• The Assessment of Escherichia coli as an Indicator of Microbial Quality of Irrigation Waters used for Produce

      Rock, Channah; Brassill, Natalie A.; McLain, Jean E.; Gerba, Charles; Nolte, Kurt (The University of Arizona., 2013)
      Escherichia coli is a bacterial species that lives in the gut of all warm-blooded animals, fish, birds as well as reptiles and is commonly used as an indicator of fecal contamination in water. This project assessed currently used culture based media for the detection of E. coli in irrigation waters used in Arizona and California, and will present recommendations towards the most reliable media for the evaluation of irrigation waters used for produce. Currently, no microbial indicator standards exist for irrigation waters used for produce production in the United States. The produce industry suggests that the recreational water standard guideline (126 E. coli/100 ml) established by the United States Environmental Protection Agency (USEPA) be used. There is concern that the false positive rate of E. coli detection may be high in these waters giving false indications of the level of risk from enteric pathogens. This project evaluated three commercially available media for E. coli detection to test irrigation waters from three agricultural areas (Yuma and Maricopa, AZ and Imperial Valley, CA) and then assessed false positive rates by utilizing Polymerase Chain Reaction (PCR) and DNA sequencing of the bacterial isolates. The media that were chosen for evaluation were (1) MI Agar, (2) IDEXX Colilert Quanti-Tray® and (3) m-ColiBlue24® broth, all evaluation media accepted by the USEPA and widely used in the monitoring of irrigation water quality by the produce industry. Four hundred and fifty 1-L irrigation water grab samples were collected between March 2012 and November 2012. The samples were analyzed for both cultural counts and water quality parameters including temperature, salinity and pH. Isolates positive and negative for E. coli were then selected and assessed utilizing PCR and DNA sequencing. The false positive rate of each method was found to be high, with MI Agar, m-ColiBlue24® broth and the IDEXX Colilert Quanti-Tray® at an accuracy of 67%, 72%, and 51% respectively. A false positive result is reported when presumptive E. coli sub cultured from the media is found to be non-E. coli through molecular analysis. Overall the IDEXX Colilert Quanti-Tray® performed at a greater rate of accuracy than the other two media evaluated, however, high false positive rates may lead to inaccurate assessment of water quality.
    • Canal Maintenance Effects on Irrigation Water Quality

      McLain, Jean E.; Obergh, Victoria Lee; McLain, Jean E.; Rock, Channah; Nolte, Kurt D.; Rivadeneira, Paula (The University of Arizona., 2015)
      Canal maintenance, involving mechanical removal of sediments and algal growth from canal basins, is necessary for sustaining the viability of the irrigation water delivery system in the Imperial Valley of California. Maintenance activities, however, disturb canal sediments laden with bacteria and can negatively impact water quality downstream. Our work quantified fecal indicator bacteria (Escherichia coli) and pathogens (Salmonella) in canal water prior to, during, and post-maintenance events. The goal of this study was to construct a post-maintenance time matrix that will allow growers downstream to estimate when canal water once again meets water quality guidelines. In addition, we assessed the water quality impacts of lining canals with concrete, which is a costly endeavor in the short term, but may be beneficial in the long term as lined canals do not require routine dredging to maintain canal integrity. During eight maintenance events from March 2013 through August 2014, 22% of 396 water samples collected exceeded the irrigation water quality guidelines (<126 MPN E. coli 100 mL-1) during canal maintenance. During summer months (July and August 2013-2014), E. coli concentrations in water samples commonly reached maximum values (>2419.6 MPN E. coli 100 mL-1), and these samples were more readily collected from unlined canal sampling sites. During winter and spring months, 80.8% of E. coli exceedances for unlined canals met guideline standards in less than 22 hours, while 19.2% of exceedances took longer (up to 48 hours) to return to acceptable levels; in lined sites, 63.6% and 36.4% met guidelines in less than 22 hours and 48 hours, respectively. Summer months showed a different trend: in unlined canal sites, 56.3% of E. coli exceedances met standards within 22 hours and 43.7% within 48 hours; in lined sites, 100% of water samples met standards in less than 22 hours. Unlined sites averaged higher temperatures overall compared to lined sites, and canal water in July (2013) was extremely warm (averaging 32.8°C) and reached human body