Matching Items (11)
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- All Subjects: Legionella
- All Subjects: Antibodies
- Creators: Abbaszadegan, Morteza
Description
Bacteria of the Legionella genus are a water-borne pathogen of increasing concern due to being responsible for more annual drinking water related disease outbreaks in the United States than all other microbes combined. Unfortunately, the development of public health policies concerning Legionella has impeded by several key factors, including a paucity of data on their interactions and growth requirements in water distribution networks, a poor understanding of potential transmission sources for legionellosis, and limitations in current methodology for the characterization of these pathogens. To address these issues, a variety of research approaches were taken. By measuring Legionella survival in tap water, association in pipe material biofilms, population dynamics in a model distribution system, and occurrence in drinking water distribution system biofilms, key aspects of Legionella ecology in drinking water systems were revealed. Through a series of experiments qualitatively and quantitatively examining the growth of Legionella via nutrients obtained from several water sources, environmental nutritional requirements and capability for growth in the absence of host organisms were demonstrated. An examination of automobile windshield washer fluid as a possible source of legionellosis transmission revealed Legionella survival in certain windshield washer fluids, growth within washer fluid reservoirs, high levels and frequency of contamination in washer fluid reservoirs, and the presence of viable cells in washer fluid spray, suggesting the potential for exposure to Legionella from this novel source. After performing a systematic and quantitative analysis of methodology optimization for the analysis of Legionella cells via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, several strains of this microbe isolated from separated and varied environmental water sampling sites were distinctly typed, demonstrating a potential application of this technology for the characterization of Legionella. The results from this study provide novel insight and methodology relevant to the development of programs for the monitoring and treatment of Legionella in drinking water systems.
ContributorsSchwake, David Otto (Author) / Abbaszadegan, Morteza (Thesis advisor) / Alum, Absar (Committee member) / Fox, Peter (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2014
Description
Granular activated carbon (GAC) filters are final polishing step in the drinking water treatment systems for removal of dissolved organic carbon fractions. Generally filters are colonized by bacterial communities and their activity reduces biodegradable solutes allowing partial regeneration of GAC's adsorptive capacity. When the bacteria pass into the filtrate due to increased growth, microbiological quality of drinking water is compromised and regrowth in the distribution system occurs. Bacteria attached to carbon particles as biofilms or in conjugation with other bacteria were observed to be highly resistant to post filtration microbial mitigation techniques. Some of these bacteria were identified as pathogenic.
This study focuses on one such pathogen Legionella pneumophila which is resistant to environmental stressors and treatment conditions. It is also responsible for Legionnaires' disease outbreak through drinking water thus attracting attention of regulatory agencies. The work assessed the attachment and colonization of Legionella and heterotrophic bacteria in lab scale GAC media column filters. Quantification of Legionella and HPC in the influent, effluent, column's biofilms and on the GAC particles was performed over time using fluorescent microscopy and culture based techniques.
The results indicated gradual increase in the colonization of the GAC particles with HPC bacteria. Initially high number of Legionella cells were detected in the column effluent and were not detected on GAC suggesting low attachment of the cells to the particles potentially due to lack of any previous biofilms. With the initial colonization of the filter media by other bacteria the number of Legionella cells on the GAC particles and biofilms also increased. Presence of Legionella was confirmed in all the samples collected from the columns spiked with Legionella. Significant increase in the Legionella was observed in column's inner surface biofilm (0.25 logs up to 0.52 logs) and on GAC particles (0.42 logs up to 0.63 logs) after 2 months. Legionella and HPC attached to column's biofilm were higher than that on GAC particles indicating the strong association with biofilms. The bacterial concentration slowly increased in the effluent. This may be due to column's wall effect decreasing filter efficiency, possible exhaustion of GAC capacity over time and potential bacterial growth.
This study focuses on one such pathogen Legionella pneumophila which is resistant to environmental stressors and treatment conditions. It is also responsible for Legionnaires' disease outbreak through drinking water thus attracting attention of regulatory agencies. The work assessed the attachment and colonization of Legionella and heterotrophic bacteria in lab scale GAC media column filters. Quantification of Legionella and HPC in the influent, effluent, column's biofilms and on the GAC particles was performed over time using fluorescent microscopy and culture based techniques.
