Browsing by Author "Falkinham, Joseph O."

Now showing 1 - 8 of 8
  • Thumbnail Image
    Assessing the Potential of Granular Activated Carbon Filters to Limit Pathogen Growth in Drinking Water Plumbing Through Probiotic Versus Prebiotic Mechanisms
    Deck, Madeline Emma (Virginia Tech, 2025-02-06)
    Legionella pneumophila (Lp) and nontuberculous mycobacteria (NTM) are opportunistic pathogens that can be transmitted via drinking water, when tiny droplets containing the bacteria are aerosolized and inhaled during activities such as showering. The resulting respiratory illnesses, Legionnaires' Disease and NTM lung disease, are among the leading sources of drinking water associated disease in the United States and other parts of the world. Lp and NTM are both difficult to control, because they establish as part of natural biofilms that form within the interiors of pipes and fixtures that deliver drinking water to the point of use. These pathogens are especially problematic within premise (i.e., building) plumbing, where intermittent use throughout the day leads to long periods of stagnation, increased water age, warmer temperatures, and depleted disinfectant residuals that exacerbate bacterial growth. The recent advent of high throughput DNA sequencing has led to the discovery that drinking water microbiomes are diverse, complex, and largely comprised of non-pathogenic microbes. This has further led researchers to hypothesize that the microbial ecology of this diverse microbiome could be harnessed as a natural means to control Lp and NTM, i.e., a "probiotic" approach, but such an approach has not yet been demonstrated. The objective of this study was to assess this hypothesis by utilizing biologically active granular activated carbon (GAC) filters, which are already a widely used drinking water treatment both at the municipal and household scale, as a means to naturally shape the microbial ecology of downstream premise plumbing and inhibit Lp and NTM proliferation. GAC has an extremely high surface area that aids removal of organic carbon via adsorption but also provides an ideal habitat for establishment of biofilms, which removes organic carbon from the water via biodegradation. Convectively-mixed pipe reactors (CMPRs) were used for replicable simulation of premise plumbing distal taps. The CMPRs consisted of four-foot-long closed polyvinyl chloride (PVC) pipe segments with the sealed bottom portion resting in a ~48 °C water bath and with the top portion plugged and exposed to the cooler, ambient atmosphere (25 °C in this study), inducing convective mixing and resulting in an internal water temperature of 37 °C. PVC was chosen because it is common in premise plumbing and generally leaches the least organic carbon among the different types of plastic pipe. Four different influent water conditions were implemented in the experimental design: 1) Untreated, dechlorinated municipal tap water with high organic carbon and low biomass; 2) GAC-treated tap water with low organic carbon and elevated, viable biomass; 3) GAC-treated + 0.22-m pore size membrane-filtered tap water to remove both nutrients and biomass; 4) GAC-treated tap water pasteurized at 70 °C with low nutrients and elevated, killed biomass. The 0.22-m pore size membrane filter simulated the use of a building scale particle filter, while pasteurization simulated water passing through a hot water heater at an elevated temperature recommended for pathogen thermal disinfection. To understand the influence of these experimental conditions on older pipes containing mature biofilms versus new pipes that leach more organics and are being freshly colonized, a set of older pipes colonized with mature ~4-year-old biofilms were compared to newly purchased pipes. Each set of pipes was tested in triplicate for the four different experimental conditions with the full volume replaced three times a week for eight months, simulating infrequently used taps containing warm, continuously mixing water thought to create conditions at a very high risk for opportunistic pathogen growth. In the aged CMPR bulk water effluents, droplet-digital-polymerase-chain-reaction measurements showed a one-log reduction of Lp and NTM when receiving GAC-treated or GAC-treated + particle-filtered influent water versus receiving dechlorinated municipal tap water or GAC-treated + pasteurized water. These findings suggest that decreased biodegradable dissolved organic carbon achieved by GAC filtration acted to suppress Lp and NTM growth, while the additional step of biomass removal by particle filtration provided a more modest benefit. In the CMPRs consisting of new pipes, concentrations of Lp and NTMs in the effluent bulk water were similar among the experimental conditions, except that the CMPRs receiving the GAC-treated + particle-filtered influent water experienced a two-log reduction in NTMs. These results demonstrate that