University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 8-2017 Experiment-Based Quantitative Modeling for the Antibacterial Activity of Silver Nanoparticles Mohammad Aminul Haque University of Arkansas, Fayetteville Follow this and additional works at:http://scholarworks.uark.edu/etd Part of theBiophysics Commons, and theNanoscience and Nanotechnology Commons Recommended Citation Haque, Mohammad Aminul, "Experiment-Based Quantitative Modeling for the Antibacterial Activity of Silver Nanoparticles" (2017). Theses and Dissertations. 2445. http://scholarworks.uark.edu/etd/2445 This Thesis is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please [email protected], [email protected]. Experiment-Based Quantitative Modeling for the Antibacterial Activity of Silver Nanoparticles A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Electrical Engineering by Mohammad Aminul Haque University of Dhaka Bachelor of Science in Applied Physics, Electronics and Communication Engineering, 2014 August 2017 University of Arkansas This thesis is approved for recommendation to the Graduate Council _______________________________ Dr. Yong Wang Thesis director _______________________________ ______________________________ Professor Morgan Ware Dr. Zhong Chen Committee member Committee member Abstract Silver (Ag) has been well known for its antimicrobial activity for a long time. Recent research showed the potential of Ag nanoparticles as emerging antimicrobial agents. However, little quantitative analysis has been performed so far to decipher the mechanism of interaction between nanoparticles and bacteria. Here, a detailed analysis based on kinetic growth assay and colony forming unit assay has been carried out to study the antimicrobial effect of Ag nanoparticles against Escherichia coli (E. coli) bacteria. It was observed that the presence of Ag nanoparticles increased the lag time of bacterial growth while not affecting the maximum growth rate significantly. Besides, they can inhibit bacterial growth in the exponential phase by killing some E. coli bacteria cells. A quantitative model was developed to describe the observed antimicrobial behaviors of Ag nanoparticles. The model can successfully predict the experimental measurements. In addition, a mathematical approach to extract the model parameters using experimental data has also been described. It is expected that the model along with the parameters will help to understand the antimicrobial activity of Ag nanoparticles. Acknowledgments I would like to express my gratitude to my advisor Dr. Yong Wang for his continuous support and mentorship throughout my MSEE study. I am also thankful to other members in my thesis committee for their suggestions. I am grateful to my parents for their support and guidance. Special thanks to my lab coworkers for their help and support during the experiments. I would also like to thank my friends for their suggestions and encouragement towards completing this thesis work. Table of Contents CHAPTER ONE ......................................................................................................................... 1 Introduction ................................................................................................................................1 1.1 History of using antimicrobial agents ....................................................................................1 1.2 Silver nanoparticles ...............................................................................................................5 1.3 Applications of nanoparticles against bacteria .......................................................................6 1.4 Mechanism of silver-bacteria interaction ...............................................................................9 1.5 Motivation of the work ........................................................................................................ 11 CHAPTER TWO ...................................................................................................................... 12 Experiment................................................................................................................................ 12 2.1 Synthesis of silver nanoparticles (AgNPs) ........................................................................... 12 2.2 Characterization of silver nanoparticles (AgNPs)................................................................. 13 2.3 Growth of bacteria ............................................................................................................... 16 2.4 Kinetic Growth Curve Experiments ..................................................................................... 17 2.5 CFU Assay and Time Kill Measurements ............................................................................ 18 CHAPTER THREE................................................................................................................... 20 Experimental observations and data analysis ............................................................................. 20 3.1 Kinetic Growth Curve assay: ............................................................................................... 20 3.2 Colony Forming Unit (CFU) assay ...................................................................................... 22 3.2 Time-kill curves: ................................................................................................................. 33 CHAPTER FOUR ..................................................................................................................... 40 Quantitative Model and Verification ......................................................................................... 40 4.1 Proposed Quantitative model: .............................................................................................. 