature (USGS Water Science School, 2018a).
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Turbidity is a measure of water clarity derived from the reflectivity of light that is scattered by material in the water (USGS Water Science School, 2018b). Materials that can cause water to have high turbidity values include clay, silt, inorganic and organic matter, algae, plankton, and other microscopic organisms.
Turbidity's relationship to TSSTurbidity and total suspended solids (TSS) are measurements used for water quality analysis. Turbidity measures the loss of transparency in a liquid, which can result from an increase in the number of suspended solids in a liquid (Hannouche, 2011, p. 2447). Turbidity measurements are a common water quality parameter and have become a standardized process (Hannouche, 2017, p. 789). TSS is a measurement of the number of sediments and organic matter in a liquid. The articles referenced in this paper measure turbidity in both nephelometric turbidity units (NTU) and Formazin Attenuation Units (FAU). TSS is typically measured as a concentration in mg/L (Line, 2013, p. 1415). Since these units are so different it is hard to convert turbidity measurements into TSS and vice versa. Instead, researchers have been looking for trends and patterns between the two variables. Over recent years, there have been many studies analyzing the relationship between turbidity and TSS, and investigating if one could be used to estimate the other and replace data collection.
While turbidity and TSS both relate to the number of suspended solids within a liquid, the uses for each water quality parameter vary and are dependent on the individual goal of the study. First, TSS measurements are a long process, taking several hours and only conducted within a lab. This is particularly challenging during rain events because sometimes it is not as useful to take TSS samples since the water conditions change rapidly. The benefit of TSS analysis is that TSS values are representative of environmental concerns. High TSS values, which typically result from resuspension and discharge of sediments, can block the gills of fish, carry bacteria, and have other detrimental effects on the water. Federal agencies place limits on the TSS concentration in bodies of water, especially close to dredging and disposal locations (Thackston, 2000, p. 1). Contrastingly, turbidity measurements can be taken quickly and provide real-time values. It is much more reflective of the current state of the water and can be measured much more frequently. Unlike TSS, turbidity does not have a strong correlation to environmental impact concerns (Thackston, 2000, p. 9-10). While both turbidity and TSS measure suspended solids in a liquid, neither measures every type. Figure 1 shows the particles that are measured with TSS and turbidity analysis. A large benefit of TSS measurements is that it records settleable solids, which turbidity does not. Since there are much more similarities in the particles measured by turbidity and TSS than differences, research has investigated the correlation between the two variables.
Nutrients commonly measured for water quality include nitrogen species like ammonia and nitrate, and phosphate. Ammonia (NH3) and nitrate (NO3-) are essential to plants but can adversely affect marine ecosystems at high concentrations (Greenberg, 1992; US EPA, n.d.a). Phosphate (PO₄³⁻) is the most common form of phosphorus utilized by marine organisms and is also a chemical required by aquatic life that can be dangerous at high concentrations (US EPA, 2013b). Too much of these forms of nitrogen and phosphorus can lead to eutrophication and an overproliferation of algae. This can ultimately lead to hypoxia, a shortage of dissolved oxygen in the water, which can kill marine organisms in higher trophic levels (Greenberg, 1992).
Light absorbance, a measure that can be taken both remotely via satellite or in a lab using a spectrometer, measures the amount of light and type of wavelength that can pass through a medium. Chemical compounds and organic materials differ in their absorption characteristics because each can absorb different wavelengths of light.
Remote Sensing for Water Quality Monitoring
As mentioned, routine on-site water sampling can be costly and logistically challenging. Fueled by the advancement in satellite imaging capabilities, the use of satellites in monitoring water quality is becoming a popular complementary tool to bolster on-site sampling efforts. Monitoring water quality with remote sensing can provide real-time water quality reports that would otherwise be inaccessible. Landsat and Sentinel satellites are two popular families of satellites that currently carry out remote sensing. Since the beginning of the program in the 1970s, a total of 9 Landsat satellites have been launched by NASA, with Landsat-9 being the newest member of the family as of 2021 (NASA, n.d.). The Sentinel satellites were designed and launched by the European Space Agency (ESA, n.d.b) to support the Copernicus Program–an Earth observation project developed by the European Union (ESA, n.d.a). Both families of satellites conduct remote sensing of Earth’s surface and gather geographical data to support a variety of research in resource management, agriculture, and natural disaster response.
The Sentinel-2 satellite is composed of two identical satellites (Sentinel-2A and Sentinel-2B) that are arranged opposite each other on the orbital (ESA, n.d.b). Similar to the purpose of OLI in Landsat-8, the Multispectral Instrument (MSI) on Sentinel-2 passively collects sunlight reflected from the Earth’s surface for different wavelength bands (ESA, n.d.b). Sentinel-2A and Sentinel-2B have trivial differences in the range of wavelengths detected by their respective bands. Both satellites have 13 different bands with a spatial resolution between 10 and 60 meters (ESA, n.d.b). 20 meters is the most common resolution among all bands (ESA, n.d.b).
One difference between Landsat-8 and Sentinel-2 is that Landsat-8 has additional capability with the installment of the TIRS. Sentinel-2 can only measure short wave infrared (SWIR) as its upper bound for detection ends at 2202.4 nm (ESA, n.d.b). In comparison, the TIRS on Landsat-8 produces two bands spanning between 10.60-11.19 μm and 11.50-12.51 μm (NASA, n.d.). These two additional bands enhance wavelength atmospheric correction and the accuracy of surface temperature and emissivity (Roy et al., 2014). Additionally, Sentinel-2 has a shorter revisit time compared to Landsat-8. Each Sentinel-2 satellite revisits every 10 days and constellation with both Sentinel-2A and Sentinel-2B occurs every 5 days (USNA, 2021). In comparison, Landsat-8 revisits every 16 days (USNA, 2021).
To conduct water quality assessments based on remote-sensing data, scientists and researchers first need to determine the source of the data. While many data sources are available, Landsat-8 and Sentinel-2 are two popular choices because of their global coverage (ESA, n.d.b; NASA, n.d.). Due to the satellites’ multispectral sensing being greatly compromised due to weather events, the raw band data has to be processed before use (Schulte to Bühne & Pettorelli, 2018). The raw Landsat-8 and Sentinel-2 data usually go through radiometric and atmospheric correction before being used to calculate water quality metrics (Bonansea et al., 2019; Gonzales et al., 2018; Y. Wang et al., 2004). The radiometric correction transforms the numeric values of each pixel to absolute measurements of radiation per unit of light (Gonzales et al., 2018). Atmospheric correction refers to the process of eliminating the atmospheric effects on the remote-sensing data (Gonzales et al., 2018). Furthermore, the data can also be preferentially selected or excluded depending on various conditions and criteria. For example, some studies only use days with little cloud coverage and no special weather events in their samples (Lim & Choi, 2015). Surrounding areas of artificial structures can also be excluded to ensure accurate analysis (Lim & Choi, 2015).