the colonization and proliferation of NTM within premise plumbing can be significantly controlled by limiting nutrients and biomass in the influent water. This work demonstrates the potential of harnessing GAC-treatment as a means to Control Lp and NTM in premise plumbing via nutrient removal. In scenarios where chemical disinfectants have been depleted, off-the-shelf GAC-treatment used as point-of-entry treatment to large buildings with recirculating plumbing could provide benefits that have previously been unrecognized. Alternatively, pasteurization in very hot water heaters could provide a short-term disinfection benefit, but eventually the nutrients embodied in the dead biomass undermine the positive influence of the nutrient removal provided by the GAC-treatment. Improved mechanistic understanding of probiotic strategies to opportunistic pathogen control would be needed to overcome inherent limitations to the approaches examined herein, if more effective control is desired in the absence of thermal or chemical disinfection.
  • Thumbnail Image
    Development and Mechanism of Action of Antimicrobial Coatings
    Behzadinasab, Saeed (Virginia Tech, 2023-07-14)
  • Thumbnail Image
    Dose Determines if Soluble Copper is a Nutrient or an Antimicrobial for Legionella pneumophila in Premise Plumbing
    Finkelstein, Rachel Briana (Virginia Tech, 2022-08-17)
    The effect of copper on Legionella pneumophila in potable water plumbing systems is dependent on its dose and water chemistry. For instance, prior research demonstrated that the presence of aluminum hydroxide from anode rods in water heaters can bind copper and render high doses non-toxic. On this basis it was also hypothesized that iron hydroxide would have similar effects and that lower levels of copper may act as a nutrient encouraging Legionella growth. Here we conducted complementary experiments at bench, microcosm and pilot-scale to evaluate the effect of copper speciation and dose on L. pneumophila. At bench-scale, the addition of 5 mg/L as Fe iron hydroxide to a solution with 1 mg/L copper decreased soluble copper from > 90% down to < 20% at pH 6.5-7. The reduction in soluble copper caused ~3-logs higher L. pneumophila culturability when iron was added with copper when compared to a condition with copper alone. In a 9-month microcosm test using simulated glass water heaters with PEX pipe, a complete range of copper doses (0, 4, 30, 250 and 2000 g/L) were tested in triplicate. Over the first phase of research covered herein, the L. pneumophila levels were low at the four lowest doses of copper, and non-detectable at the highest dose. Moreover, total cell counts were highest at 250 g/L copper, lowest at 2000 g/L copper, and in between these extremes at the lower copper doses. This ongoing experiment will continue for months after this thesis was complete. Pilot-scale experiments were conducted with anode rods removed from tank water heaters, to examine the effects of unprotected corrosion of the steel on iron release and Legionella pneumophila levels in systems with 1) added copper (WH-Cu), 2) copper and phosphate corrosion inhibitor (WH-Cu+PO4-3), 3) phosphate corrosion inhibitor alone (WH-PO4-3) and 4) a control with neither copper nor inhibitor (WH-Control). While there were slight differences in iron between the conditions, the iron concentration in the water of the tanks did not dramatically increase compared to when the powered anodes were still installed and reducing corrosion. Because the iron level was usually < 0.1 mg/L, the released iron dose was not high enough to reduce soluble copper. In fact, oddly, soluble copper increased by 37-183%, mostly likely because the installed anode rods were suppressing copper release. Consequently, with the anode rods removed, the dose of 2000 g/L copper still had a strong antimicrobial effect. The levels of L. pneumophila increased in the order WH-Cu (2.6-logs CFU/L) < WH-PO4-3 (5.1-logs CFU/L) ≈ WH-Control (5.1-logs CFU/L). The addition of phosphate precipitated some of the added copper, such that the condition with copper and phosphate [WH-Cu+PO4-3 (4.2-logs CFU/L)] had L. pneumophila in between the condition with copper alone and that with no added copper. When the copper dose in the pilot rig was reduced to 1000 g/L, Legionella increased somewhat in the system with added copper compared to the control, and L. pneumophila increased in the water heaters in the order WH-Cu (3.4-logs CFU/L) < WH-Cu+PO4-3 (4.3-logs CFU/L) < WH-PO4-3 (4.9-logs CFU/L) ≈ WH-Control (5.0-logs CFU/L). Overall, an antimicrobial effect of copper was maintained in the water heaters even after the anodes were removed and iron in the water increased slightly. If iron corrosion and release to water were much higher without the anode rods than observed in this study, it would be predicted that the higher iron would have reduced the copper antimicrobial effect.