The results indicated gradual increase in the colonization of the GAC particles with HPC bacteria. Initially high number of Legionella cells were detected in the column effluent and were not detected on GAC suggesting low attachment of the cells to the particles potentially due to lack of any previous biofilms. With the initial colonization of the filter media by other bacteria the number of Legionella cells on the GAC particles and biofilms also increased. Presence of Legionella was confirmed in all the samples collected from the columns spiked with Legionella. Significant increase in the Legionella was observed in column's inner surface biofilm (0.25 logs up to 0.52 logs) and on GAC particles (0.42 logs up to 0.63 logs) after 2 months. Legionella and HPC attached to column's biofilm were higher than that on GAC particles indicating the strong association with biofilms. The bacterial concentration slowly increased in the effluent. This may be due to column's wall effect decreasing filter efficiency, possible exhaustion of GAC capacity over time and potential bacterial growth.
ContributorsSharma, Harsha (Author) / Abbaszadegan, Morteza (Thesis advisor) / Alum, Absar (Committee member) / Fox, Peter (Committee member) / Arizona State University (Publisher)
Created2014
Description
Research in microbial biofuels has dramatically increased over the last decade. The bulk of this research has focused on increasing the production yields of cyanobacteria and algal cells and improving extraction processes. However, there has been little to no research on the potential impact of viruses on the yields of these phototrophic microbes for biofuel production. Viruses have the potential to significantly reduce microbial populations and limit their growth rates. It is therefore important to understand how viruses affect phototrophic microbes and the prevalence of these viruses in the environment. For this study, phototrophic microbes were grown in glass bioreactors, under continuous light and aeration. Detection and quantification of viruses of both environmental and laboratory microbial strains were measured through the use of a plaque assay. Plates were incubated at 25º C under continuous direct florescent light. Several environmental samples were taken from Tempe Town Lake (Tempe, AZ) and all the samples tested positive for viruses. Virus free phototrophic microbes were obtained from plaque assay plates by using a sterile loop to scoop up a virus free portion of the microbial lawn and transferred into a new bioreactor. Isolated cells were confirmed virus free through subsequent plaque assays. Viruses were detected from the bench scale bioreactors of Cyanobacteria Synechocystis PCC 6803 and the environmental samples. Viruses were consistently present through subsequent passage in fresh cultures; demonstrating viral contamination can be a chronic problem. In addition TEM was performed to examine presence or viral attachment to cyanobacterial cells and to characterize viral particles morphology. Electron micrographs obtained confirmed viral attachment and that the viruses detected were all of a similar size and shape. Particle sizes were measured to be approximately 50-60 nm. Cell reduction was observed as a decrease in optical density, with a transition from a dark green to a yellow green color for the cultures. Phototrophic microbial viruses were demonstrated to persist in the natural environment and to cause a reduction in algal populations in the bioreactors. Therefore it is likely that viruses could have a significant impact on microbial biofuel production by limiting the yields of production ponds.
ContributorsKraft, Kyle (Author) / Abbaszadegan, Morteza (Thesis advisor) / Alum, Absar (Committee member) / Fox, Peter (Committee member) / Arizona State University (Publisher)
Created2014
Description
This study was devised to elucidate key information concerning the potential risk posed by Legionella in reclaimed water. A series of biological experiments and a recharge basin soil column study were conducted to examine the survival, growth, and transport of L. pneumophila through engineered reclaimed water systems. A pilot-scale, column study was set up to measure Legionella transport in the columns under Arizona recharge basin conditions. Two columns, A and B, were packed to a depth of 122 cm with a loamy sand media collected from a recharge basin in Mesa, Arizona. The grain size distribution of Column A differed from that of Column B by the removal of fines passing the #200 sieve. The different soil profiles represented by column A and B allowed for further investigation of soil attributes which influence the microbial transport mechanism. Both clear PVC columns stand at a height of 1.83 m with an inner diameter of 6.35 cm. Sampling ports were drilled into the column at the soil depths 15, 30, 60, 92, 122 cm. Both columns were acclimated with tertiary treated waste water and set to a flow rate of approximately 1.5 m/d. The columns were used to assess the transport of a bacterial indicator, E. coli, in addition to assessing the study's primary pathogen of concern, Legionella. Approximately, 〖10〗^7 to 〖10〗^9 E. coli cells or 〖10〗^6 to 〖10〗^7Legionella cells were spiked into the columns' head waters for each experiment. Periodically, samples were collected from each column's sampling ports, until a minimum of three pore volume passed through the columns.
