Borrelia burgdorferi is the etiological agent of Lyme disease. Not much is known about the susceptibility of this organism to chemical disinfection. Current antimicrobial susceptibility test methods, such as those published by the American Society for Testing and Materials (ASTM), usually require assessment of the number of colony forming units (cfu) of growing organisms on plates following exposure to an agent. For fast-growing organisms, plates are ready for counting 1-2 days post plating, while several weeks may be needed for slower growing organisms. Spirochetes, like B. burgdorferi are difficult to grow on solid media and typically require long incubation periods, sometimes up to several weeks, to generate visible colonies. These issues make B. burgdorferi cfu assessment by plate counting difficult and unreliable. Furthermore, Borrelia have a demonstrated capacity for pleomorphic forms, and can exist in spirochete, round body, or biofilm forms, depending on culture conditions. Plate counts, by nature, do not allow for assessment of morphological form changes. Additionally, the susceptibility of B. burgdorferi pleomorphic forms to chemical disinfectants has not been tested. In this study, we used the SYBR GREEN I/Propidium Iodide (SG I/PI) viability assay to rapidly estimate the percent kill of B. burgdorferi pleomorphic forms to chemical disinfection. Planktonic spirochete populations in 30-second treated samples showed viability percent values of: >95% for Hanks balanced salt solution (HBSS), ~60% for distilled deionized H2O (dd H2O), <5% for ACS 200, and 1% for 1% glutaraldehyde (GTA). Solutions containing 70% ethanol (ETH) and 1% hypochlorite (HC) showed no viable spirochetes following treatment. The percent of live round body cells following different treatments were: >99% for HBSS and <25% for dd H2O. ACS 200, 1% GTA, and 70% ETH treatments resulted in <1% live round body forms, whereas HC showed no live round cell forms. The susceptibility of B. burgdorferi biofilms to various treatments was also assayed using a SG I/PI viability stain after 30-minute contact times. The percent of viable organisms (green) in the treated biofilms was estimated by microscopic observations. HBSS controls showed >98% of bacteria in the biofilm were alive, while treated biofilms showed the following percent viabilities: ACS 200 - ~2%, 1% HC - <1%, 5% HC - <1%, 1% GTA - ~10%, 70% ETH - ~ 2%, and dd H2O ~40%. These techniques merged standardized assessment of antimicrobial activity in liquid culture using an ASTM-type kill-time procedure with viability techniques used in antibiotic susceptibility testing to rapidly evaluate the percent kill of B. burgdorferi pleomorphic forms in vitro following disinfectant exposure. These results showed that B. burgdorferi biofilm forms are orders of magnitude more resistant to chemical disinfection than other morphological forms of this organism.

In many drinking water systems in the United States, disinfectant is added to water as it leaves the plant to maintain a residual concentration in the distribution system. The disinfectant residual is maintained to inactivate contamination that enters the distribution system, to control biofilms, and to act as a sentinel for contamination in the distribution system. A model was developed to evaluate the potential effectiveness of the disinfectant residual at inactivating contamination. The model was used to examine contamination of a hypothetical distribution system through backpressure at a cross-connection under different operating conditions. The dilution and pathway of the hypothetical contaminant were examined as the contaminant moved through the system. Disinfection and inactivation kinetic relationships were used to model the inactivation of the contaminant in the system by the amount of disinfectant present. The model showed that both chlorine and chloramines in each decay and inactivation condition considered provided some benefit over no disinfectant at all when examining susceptible organisms. Chlorine, under medium and low decay conditions, provided the best inactivation. Where 29.8% of total node time steps received a contamination of concern in the absence of disinfectant residual, as low as 4.8% of total node time steps received a contamination of concern in the presence of disinfectant residual. Chloramines was found to persist longer in the distribution system, but resulted in much lower inactivation compared to chlorine. Disinfectant doses typical of common distribution system operation were able to reduce the impact of contamination once it entered the distribution system but, except for four cases, were unable to prevent contamination from spreading within the distribution system. Therefore, it was concluded that presence of a disinfectant residual will reduce the total number of exposure opportunities from a contamination event, but cannot be relied upon to eliminate the chance of exposure resulting from contamination. / Master of Science