temperature (37°C) at several unlined sites, a temperature at which enteric bacteria are known to thrive. Culturable Salmonella were detected in water samples collected in summer, with 22.2% of Salmonella-positive samples within 1°C of human body temperature. E. coli concentrations were significantly correlated with temperature and pH in unlined canals only. Unlined canals showed 15.2% of water samples were Salmonella-positive during summer maintenance whereas 1.7% of lined canals were positive. Salmonella significantly correlated with pH in lined canals. Fecal indicators (E. coli) did not predict pathogen (Salmonella) presence. Molecular methods (qPCR) suggested far higher levels of Salmonella when compared to cultural methods, with molecular markers for Salmonella exceeding culturing by more than 600%. The results of this work suggest that growers should exercise caution when irrigating after canal maintenance events, and to be completely certain of acceptable irrigation water quality, should wait for 48 hours following the onset of maintenance (typically 24 hours following the re-introduction of water to the channels) prior to irrigating crops. Further, irrigation district guidelines may consider: 1) disposing of the“first flush”of canal water following maintenance into nearby open areas, rather than sending poor-quality water into the irrigation canal system; 2) collect sediments and algae deposited on canal banks and transport to a secondary location to prevent precipitation runoff and re-introduction of bacteria-laden sediments to canals, and 3) consider the long-term costs and benefits of canal lining.
    • Development Of Biosensors For Detection Of Pathogens In Complex Sample Matrices

      Yoon, Jeong-Yeol; Angus, Scott V.; Yoon, Jeong-Yeol; Slack, Donald C.; Reynolds, Kelly (The University of Arizona., 2014)
      Protozoa and bacteria can easily cause disease in humans, specifically E. coli, Plasmodium falciparum, and Cryptosporidium parvum. These three pathogens are associated with large public health concerns that span the globe. The variety of locations in which these can be found is extremely high. Cryptosporidium spp. are extremely resilient when in oocyst form, P. falciparum is in the Anopheles spp. mosquito, while E. coli can be found on anything from food and water, to the skin and gut. The diverse range of locations these can be found in means that a portable sensor for their detection is necessary. In detecting Cryptosporidium, microscopy is the preferred method of identification currently. This requires a trained lab technician as well as calibrated and expensive optical equipment. Technician error can lead to false negative or positive diagnoses as well as sample destruction. A method to remove this technician interaction is thus necessary. This method must allow for objective results that are not open to interpretation. Particle immunoagglutination assays with Mie scatter allow for such an approach using inexpensive components. Particle immunoagglutination relies on the principles of antibody-antigen interaction and antibody conjugated latex particles. Using highly carboxylated latex particles, it is possible to attach IgG antibodies that are specific to a target antigen. Mie scatter is governed by particle size rather than wavelength as other forms of scatter. These two combined allow for an increase in light scatter based on particle size. This is correlated in a linear manner as long as the number of antibody-conjugated particles is higher than the number of antigens. Microfluidics is an ever growing field in the field of lab-on-a-chip that works very well with particle immunoagglutination. In this paper, a method to rapidly identify the presence of Cryptosporidium using microfluidics and particle immunoagglutination is discussed and analyzed. This method allows for a low detection limit of 1-10 oocysts/sample and an assay time of approximately 10 minutes. Results are displayed on a computer screen as the value of light scatter intensity and, when compared to a standard curve, is an objective way to identify the concentration and presence of oocysts in a diverse range of samples. These samples include PBS, pool water, and sump water. This system also works with P. falciparum, which causes malaria in rural and urban poor regions of the world. With the low income and remote nature of these locations, a portable microfluidic device is necessary. Smartphones allow for a portable microfluidic device that can detect P. falciparum antigens in 10% whole blood. This system is capable of detecting as little as 1 pg/mL antigen. The microfluidic chip is inexpensive and disposable, allowing for a portable and inexpensive system. Using a single smartphone, a lab technique requiring a spectrometer, light source, and laptop can be made portable and less expensive, while