The pilot-scale, column study produced novel results which demonstrated the mechanism for Legionella to be transported through recharge basin soil. E. coli was transported, through 122 cm of the media in under 6 hours, whereas, Legionella was transported, through the same distance, in under 30 hours. Legionella has been shown to survive in low nutrient conditions for over a year. Given the novel results of this proof of concept study, a claim can be made for the transport of Legionella into groundwater aquifers through engineering recharge basin conditions, in Central Arizona.
The pilot-scale, column study produced novel results which demonstrated the mechanism for Legionella to be transported through recharge basin soil. E. coli was transported, through 122 cm of the media in under 6 hours, whereas, Legionella was transported, through the same distance, in under 30 hours. Legionella has been shown to survive in low nutrient conditions for over a year. Given the novel results of this proof of concept study, a claim can be made for the transport of Legionella into groundwater aquifers through engineering recharge basin conditions, in Central Arizona.
ContributorsMcBurnett, Lauren Rae (Author) / Abbaszadegan, Morteza (Thesis advisor) / Alum, Absar (Committee member) / Fox, Peter (Committee member) / Arizona State University (Publisher)
Created2014
Description
Legionella pneumophila is a waterborne pathogen that causes Legionnaires' disease, an infection which can lead to potentially fatal pneumonia. In a culture-based technique, Legionella is detected using buffered charcoal-yeast extract (BCYE) agar supplemented with L-cysteine, Iron salt and antibiotics. These supplements provide essential and complex nutrient requirements and help in the suppression of non-target bacteria in Legionella analysis. Legionella occurs naturally in freshwater environments and for their detection; a sample is plated on solid agar media and then incubated for several days. There are many challenges in the detection of Legionella in environmental waters and the built environments. A common challenge is that a variety of environmental bacteria can be presumptively identified as Legionella using the culture-based method. In addition, proper identification of Legionella requires long incubation period (3-9 days) while antibiotics used in BCYE agar have relatively short half-life time. In order to overcome some of the challenges, Legionella has been genetically modified to express reporter genes such Green Fluorescent Protein (GFP) that can facilitate its detection in process validation studies under controlled laboratory conditions. However, such studies had limited success due to the instability of genetically modified Legionella strains. The development of a genetically modified Legionella with a much rapid growth rate (1-2 days) in simulated environmental systems (tightly-controlled water distribution system) is achieved. The mutant Legionella is engineered by transforming with a specific plasmid encoding CymR, LacZ and TetR genes. The newly engineered Legionella can grow on conventional BCYE agar media without L-Cysteine, Iron salt and only require one antibiotic (Tetracycline) to suppress the growth of other microorganisms in media. To the best of our knowledge, this is the first report of L. pneumophila strain capable of growing without L-Cysteine. We believe that this discovery would not only facilitate the study of the fate and transport of this pathogen in environmental systems, but also further our understanding of the genetics and metabolic pathways of Legionella.