The net present replacement value of all publicly and privately owned potable water pipes in the U.S. is on the order of $2.4 trillion dollars, and costs associated with deteriorating iron pipes is billions of dollars per year. Problems arising from iron corrosion include reduced lifetime of the material, scale buildup and energy loss, nonuniform corrosion and leaks, catastrophic failure, "red water," disinfectant loss and bacterial re-growth. Iron corrosion is a very complicated process and is affected by many factors. This research focused on the effect of disinfectant type, sulfate/chloride ratios, nitrate concentration, and magnesium hardness on iron corrosion. For the waters tested, chlorine better controlled red water and microbial activity in the bulk solution than chloramine. Changes in the sulfate/chloride ratio did not have a large effect on iron corrosion. High levels of nitrate increased the rate of chlorine decay as a result of free ammonia formation, and also increased the release of iron. Increased magnesium and zinc decreased the red water caused by high silicate. Microbiological activity is important in iron corrosion, and control of re-growth in water distribution systems is a major challenge for water utilities. A separate study examined the inter-relationship between iron corrosion and bacterial re-growth, with a special focus on the potential of iron pipe to serve as a source of phosphorus. Under some circumstances corroding iron and steel may serve as a source for all macronutrients necessary for bacterial re-growth including fixed carbon, fixed nitrogen and phosphorus. Conceptual models and experimental data illustrate that levels of phosphorus released from corroding iron are significant relative to that necessary to sustain high levels of biofilm bacteria. Consequently, it may be more difficult to limit re-growth on iron surfaces by limiting phosphorus in the bulk water. / Master of Science

The primary aim of this investigation was to identify and assess the interactions (synergies and antagonisms) that exist between 20 minute detergent and 5 minute disinfectant treatments upon three factory isolated strains of surface adhered (1-hour attached) and surface adapted (24-hour biofilm) populations of Escherichia coli and Listeria monocytogenes, plus a comparison with vero-toxin producing strains of E. coli, when used as part of a cleaning and disinfection regime. The detergents chosen for assessment were two non-ionic (91/4 - Alcohol Ethoxylate and KCL5 - Polyethoxylated Alcohol), two anionic (LX28 - Sodium Lauryl Sulphate and Nec28 - Sodium Laurylether Sulphate) and two novel bismuth thiols (BisEDT - 1:1 Bismuth nitrate 1,2-ethanedithiol and BisTOL - 2:1 Bismuth nitrate 3,4-dimercaptotoluene), developed at Winthrop University Hospital, New York. The disinfectants chosen for assessment were a quaternary ammonium compound (BAC - Benzyl alkonium Chloride) and a chlorine releasing agent (NaDCC - Sodium Dichloroisocyanurate). The investigation showed that there were no specific cleaning and disinfection regimes that will adequately target both E. coli and L. monocytogenes strains. It was also concluded that to maximise the removal and disinfection of persistent strains of a given microorganism, it may be necessary to design a regime to specifically target not just the species, but the strain involved and where possible requires mechanical cleaning. The novel bismuth thiols were seen to be promising detergents to aid in the removal of E. coli strains and warrant further attention for future studies. Finally, an investigation to identify possible mechanisms of resistance to disinfectant treatments following detergent treatment, showed that different detergents can induce expression of the stress response proteins, HSP60 and HSP70, at differing levels of expression after the same contact time and against different states of adherent populations, i.e. 1-hour attached or 24-hour biofilm populations.

Chlorine as a disinfectant reacts with natural organic matter to produce undesired and possibly carcinogenic halogenated disinfection by-products (DBPs), which are regulated by the U.S. Environmental Protection Agency under the Disinfectant/Disinfection By-products Rule (DBPR). In order to comply with the increasingly stringent regulations, alternative disinfectants such as ozone, UV irradiation, and chloramines have been investigated. Unfortunately, these alternatives have their own limitations and disadvantages as well. Sonication is another alternative that has not yet received adequate research. The hydroxyl radicals, tensile stresses, and fluid shear generated during sonication may inactivate microorganisms. The goals of this research were to evaluate the effectiveness of sonication alone and combined sonication and chlorination for inactivation of E. coli. Four stages of disinfection experiments were conducted: chlorine alone, sonication alone, combined sonication and chlorination, and heating alone. Experiments were conducted in laboratory prepared phosphate buffered saline. The variables tested included the chlorine dose, chlorine contact time, sonication time, sonication system (probe or bath), sonication power-to-volume ratio, and sonication frequency. E. coli was enumerated by use of pour plates and/or membrane filtration before and after disinfection. Substantial temperature and turbidity increases were recorded after sonication, especially at 900 W/L. After 10 minutes of sonication at 900 W/L, the temperature and turbidity of the experimental solution rose up to 77oC and 23 NTU, respectively. At both 180 W/L and 900 W/L, sonication alone demonstrated little inactivation (less than 1 log10) of E. coli for temperatures below 60oC and greater than 7 log10 inactivation at temperatures over 60oC. The results from heating only experiments confirmed that temperature was responsible for the inactivation rather than other ultrasonic wave effects Sequential application of sonication and chlorination was ineffective at inactivating E. coli. Chlorination alone achieved higher levels of E. coli inactivation than the combination of both disinfectants. When sonication and chlorination were applied simultaneously, the inactivation was greater than the additive effect of two disinfectants, indicating that there were synergistic effects between sonication and chlorination. For example, at 900 W/L, chlorination alone at 0.6 mg/L for 2 minutes provided 1.2 log10 inactivation and sonication for 2 minutes alone provided less than 1 log10 inactivation of E. coli. When the two disinfectants were applied simultaneously, 4.5 log10 was achieved. Sonication may have weakened the cell membranes, causing them to be more susceptible to chlorine disinfection.