40 4.2 Application in experimental condition for Verification ........................................................ 43 4.3 Parameter extraction and Application .................................................................................. 47 CHAPTER FIVE ...................................................................................................................... 59 Discussion and Conclusion ........................................................................................................ 59 References ................................................................................................................................ 61 Appendix .................................................................................................................................. 67 List of Published Papers 1. M. Haque, R. Imamura, G. A. Brown, T. Marcelle, J. Chen, and Y. Wang, “An Experiment- Based Quantitative Model Describing Antimicrobial Activity of Silver Nanoparticles on Escherichia coli,” Sci. Rep., 2017, under review. CHAPTER ONE Introduction 1.1 History of using antimicrobial agents Different plants were used to heal wounds thousands of years ago [1]. Allantoin is found in Comfrey (Symphytum officinale) which is antibacterial as well as a healing agent [1]. Leaves of St John's wort (Hypericum perforatum) are also supposed to have some healing effect in perforating wound [1]. Grasses have always been used as bandages for soothing [1]. Honey, animal fats and butter were later used in wound treatment [1]. Antagonistic behavior of some microorganisms against bacteria was first observed by William Roberts (1874) and John Tyndall (1876) [2]. Roberts reported inhibition of bacteria on Penicillium glaucum covered media and Tyndall was able to explain the hostility between bacteria and moulds (multicellular filament fungus) [2]. In 1897, Ernest Duchesne, while working towards his PhD, observed the antimicrobial activity of Penicillium glaucum [3]. This is the first known published research on the antimicrobial activity of moulds [3]. In 1928, Sir Alexander Fleming proposed the existence of penicillin in Penicillium chrysogenum secretion preventing the growth of bacteria [4]. The improvement in this field encouraged the scientists to pursue further research for more antibiotics [5]. In 1939, Rene Dubos was able to synthesize the first naturally derived antibiotic, tyrothricin [6]. It contained gramicidin and tyrocidine [6]. Tyrocidine attacked both gram- positive and gram-negative bacteria whereas gramicidin was able to inhibit gram-positive bacteria [6]. In fact, gramicidin was used to treat wounds and ulcers during World War II [6]. However, it was not suitable against systemic infections due to toxicity [6]. 1 Fig. 1.1: A magnified electron microscopic image of E. coli [7] E. coli, discovered by Theodor Escherich in 1885, is a rod-shaped gram-negative bacterium frequently found in lower intestine of warm-blooded lives [8]. Although most of them are found inside the body, they are capable of surviving outside the body [9]. They are 2 μm long, 0.5-2 μm in diameter while the cell volume is 0.6-0.7 μm3 [9]. Most frequently used temperature for E. coli growth is 37 0C [9]. E. coli can transfer DNA from generation to generation through conjugation, transduction or transformation [9]. Bacterial strains (sub-type of bacteria having exclusive properties to differentiate from other strains) are host specific [9]. By knowing which strain is present in human body, it can be determined where the contamination arises from (for instance, from another human or animal) [9]. Although use of temperature beyond 37 0C is not recommended for E. coli growth, protocol has been developed to grow E. coli (DH5alpha) up to 49 0C [10]. It means that E. coli (DH5alpha) can go through mutation that enables them to grow at temperature beyond 37 0C [10]. E. coli can grow in any medium (for instance, LB) containing ammonium phosphate, sodium chloride, magnesium sulfate, potassium phosphate, glucose and water. Both aerobic and anaerobic respiration can drive the growth of E. coli [11]. E. coli is termed as facultative anaerobic. It uses oxygen for growth wherever it is present. However, it can 2 still grow through anaerobic respiration if oxygen is present. Therefore, its growth is accelerated if water increases in the environment [12]. Many of the E. coli bacteria are nonpathogenic and can be good source of vitamin K thereby 2 benefitting the host [13]. However, some can cause food contamination and food poisoning [14]. Harmful strains can cause gastroenteritis, urinary tract infections, neonatal meningitis etc [15]– [17]. Gastroenteritis is a complex biological response of stomach and small intestine to pathogens or damaged cells [18]. Vomiting, diarrhea, abdominal pain and fever are some of the common syndromes [18]. When part of the urinary tract (kidneys, bladders, ureters and urethra) is affected by bacterial infection, it is termed as urinary tract infection [19]. Bladder infection (cystitis) occurs when lower urinary tract is affected whereas infection in the upper urinary tract is called kidney infection (pyelonephritis) [20]. Pain with urination, frequent urination and fever are some symptoms of urinary tract infection [19], [20]. Neonatal meningitis is a complex response of the meninges (membranes protecting brain and spinal cords) and a serious medical problem in infants [21]. The possible symptoms are fever, poor appetite, vomiting, diarrhoea, neck rigidity, jaundice etc. [15]–[17], [22]. Properly cooking food, use of gloves, clean drinking water, pasteurization etc are some of the ways to prevent contamination [9]. E. coli has been widely used in research for the following reasons:  E. coli has small genome size with respect to eukaryotes. They have about 4400 genes whereas humans have almost 30000 genes [23], [24]. 3