  • Thumbnail Image
    Facile Implementation of Antimicrobial Coatings through Adhesive Films (Wraps) Demonstrated with Cuprous Oxide Coatings
    Behzadinasab, Saeed; Williams, Myra D.; Falkinham, Joseph O.; Ducker, William A. (MDPI, 2023-05-17)
    Antimicrobial coatings have a finite lifetime because of wear, depletion of the active ingredient, or surface contamination that produces a barrier between the pathogen and the active ingredient. The limited lifetime means that facile replacement is important. Here, we describe a generic method for rapidly applying and reapplying antimicrobial coatings to common-touch surfaces. The method is to deposit an antimicrobial coating on a generic adhesive film (wrap), and then to attach that modified wrap to the common-touch surface. In this scenario, the adhesion of the wrap and antimicrobial efficacy are separated and can be optimized independently. We demonstrate the fabrication of two antimicrobial wraps, both using cuprous oxide (Cu2O) as the active ingredient. The first uses polyurethane (PU) as the polymeric binder and the second uses polydopamine (PDA). Our antimicrobial PU/Cu2O and PDA/Cu2O wraps, respectively, kill >99.98% and >99.82% of the human pathogen, P. aeruginosa, in only 10 min, and each of them kill >99.99% of the bacterium in 20 min. These antimicrobial wraps can be removed and replaced on the same object in <1 min with no tools. Wraps are already frequently used by consumers to coat drawers or cars for aesthetic or protective purposes.
  • Thumbnail Image
    A Framework for Controlling Opportunistic Pathogens in Premise Plumbing Considerate of Disinfectant Concentration x Time (CT) and Shifts in Microbial Growth Phase
    Odimayomi, Tolulope Olufunto (Virginia Tech, 2025-01-02)
    Opportunistic pathogens (OPs) can naturally colonize premise (i.e., building) plumbing and are the leading cause of disease associated with potable water in the U.S. and many other countries. While secondary disinfectant is added by utilities prior to water distribution through pipes, the residual in water at the property line is sometimes insufficient to suppress OP growth. Conditions encountered in premise plumbing can further diminish disinfectant in water after it crosses the property line. This dissertation examines how multiple factors at play in drinking water distribution systems and premise plumbing influence OP growth in order to inform development of rational guidance to reduce incidence of waterborne illness. Operating an at-scale cross-linked polyethylene (PEX) plumbing system with one water flush per day, influent chloramine always decayed within four hours in stagnant pipes containing mature biofilms, which is 2-3 orders of magnitude faster than in the same water not contacting pipes. Chloramine often followed second order decay kinetics, though decay rate coefficients were highly variable with some taps eventually transitioning from second to first order decay over time or with increasing influent chloramine concentration. The rate of chloramine decay was unexpectedly reduced in the water heater tank compared to room temperature pipes, possibly due to lower surface-area-to-volume ratio and higher temperature within the tank. A complementary glass jar experiment confirmed that, contrary to expectations, chloramine could decay slower at the higher temperature of 37-39°C maintained in the water heater, compared to the cooler 19-30°C typical of the pipes. These findings demonstrate the need for disinfectant decay models specific to conditions encountered in premise plumbing. Nitrification, a key microbial process that can catalyze chloramine decay, was typically complete within 24 hours after water entered the stagnant pipes. Counterintuitively, the water