Melioidosis is a neglected tropical disease that continues unabated in many countries, particularly in Southeast Asia. There is no vaccine and antimicrobial treatment is expensive and complicated. Virulence models are important tools used to investigate genes involved in pathogenesis. Galleria mellonella is the larvae of the wax worm moth that has been used to model various infections. Based on previous studies, we attempted to establish an infection model using Burkholderia pseudomallei and Burkholderia thailandensis, a related species which is avirulent in humans. Injections of various forms of these species (fresh and frozen) were used to develop Kaplan-Meier plots. We also tested Burkholderia cepacia, Burkholderia vietnamiensis, Burkholeria ambifaria, and Burkholderia multivorans to understand how they affect the larvae. We found that larvae injected with B. pseudomallei and B. thailandensis did not accurately model the respective infections these species cause in humans, while the other non-virulent species did not produce disease, as expected. We conclude that G. mellonella is not an appropriate infection model for B. pseudomallei and B. thailandensis. Healthcare-associated infections (HAI) are on the rise, and place a heavy burden on our healthcare system each year. Disinfectants used in healthcare settings can reduce HAIs, but first must be evaluated for proper efficacy. To date there are few statistical models that are useful in comparing disinfectant test methods. We conducted a head-to-head comparison of two common test methods, suspension and surface, using Clostridium difficile spores as the test organism. A novel statistical method was developed to evaluate which test method better predicted disinfectant performance. An activated disinfectant that gradually lost activity over time was used in these evaluations. Results showed that the suspension test method was less variable, and was a better predictor of disinfectant efficacy over time.

This research examined the impacts of water quality, phosphate-based corrosion inhibitors and pipe wall exposure on free chlorine (HOCl)/chloramine (NH2Cl) degradation and haloacetic acid (HAA)/N-nitrosodimethylamine (NDMA) formation in simulated distribution system water mains and household plumbing at bench-scale and pilot scale. In bench-scale bottle tests, the reactivity of fresh/pre-corroded pipe materials with HOCl/NH2Cl in decreasing order was: ductile iron, copper, lead. The addition of phosphate-based corrosion inhibitors generally increased HOCl/NH2Cl degradation for fresh iron coupons, but decreased HOCl/NH2Cl decay only for fresh copper coupons. Generally, these corrosion inhibitors did not impact HAA formation. Copper corrosion products, including Cu(II), Cu2O, CuO and Cu2(OH)2CO3, catalyzed HAA and NDMA formation. For HAAs, copper catalysis increased with increasing pH from 6.6 to 8.6 and/or increasing concentrations of these copper corrosion products. Interactions of copper with natural organic matter (NOM), likely by complexation, and the subsequent increase in the reactivity of NOM were proposed to be the primary reason for the increased HAA formation.NDMA formation increased with increasing Cu(II) concentrations, DMA concentrations, alkalinity and hardness but was inhibited by the presence of NOM. The transformation of NH2Cl to dichloramine (NHCl2) and complexation of copper with DMA were proposed to be involved in elevating the formation of NDMA at pH 7.0. Finally, in pilot-scale modified pipe loop tests, copper catalysis of NDMA formation was confirmed, especially under laminar flow conditions, and iron was shown to possibly catalyze NDMA formation under turbulent conditions. Orthophosphate increased the catalytic effects of iron but decreased copper catalysis on NDMA formation by either modifying the properties of the iron-associated suspended particles or reducing the dissolved metal concentrations. Orthophosphate increased chloramine decay when in contact with iron, likely by promoting nitrite formation, but orthophosphate decreased chloramine decay for copper and lead by reducing the availability of metal corrosion products.