ContributorsAloraini, Saleh Ali A (Author) / Abbaszadegan, Morteza (Thesis advisor) / Fox, Peter (Committee member) / Alum, Absar (Committee member) / Arizona State University (Publisher)
Created2018
Description
Background: Measles virus (MV) infections are the main cause of vaccine-preventable death in children younger than 5 years. The World Health Organization (WHO) has estimated there are over 20 million cases of measles every year. Currently, diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. These commercial assays measure reactivity against the immunodominant N antigen and can have a false negative rates of 20-30%. Centralized testing by clinical labs can delay rapid screening in an outbreak setting. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to five individual MV proteins representing 85% of the measles proteome. Methods: MV genes were subcloned into pANT_cGST vector to generate C-terminal GST fusion proteins. Single MV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured using a sandwich ELISA for GST expression using relative light units (RLUs) as readouts. Single MV antigens were used as bait to determine the IgG-dependent reactivity in 12 serum samples obtained from immunized animals with previously determined neutralization titer (NT) and the correlation between NT and ELISA reactivity was determined. Results: Protein expression of five measles genes of interest, M, N, F, H, and L, was measured. L exhibited the strongest protein expression with an average RLU value of 4.34 x 10^9. All proteins were expressed at least 50% greater than control (2.33 x 10^7 RLU). As expected, reactivity against the N was the highest, followed by reactivity against M, F, H and L. The best correlation with NT titer was reactivity against F (R^2 = 0.62). Conclusion: These data indicate that the expression of single MV genes M, N, F, H, and L are suitable antigens for serologic capture analysis of measles immunity.
ContributorsMushtaq, Zuena (Author) / Anderson, Karen (Thesis director) / Reyes del Valle, Jorge (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
This project aims to address the current protocol regarding the diagnosis and treatment of traumatic brain injury (TBI) in medical industries around the world. Although there are various methods used to qualitatively determine if TBI has occurred to a patient, this study attempts to aid in the creation of a system for quantitative measurement of TBI and its relative magnitude. Through a method of artificial evolution/selection called phage display, an antibody that binds highly specifically to a post-TBI upregulated brain chondroitin sulfate proteoglycan called neurocan has been identified. As TG1 Escheria Coli bacteria were infected with KM13 helper phage and M13 filamentous phage in conjunction, monovalent display of antibody fragments (ScFv) was performed. The ScFv bind directly to the neurocan and from screening, phage that produced ScFv's with higher affinity and specificity to neurocan were separated and purified. Future research aims to improve the ScFv characteristics through increased screening toward neurocan. The identification of a highly specific antibody could lead to improved targeting of neurocan post-TBI in-vivo, aiding researchers in quantitatively defining TBI by visualizing its magnitude.
ContributorsSeelig, Timothy Scott (Author) / Stabenfeldt, Sarah (Thesis director) / Ankeny, Casey (Committee member) / Barrett, The Honors College (Contributor) / Harrington Bioengineering Program (Contributor)
Created2015-05
Description
Measles is a contagious, vaccine-preventable disease that continues to be the leading
cause of death in children younger than the age of 5 years. While the introduction of the Measles, Mumps, and Rubella vaccine (MMR) has significantly decreased morbidity and mortality rates worldwide, vaccine coverage is highly variable across global regions. Current diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. Commercially available Diamedix Immunosimplicity® Measles IgG test kit has been shown to have 91.1% sensitivity and 93.8% specificity, with a positive predictive value of 88.7% and a negative predictive value of 90.9% on the basis of a PRN titer of 120. There is an increasing need for rapid screening for measles specific immunity in outbreak settings. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to three individual measles virus (MeV) proteins.
Measles virus (MeV) genes were subcloned into the pJFT7_nGST vector to generate N- terminal GST fusion proteins. Single MeV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured with mouse anti-GST mAb and sheep anti-mouse IgG. Relative light units (RLUs) as luminescence was measured. Antibodies to MeV antigens were measured in 40 serum samples from healthy subjects.
Protein expression of three MeV genes of interest was measured in comparison with vector control and statistical significance was determined using the Student’s t-test (p<0.05). N expressed at the highest level with an average RLU value of 3.01 x 109 (p<0.001) and all proteins were expressed at least 50% greater than vector control (4.56 x 106 RLU). 36/40 serum samples had IgG to N (Ag:GST ratio>1.21), F (Ag:GST ratio>1.92), or H (Ag:GST ratio> 1.23).