heater had a relatively lower rate of nitrification along with some detectable denitrification. This work also showed that oxygen, essential for aerobic microbial growth, can permeate through walls of PEX pipe and enter into the water from the atmosphere of the building. Considering the unique array of conditions that were found to influence the persistence of disinfectants in premise plumbing, a new approach was proposed for managing OP risk, referred to herein as the "CT framework." CT was defined as the integral of the chlorine concentration (C) at a point in the premise plumbing versus water retention time (T). Legionella pneumophila was not detectable in pipes with a CT > 78 mg*min/L over a 24 hour period, which is comparable to reported CT thresholds for 3-log inactivation of biofilm-associated L. pneumophila in batch experiments. There was a tradeoff between control of L. pneumophila and Mycobacterium avium in the water heater, as M. avium increased by >1 log as influent chloramine and CT increased, while L. pneumophila decreased by >1.5 logs. Further research is needed to elucidate the influence of factors such as water storage tank hydrodynamics and sediment on the persistence of different OPs. Building water retention time was also found to be an overarching variable that governs microbial growth in some circumstances in premise plumbing. Total cell counts and L. pneumophila occurrence mirrored expected trends based on the classic microbial growth curve with phases of lag, exponential growth, stationary growth, and decay. The location in the plumbing system where each phase dominated depended on water retention time, disinfectant level, and temperature. The microbial growth curve considerations add an additional dimension to the CT framework for predicting L. pneumophila growth potential in premise plumbing. Specifically, elevated heat or chloramine, was able to temporarily suppress or even eliminate growth, but the phases of classic microbial growth could be restarted once disinfectant or very high temperatures were absent. Total cell counts and L. pneumophila typically peaked at a building water retention time of 7 days, demonstrating that once a week flushing guidance to protect public health may not be advantageous in all situations. Collectively, this work offers fundamental and practical insights into factors driving disinfectant decay and microbial proliferation in premise plumbing, offering a modified CT and microbial growth concept framework to help guide the management of OPs in premise plumbing.
  • Thumbnail Image
    Robust and Transparent Silver Oxide Coating Fabricated at Room Temperature Kills Clostridioides difficile Spores, MRSA, and Pseudomonas aeruginosa
    Hosseini, Mohsen; Huang, Jinge; Williams, Myra D.; Gonzalez, Gerardo Alexander; Jiang, Xiuping; Falkinham, Joseph O.; Ducker, William A. (MDPI, 2023-12-31)
    Antimicrobial coatings can inhibit the transmission of infectious diseases when they provide a quick kill that is achieved long after the coating application. Here, we describe the fabrication and testing of a glass coating containing Ag2O microparticles that was prepared from sodium silicate at room temperature. The half-lives of both methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa on this coating are only 2–4 min. The half-life of Clostridioides difficile spores is about 9–12 min, which is extremely short for a spore. Additional tests on MRSA demonstrate that the coating retains its antimicrobial activity after abrasion and that an increased loading of Ag2O leads to a shorter half-life. This coating combines the properties of optical transparency, robustness, fast kill, and room temperature preparation that are highly desirable for an antimicrobial coating.