maintaining sensitivity and specificity. In order to identify biological agents, there are commonly 3 methods for doing so: PCR, culturing, and ELISA. Culturing can take more than 24 hours, but results in a high signal to initial target ratio, while ELISA has poor sensitivity due to a 1:1 signal to target ratio, though is much quicker than culturing at usually 3 hours or less. PCR manages to solve both these problems by exponentially increasing the number of copies of target genetic material in a relatively short time frame of 1-3 hours. PCR relies on 4 basic components: target genetic material, primers to set a start and end location for duplication, polymerase to add base pairs to the strand beginning at the primers, and heat. PCR has worked very well during the past 31 years. It has worked so well that it is often the gold standard. However, there are flaws built into today's systems. These largely come in the form of inefficient heat transfer via conduction and large sample volumes due to unnecessary additions of nuclease free water (NFW). Both of these can be easily overcome by droplet PCR. Droplet PCR relies on small sample volumes of between 8 and 12 μL and convection in oil rather than conduction through plastic. In this study, it was found that droplet PCR could be performed on genomic E. coli DNA in as little as 15 minutes for 30 cycles. Sensitivity was also analyzed and found to be 2.62 pg DNA/μL or about 5 x 10² cfu/sample. PCR has a theoretical lower limit of 1 copy of genetic material and this is only 2 orders of magnitude above that. The system was also tested for portability and resistance to shock and vibration. It was found that the surface heated, thermocouple guided system is more shock and vibration resistant than standard wire guided, hanging droplet PCR systems. It was also found that the use of coconut oil allows for the system to be transported without fear of the contents spilling out and contaminating other samples. This is because of coconut oil's high melting temperature.
    • Evaluation of the Uptake of Microbial Surrogates by Leafy Greens Using Subsurface Irrigation

      Slack, Donald C.; Reyes Esteves, Rocio Guadalupe; Gerba, Charles P.; Pepper, Ian L.; Betancourt, Walter Q. (The University of Arizona., 2020)
      Foodborne outbreaks have been traced to contaminated irrigation water. The use of contaminated water to irrigate crops by surface, sprinkler, or conventional drip irrigation may pose a public health hazard due to the potential presence of pathogens. It is immediately apparent that crops that come in direct contact with contaminated irrigation water can become contaminated. Thus, this study assessed a subsurface irrigation system quantitatively with romaine lettuce in terms of crop contamination when microbial-contaminated water was used for irrigation. A cropping system for bottom watering pots at the greenhouse and a subsurface drip irrigation (SDI) system commonly used for lettuce production in Arizona was modeled with the well-known software, HYDRUS 2D/3D, to ensure that the soil surface does not become wet and consequently contaminated. Experiments were conducted during the full growth cycle of romaine lettuce to fulfill the study purposes. Escherichia coli ATCC 25922 and bacteriophage MS2 were used as surrogate microorganisms for this study. No microbial contamination of produce occurred in romaine lettuce irrigated with contaminated irrigation water utilizing bottom watering pots. Maintenance of a dry soil surface appeared to avoid crop contamination in subsurface irrigated lettuces. Subsurface irrigation systems with proper management can be excellent strategies to not only conserve water resources but to reduce or eliminate contamination of leafy green vegetables when harmful microbes are present in the irrigation water. In the second aspect of this study, microbial contamination of lettuce plants irrigated by surface irrigation was investigated to aid in the development of irrigation water quality standards for enteric bacteria. Surface irrigation was evaluated with the use of Escherichia coli O157:H7 transfer data to the plants. The Beta-Poisson model was used to estimate the microbial risk of annual pathogen ingestion. The risk of infection was found to be variable depending on the E. coli concentration in water, and the pathogen transfer to the crop days post-exposure. The worst-case scenario, in which produce could be harvested and consumed the day after the last irrigation event, and maximum exposure is assumed, indicated that concentrations of 104 CFU (colony forming units) /ml in 30-day-old lettuce plants would result in an annual risk of 1:10,000 if the crop would be consumed. Similarly, concentrations of 103 CFU/ml, and 104 CFU/ml in 12-day-old lettuce would result in an annual risk of 1:10,000 if the crop would be ingested.