These data indicate that the in vitro expression of MeV antigens, N, F, and H, were markedly improved by subcloning into pJFT7_nGST vector to generate N-terminal GST fusion proteins. The expression of single MeV genes N, F and H, are suitable antigens for serologic capture analysis of measles-specific antibodies. These preliminary data can be used to design a more intensive study to explore the possibilities of using these MeV antigens as a diagnostic marker.
cause of death in children younger than the age of 5 years. While the introduction of the Measles, Mumps, and Rubella vaccine (MMR) has significantly decreased morbidity and mortality rates worldwide, vaccine coverage is highly variable across global regions. Current diagnostic methods rely on enzyme immunoassays (EIA) to detect IgM or IgG Abs in serum. Commercially available Diamedix Immunosimplicity® Measles IgG test kit has been shown to have 91.1% sensitivity and 93.8% specificity, with a positive predictive value of 88.7% and a negative predictive value of 90.9% on the basis of a PRN titer of 120. There is an increasing need for rapid screening for measles specific immunity in outbreak settings. This study aims to develop a rapid molecular diagnostic assay to detect IgG reactive to three individual measles virus (MeV) proteins.
Measles virus (MeV) genes were subcloned into the pJFT7_nGST vector to generate N- terminal GST fusion proteins. Single MeV cistrons were expressed using in vitro transcription/translation (IVTT) with human cell lysate. Expression of GST-tagged proteins was measured with mouse anti-GST mAb and sheep anti-mouse IgG. Relative light units (RLUs) as luminescence was measured. Antibodies to MeV antigens were measured in 40 serum samples from healthy subjects.
Protein expression of three MeV genes of interest was measured in comparison with vector control and statistical significance was determined using the Student’s t-test (p<0.05). N expressed at the highest level with an average RLU value of 3.01 x 109 (p<0.001) and all proteins were expressed at least 50% greater than vector control (4.56 x 106 RLU). 36/40 serum samples had IgG to N (Ag:GST ratio>1.21), F (Ag:GST ratio>1.92), or H (Ag:GST ratio> 1.23).
These data indicate that the in vitro expression of MeV antigens, N, F, and H, were markedly improved by subcloning into pJFT7_nGST vector to generate N-terminal GST fusion proteins. The expression of single MeV genes N, F and H, are suitable antigens for serologic capture analysis of measles-specific antibodies. These preliminary data can be used to design a more intensive study to explore the possibilities of using these MeV antigens as a diagnostic marker.
ContributorsMushtaq, Zuena (Author) / Anderson, Karen (Thesis advisor) / Blattman, Joseph (Committee member) / Lake, Douglas (Committee member) / Arizona State University (Publisher)
Created2016
Description
Legionella is a gram-negative bacterium with the ability for human infection by inhalation or aspiration of water containing the bacteria. Legionella live in aquatic environments and have been identified in cooling towers, humidifiers and respiratory therapy treatments, among others. Infection with Legionella bacteria leads to Legionnaire’s Disease or Pontiac Fever (Edelstein, 1993). Information regarding the means of aerosolization of Legionella bacteria has not yet been reported, therefore the relevance of experimentation was defined. The objective of this study is to determine the modes by which bacteria may be aerosolized under laboratory conditions. Specifically, to measure the amount of bacteria transported over a specific distance in a given amount of time and determine the most effective mode of bacterial aerosolization. Three methods of bacterial aerosolization were tested, these included an electric paint sprayer, an air paint sprayer and a hand-held spray bottle. E. coli was used as a surrogate for Legionella in experimentation due to its similar bacterial properties. Both bacteria are gram-negative, aerobic bacilli while Legionella is approximately 2 μm in length (Botzenhart, 1998), and E. coli is between 1 and 3 μm in length (Reshes, 2007). The accessibility and non-pathogenicity of E. coli also served as factors for the substitution.