  • Thumbnail Image
    Surface Coatings for Antimicrobial Activity and Fast Evaporation
    Hosseini, Mohsen (Virginia Tech, 2024-05-29)
    Coatings play a pivotal role in everyday life and across various industries. They offer protection, corrosion resistance, insulation, optical improvements, aesthetics, etc. This study investigates the design, fabrication, characterization and evaluation of surface coatings in two areas: antimicrobial activity and fast evaporation. The COVID-19 pandemic underscored the necessity for coatings that mitigate microbial transmission through surfaces, alleviating both contagion and personal fears. The first part of this study presents the design, development, and evaluation of antimicrobial coatings that efficiently inactivate 99.9% of SARS-CoV-2 virus and kill more than 99.9% of pathogenic bacteria such as Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa within one hour. Prioritizing rapid infectivity reduction, we designed and fabricated several coatings using silver oxide (Ag2O), cupric oxide (CuO), and zinc oxide (ZnO) particles as active ingredients. Applying small quantities of micron-sized opaque particles onto a surface yields a transparent film. Although Ag2O particles are inherently opaque, they possess potent antimicrobial properties. Consequently, incorporating small quantities of Ag2O into the coating results in the desired antimicrobial activity while maintaining transparency. Transparent antimicrobial coatings are a necessity for applications such as touchscreens, offering the benefit of reducing disease transmission while maintaining the aesthetic appeal of surfaces. We employed a variant of the Stöber process to bind Ag2O particles to the substrate using a silica matrix. To improve this coating method, we employed room-temperature spin-coating of a suspension of Ag2O/sodium silicate solution on the substrate, eliminating reactions with toxic chemicals in Stöber process and subsequent heat treatment. Two key features of the improved coating are its high robustness and its capability to kill 98.6% of Clostridioides difficile endospores in 60 minutes. On the other hand, CuO and ZnO particles exhibit mild antimicrobial properties; thus, their activity could be enhanced by a porous coating. When an infected droplet lands on such a coating, it is imbibed into the porous structure, where diffusion distances are smaller, and there is a larger active area to inactivate the virus or kill the bacteria. Furthermore, porosity facilitates faster droplet drying, leading to the concentration of cupric and zinc ions in the droplet, which are designed to be toxic to microbes. The second major topic of this thesis is the development, and evaluation of porous coatings for fast evaporation. At low Bond numbers, droplet evaporation is slow on an impermeable surface. We investigated whether application of a thin, porous coating leads to faster droplet evaporation. The droplet will imbibe quickly, but progress normal to the interface will be limited to the thickness of the coating. Therefore, the liquid will spread laterally into a broad disk to expose a large liquid–vapor interface for evaporation. As a result, the evaporation of a droplet is enhanced by a factor of 7–8 on the thin porous coatings. Factors such as coating thickness, pore size and distribution, and the contact angle of the coating, as well as ambient conditions like temperature and relative humidity, could affect the droplet evaporation rates by modifying the droplet's imbibition process and the evaporation driving force. While decreasing the coating thickness and increasing pore size and distribution promoted evaporation, the impact of contact angle is insignificant. Confocal microscopy observations of a coating composed of particles with varying sizes depicted liquid migration along the top of the coating and the edges of the interface. We developed and validated an equation to estimate the rate of evaporation. The rate correlated with the radius of the imbibition area, with higher temperatures and lower humidity further augmenting evaporation.