    • Every Last Drop: Synthesizing the Microbial Quality of Harvested Rainwater and Garden Soils to Inform Environmental Justice Communities

      Ramirez-Andreotta, Monica D.; Moses, Arthur; McLain, Jean E.; Kilungo, Aminata P. (The University of Arizona., 2021)
      Water scarcity has long plagued human civilization, but arid environments such as the American Southwest face an exceptionally difficult climate fight. The states of that make up southwestern United States all primarily depend on the same major water source, the Colorado River, and are at the mercy of its reservoirs to meet their demand. While the switch to Colorado water has allowed for some recovery in groundwater in the region, the lack of precipitation in the coupled with 20th century population growth makes recharging aquifers difficult. As such, many states, such as Arizona, have turned to water conservation plans to meet the growing demand in water, as well as to improve the quality of their own aquifers. One popularly embraced method in Arizona is harvesting rainwater, or the collecting rainfall in cisterns or catchment devices for later use, particularly when it comes to crop and garden irrigation. However, the communities of Dewey-Humboldt, Globe/Miami, Hayden/Winkelman, and Tucson, Arizona, face an additional uphill battle as border communities to Superfund and Toxic Release Inventory sites. These communities have concerns about the quality of their harvested rainwater, and garden soils. By using a co-created citizen science framework, Project Harvest, works directly with community members to sample and participate in the scientific process, answering questions and concerns they may have about their harvested rainwater and garden soils. This dissertation research seeks to understand the socio-demographic factors influencing water conservation infrastructure, as it pertains to rainwater as an alternative water source, to determine the microbial quality of harvested rainwater, and to determine if a lower cost testing method is satisfactory to determine water quality.
    • Optimization of Growth Conditions in Minimal Media for Production of Nucleolin Protein

      Yang, Danzhou; Bruce Carver, Megan; Brewer, William Ryan; Yang, Danzhou; Bruce Carver, Megan; College of Pharmacy, The University of Arizona (The University of Arizona., 2016)
      Objectives: The purpose of this experiment is to compare NUC1234 expression levels from E. coli cultures by manipulating cell cultures according to the optical density (OD595) at which protein expression is induced, and the harvest time after induction. Methods: E. coli BL21(DE3) cells transformed with DNA to produce NUC1234 were plated and then grown in minimal media for protein expression. They were induced at a variety of optical densities and harvested at different times post-induction. Protein quantities from each sample were then compared using a protein determination assay and an SDS-PAGE gel. Results: The sample induced at an OD595 of 0.5 yielded a lower concentration of protein (0.874409mg/ml) compared to other samples; however, it visualized as a stronger band in the SDS-PAGE gel. The sample harvested at 6 hours yielded the largest protein concentration (1.347215mg/ml) among all active samples and appeared as bold as, or bolder than, those harvested at other times. Conclusions: The results of the protein assay study and gel visualization suggest that the optimal conditions for the production of NUC1234 are growth to an OD595 of 0.5 before induction of protein expression and a harvest time of 6 hours after induction.
    • Reduction of Bacterial and Viral Indicators in Laundry Graywater by Solar Disinfection

      Slack, Donald C.; Terrazas Onofre, Maria Liliana; Slack, Donald C.; Yitayew, Muluneh; Gerba, Charles P; Lopez, Gerardo U. (The University of Arizona., 2016)
      Current competitive status among potable and non-potable use makes the water reuse mandatory. Presently, water reuse is common only for reclaimed water coming from municipal or industrial water treatment plants. In those facilities, the treatment includes disinfection. The disinfection methods widely used are chlorination and Ultra Violet (UV) lamps adapted to the conditions of large volume of municipal and industrial systems. This study proposes a disinfection method adequate to the household level to reuse graywater. The method is called solar disinfection (SODIS), which allows the reuse of graywater even though it contains fecal contamination. In this research, natural sun radiation as a free source of heat and UV radiation was utilized. In a first stage, periods of sun exposure, graywater depth, and cell covers as external factors were studied. In later stages, the graywater temperature (GWT) and the UV radiation effects on the reduction of the microbial indicators were observed. Results showed that graywater depth of five cm had a statistical significant reduction rather than ten cm depth (p = 0.0035). Plexiglas and poly-vinyl chloride (PVC), as transparent covers, had a statistical significant reduction (p<0.00001) due to the greenhouse effect increasing the GWT. The black cover had the lowest GWT and reduction of the bacterial and viral indicators. This research found different behavior between bacteria and virus reduction by graywater solar disinfection. In order to reduce the concentration of total coliforms, Escheriquia coli (E. coli) and enterococcus to non-detectable levels (<1.0 most probable number, MPN 100 ml⁻¹), a combination of GWT >45 °C, and UV radiation >24 W m⁻² was required. In contrast, coliphage MS-2, as viral indicator, was resistant to different UV radiation magnitudes (up to 50 W m⁻²), but with a GWT >55 °C non-detectable levels (<1.0 plaques forming units, PFU) were reached.