In order to measure the aerosolization efficiency of each spray method, an air sampler was placed opposite to the position of the sprayer, on either side of a sealed box. Each sprayer was filled with E. coli concentrated at 104 CFU/ml in a PBS solution and sprayed for a time span of 1 and 5 seconds. For each of these time intervals an air sample was collected immediately following the spray as well as 5 minutes after the spray. Compared to the other two methods, the air spray method consistently showed the highest number of bacterial cells aerosolized. While all three methods resulted in the aerosolization of bacteria, the results determined the Air Spray method as the most efficient means of bacterial aerosolization. In this study, we provide a practical and efficient method of bacterial aerosolization for microbial dispersion in air. The suggested method can be used in future research for microbial dispersion and transmission studies.
In addition, a humidifier was filled with a spiked solution of E. coli and operated for a period of 1 and 5 seconds at its maximum output. Air samples were collected after 0 and 5 minutes. Immediately after the humidifier operation was stopped a small number of colonies were detected in the air sample and no colonies were detected in the air sample collected after a 5-minute elapsed time. This experiment served as a proof of concept for airborne pathogen’s transmission by a humidifier.
In order to measure the aerosolization efficiency of each spray method, an air sampler was placed opposite to the position of the sprayer, on either side of a sealed box. Each sprayer was filled with E. coli concentrated at 104 CFU/ml in a PBS solution and sprayed for a time span of 1 and 5 seconds. For each of these time intervals an air sample was collected immediately following the spray as well as 5 minutes after the spray. Compared to the other two methods, the air spray method consistently showed the highest number of bacterial cells aerosolized. While all three methods resulted in the aerosolization of bacteria, the results determined the Air Spray method as the most efficient means of bacterial aerosolization. In this study, we provide a practical and efficient method of bacterial aerosolization for microbial dispersion in air. The suggested method can be used in future research for microbial dispersion and transmission studies.
In addition, a humidifier was filled with a spiked solution of E. coli and operated for a period of 1 and 5 seconds at its maximum output. Air samples were collected after 0 and 5 minutes. Immediately after the humidifier operation was stopped a small number of colonies were detected in the air sample and no colonies were detected in the air sample collected after a 5-minute elapsed time. This experiment served as a proof of concept for airborne pathogen’s transmission by a humidifier.
ContributorsJohnson, Chelsea Elizabeth (Author) / Abbaszadegan, Morteza (Thesis director) / Stout, Valerie (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2015-12
Description
The study was to analyze the extent of bacterial transport in a two-dimensional tank under saturated conditions. The experiments were done in a 2-D tank packed with 3,700 in3 of fine grained, homogenous, chemically inert sand under saturated conditions. The tank used for transport was decontaminated by backwashing with 0.6% chlorine solution with subsequent backwashing with chlorine-neutral water (tap water and Na2S2O3) thus ensuring no residual chlorine in the tank. The transport of bacteria was measured using samples collected from ports at vertical distances of 5, 15 and 25 inches (12.7, 38.1 and 63.5 cm) from the surface of the sand on both sides for the 2-D tank. An influent concentration of 105 CFU/mL was set as a baseline for both microbes and the percolation rate was set at 11.37 inches/day using a peristaltic pump at the bottom outlet. At depths of 5, 15 and 25 inches, E. coli breakthroughs were recorded at 5, 17 and 28 hours for the ports on the right side and 7, 17 and 29 hours for the ports on the left sides, respectively. At respective distances Legionella breakthroughs were recorded at 8, 22 and 35 hours for the ports on the right side and 9, 24, 36 hours for the ports on the left side, respectively which is homologous to its pleomorphic nature. A tracer test was done and the visual breakthroughs were recorded at the same depths as the microbes. The breakthroughs for the dye at depths of 5, 15 and 25 inches, were recorded at 13.5, 41 and 67 hours for the ports on the right side and 15, 42.5 and 69 hours for the ports on the left side, respectively. However, these are based on visual estimates and the physical breakthrough could have happened at the respective heights before the reported times. This study provided a good basis for the premise that transport of bacterial cells and chemicals exists under recharge practices.
ContributorsMondal, Indrayudh (Author) / Abbaszadegan, Morteza (Thesis advisor) / Dahlen, Paul (Committee member) / Delgado, Anca (Committee member) / Arizona State University (Publisher)
Created2019