  • Thumbnail Image
    Towards Optimization of Residual Disinfectant Application for Mutual Control of Opportunistic Pathogens and Antibiotic Resistance in In-Building Plumbing
    Cullom, Abraham Charles (Virginia Tech, 2023-07-13)
    Opportunistic premise (i.e., building) plumbing pathogens (OPPPs) and antibiotic resistant bacteria are emerging microbial concerns in drinking water. OPPPs, such as Legionella pneumophila, are the leading cause of drinking water disease in many developed countries. Contributing factors include the relative success in controlling fecal pathogens, the presence of complex building plumbing systems that create habitats for OPPPs, and the relative resistance of OPPPs to disinfectants, and aging populations that are susceptible to infection. Concurrently, drinking water is increasingly being scrutinized as a potential environment that is conducive to horizontal gene transfer of antibiotic resistance genes (ARGs), selection pressure for enhanced survival of resistant bacteria, and a route of transmission of antibiotic resistant pathogens. While maintaining a disinfectant residual is an established approach to controlling OPPPs in premise plumbing, some studies have indicated that co-resistance and cross-resistance to disinfectants can increase the relative abundances of resistant bacteria and ARGs. Thus, there may be trade-offs to controlling both OPPPs and antibiotic resistance in premise plumbing that call for controlled study aimed at optimizing residual disinfection application for this purpose. A critical review of the scientific literature in Chapter 2 revealed that premise plumbing is a biologically and chemically complex environment, in which the choice of pipe material has cascading effects on water chemistry and the corresponding premise plumbing microbiome. This, in turn, has broad implications for the control of OPPPs, which need to be elucidated through controlled experiments in which worst case premise plumbing conditions are held constant (e.g., warm temperature), while other variables are manipulated. Chapter 3 introduces the convectively-mixed pipe reactors (CMPRs) as a novel low-cost, small footprint approach to replicably conduct such experiments. The CMPRs were demonstrated to effectively simulate key chemical and biological phenomena that occur in distal reaches of premise plumbing. In Chapter 4, the CMPRs were leveraged to study the interactive effects of four disinfectants (chlorine, monochloramine, chlorine dioxide, and copper-silver ionization) and three pipe materials (PVC copper, and iron). The CMPRs were inoculated with two antibiotic-resistant OPPPs: Pseudomonas aeruginosa and Acinetobacter baumannii. It was found that pipe-material (PVC or PVC combined with iron or copper) profoundly impacted the water chemistry in a manner that dictated disinfection efficacy. In Chapter 5, we applied shotgun metagenomic shotgun sequencing to evaluate effects of the combination of pipe material and disinfectant type on the wider microbial community, especially their ability to select for or reduce ARGs. In Chapter 6, we used CMPRs and metagenomic sequencing in a study comparing Dutch drinking water practices to our prior testing in an American system. Dutch drinking water is of interest because of lack of historical use of disinfectants was hypothesized to result in a microbial community that is relatively depleted of ARGs or mobile genetic elements, which can enhance spread of ARGs as disinfectants are applied. Generally, it was found that OPPPs required higher doses of disinfectants for inactivation than the general microbial community, sometimes concentrations approaching the regulatory limits in the US (e.g., 4 mg/L of total chlorine). Even successful reductions were modest, typically ~1-log, and failed to eliminate either P. aeruginosa or A. baumannii. Moreover P. aeruginosa, A. baumannii, and non-tuberculous mycobacteria varied substantially in their preference for pipe material and susceptibility to disinfectants. We found that disinfectants tended to increase the relative abundance of OPPPs, ARGs, and mobile genetic elements. Disinfectants were sometimes associated with net increases in levels of these pathogens and genes when applied at low levels (e.g., 0.1 mg/L of monochloramine), which effectively acted to reduce competition from less resistant and non-pathogenic taxa. When a low dose of monochloramine was applied to PVC CMPRs in the US, we estimated from metagenomic sequencing data that this water contained roughly 100,000 cells per milliliter of taxa known to contain pathogenic members. The Dutch drinking water exhibited more diverse microbial communities and lower relative abundances of taxa containing pathogens. ARGs were two times proportionally more abundant in CMPRs operated in the US without disinfectant than in the corresponding CMPRs operated in the Netherlands. The findings of this dissertation can help to optimize the application of in-building disinfectant addition for addressing concerns related both to OPPPs and antibiotic resistance. The studies herein highlight the necessity of developing comprehensive OPPP and antibiotic resistance control strategies that emphasize not just disinfectant dose, but other key control parameters such as contact time, hydraulics, and temperature. The functional diversity of OPPPs, antibiotic resistant bacteria, and the background premise plumbing microbiome further necessitates broad, holistic programs for monitoring and control.