    • Risk Perception, Drinking Water Source and Quality in a Low-Income Latino Community along the U.S.-Mexico Border

      Beamer, Paloma I.; Victory, Kerton Richard; Beamer, Paloma I.; Burgess, Jefferey L.; Cabrera, Nolan L.; Reynolds, Kelly A.; Thomson, Cynthia A. (The University of Arizona., 2014)
      Previous studies have shown that low-income Latinos frequently drink bottled water instead of tap water and might be at increased risk for dental caries from unfluoridated bottled water. The goals of this study were to: 1) determine if a difference exists between grab samples of municipal tap water and bottled water used by families in Nogales, Arizona in chemical and microbial contaminants; 2) understand the risk perceptions these low-income families have about both their publically supplied tap water, bottled/vended water; and 3) examine whether families have adequate fluoride levels in their drinking water for optimal dental health. Tap and bottled water samples were collected from thirty of these households for analysis of drinking-water contaminants regulated by the U.S. Environmental Protection Agency (USEPA). Bottled water included small (0.5 L) and large (3.8 L), individually sealed water bottles as well as water vended into reusable containers from self-service vending machines or from water stores. Some of the large bottles were purchased sealed, but had been previously opened and partially consumed by study participants. In addition to the 30 households mentioned above, an additional sixty low-income Latino households, who primarily drink bottled water, were also recruited for this study to complete a questionnaire on risk perceptions related to drinking water consumption, water storage practices, and fluoride supplementation. There were no significant differences in the concentration of chemical contaminants between tap, bottled and vended water, and none of the chemical concentrations exceeded USEPA regulations. Escherichia coli (E. coli) was confirmed in 3% (1/30) of tap, 0% (0/8) of sealed bottled, 40% (2/5) of previously opened bottled, and 35% (6/17) of vended water samples. One of the 3 samples collected directly from a vending machine had an E. coli concentration of 0.33 CFU/100 mL. None of the 10 samples collected directly from sealed bottles purchased directly from the store had any E. coli detected. While no significant differences were observed in the chemical quality of water samples, results suggest that secondary contamination in the home contributes to higher levels of E.coli in water from partially consumed larger water bottles or water stored in reusable containers. Families who reported cleaning large reusable water containers with soap or hot water had significantly lower E.coli (p=0.003) in their drinking water than those who rinsed with only water. Respondents viewed bottled water to be significantly safer to consume than tap water (p<0.001). On a Likert scale from 1 (low risk) to 5 (high risk), "drinking tap water in Nogales, Arizona" received an average score of 4.7, which was significantly higher than the average perceived risk of smoking (µ= 3.5, p<0.001) or of "drinking tap water in San Francisco, California" (µ= 3.4, p<0.001), and as risky as "drinking and driving" (µ= 4.8, p=1.00) and "drinking tap water in Nogales, Sonora, Mexico" (µ= 4.8, p=1.00). Additionally, 98% (88/90) of respondents feared that drinking local tap water could result in illness. The majority of respondents (79%, 71/90) did not drink their tap water because of fear of contamination and would drink their tap water if they knew it was safe, regardless of the taste (73%, 66/90).Fluoride was detected in 53% (16/30) of tap-water samples and only in one bottled-water sample. However, only 27% (8/30) of tap-water samples had fluoride levels within the recommended range (0.7-1.2 mg/L) by the American Dental Association for optimally fluoridated water. From multiple logistic regression, children in a household were less likely to have cavities if their parents reported using fluoridated mouthwash (p<0.001) or visited the dentist yearly (p<0.001). None of the participants reported discussing the type of water they drank or fluoride supplementation with any healthcare providers. Results suggest that secondary contamination in the home is contributing to significantly higher levels of bacteria in vended water stored in reusable containers. In addition, fear of illness from contamination of tap water is an important contributing factor to increased use of bottled water. Interventions could be developed to educate families about how to keep their reusable water containers clean, reduce perceived risks associated with tap-water consumption, and emphasize the importance of fluoride supplementation if families continue drinking bottled water.