This research examined the impacts of water quality, phosphate-based corrosion inhibitors and pipe wall exposure on free chlorine (HOCl)/chloramine (NH2Cl) degradation and haloacetic acid (HAA)/N-nitrosodimethylamine (NDMA) formation in simulated distribution system water mains and household plumbing at bench-scale and pilot scale. In bench-scale bottle tests, the reactivity of fresh/pre-corroded pipe materials with HOCl/NH2Cl in decreasing order was: ductile iron, copper, lead. The addition of phosphate-based corrosion inhibitors generally increased HOCl/NH2Cl degradation for fresh iron coupons, but decreased HOCl/NH2Cl decay only for fresh copper coupons. Generally, these corrosion inhibitors did not impact HAA formation. Copper corrosion products, including Cu(II), Cu2O, CuO and Cu2(OH)2CO3, catalyzed HAA and NDMA formation. For HAAs, copper catalysis increased with increasing pH from 6.6 to 8.6 and/or increasing concentrations of these copper corrosion products. Interactions of copper with natural organic matter (NOM), likely by complexation, and the subsequent increase in the reactivity of NOM were proposed to be the primary reason for the increased HAA formation.NDMA formation increased with increasing Cu(II) concentrations, DMA concentrations, alkalinity and hardness but was inhibited by the presence of NOM. The transformation of NH2Cl to dichloramine (NHCl2) and complexation of copper with DMA were proposed to be involved in elevating the formation of NDMA at pH 7.0. Finally, in pilot-scale modified pipe loop tests, copper catalysis of NDMA formation was confirmed, especially under laminar flow conditions, and iron was shown to possibly catalyze NDMA formation under turbulent conditions. Orthophosphate increased the catalytic effects of iron but decreased copper catalysis on NDMA formation by either modifying the properties of the iron-associated suspended particles or reducing the dissolved metal concentrations. Orthophosphate increased chloramine decay when in contact with iron, likely by promoting nitrite formation, but orthophosphate decreased chloramine decay for copper and lead by reducing the availability of metal corrosion products.

The 2014 West Africa Ebola outbreak saw a substantial number of healthcare workers (HCWs) being infected, despite the use of personal protective equipment (PPE). PPE is intended to protect HCWs when caring for patients with Ebola virus disease (EVD), but PPE may play a role in the spread of Ebola in healthcare environments. Before the removal of PPE, chemical disinfection may prevent the transfer of pathogens to HCWs, but the efficacy of common disinfectants against enveloped viruses, such as Ebola, on PPE surfaces is relatively unknown. The purpose of this study is to assess the efficacy of two common disinfectants, chlorine bleach (Clorox® bleach) and quaternary ammonium (Micro-Chem Plus®), used in healthcare settings for inactivation of enveloped viruses on PPE. The virucidal activity of the two disinfectants were tested against bacteriophage Φ6, an enveloped, non-pathogenic surrogate for enveloped viruses, on Tyvek suit surfaces. Virus was dried onto Tyvek suit surface, exposed to the disinfectants at use-dilution for a contact time of one minute, and the surviving virus was quantified using a double agar layer (DAL) assay. The Clorox® bleach and Micro-Chem Plus® produced a >3.21 log10 reduction and >4.33 log10 reduction, respectively, in Φ6 infectivity. The results of this study suggest that chlorine bleach and quaternary ammonium are effective in the inactivation of enveloped viruses on Tyvek suit surfaces. Chemical disinfection of PPE should be considered as a viable method to reduce the spread of pathogenic, enveloped viruses to HCWs, patients, and other environmental surfaces in healthcare settings.

This research used the tracer study method to determine more accurate disinfectant contact times in the City of Carbondale Water Treatment Plant. In this investigation, three clarifiers were selected to be the experimental segments. Water samples coming from each clarifier and the confluence of the three clarifiers were analyzed to obtain the disinfectant contact times. Based on a continuous source of infinite duration model and the tool "Solver" in Microsoft Excel, this study optimized the experimental data. The experimental data and the optimized data were then analyzed to determine the disinfectant contact times using the graphical method and the numerical method. The disinfectant contact times obtained between using the experimental data and the optimized data, and between using the graphical method and the numerical method were then compared. The results from the numerical method on the optimized data were most accurate and reliable according to these comparisons. The results suggested that the disinfectant contact times were 192.7 min for the first clarifier when its flow rate was 5.72 ML/d; 161.46 min for the second clarifier when its flow rate was 5.99 ML/d; 156.06 min for the third clarifier when its flow rate was 6.26 ML/d; and 168.48 min for the confluence of all clarifiers when its flow rate was 17.5 ML/d, respectively. The feed flows increase down the distribution manifold changing the disinfectant contact times for the clarifiers.