Synthesis of Cobalt Aluminum Oxide and Aluminum Oxide Nanoparticles and Evaluation of their

Antimicrobial Activity Against Some Pathogens

By Esraa Khodor El-Hadidi

Thesis

Submitted in Partial Fulfillment of the Requirements for

the Degree of Master of Science in Biology

Department of Biological Sciences

Faculty of Science

2020

Synthesis of Cobalt Aluminum Oxide and Aluminum Oxide Nanoparticles and Evaluation of their

Antimicrobial Activity Against Some Pathogens

By Esraa Khodor El-Hadidi

Submitted in Partial Fulfillment of the Requirements for

the Degree of Master of Science in Biology

Department of Biological Sciences

Faculty of Science

Supervised by Prof. Dr. HodaYusef Prof. Dr. Ramadan Awad Professor of Microbiology Professor of Material Science

Biological Sciences Department Dean of Faculty of Science

Faculty of Science Faculty of Science

Beirut Arab University Beirut Arab University

2020

iv

Abstract

With the increasing concern for human health and quality of life, the uses of

nanoparticles for disinfection become more and more important. There is a special

interest in induced bactericidal effect of Aluminum Oxide (Al2O3), Cobalt Aluminum

oxide (CoAl2O4), and Aluminum doped Zinc Oxide (Zn0.9Al0.1O) nanoparticles in the

area of coated ceramic and paints production for hospitals and health cares to reduce

the spread of nosocomial infection. The current study outlines the synthesis of Al2O3,

CoAl2O4, and Zn0.9Al0.1O Nps nanoparticles by co-precipitation method and

subsequently the antimicrobial potentials of these nanoparticles was evaluated against

several pathogenic bacteria along with two fungi, mostly nosocomial pathogens. The

formation and the characterization of Al2O3, CoAl2O4, and Zn0.9Al0.1O nanoparticles

were confirmed by UV-Vis Spectroscopy, X-Ray Diffraction (XRD), Fourier

Transform Infra-Red Spectroscopy (FTIR), and Transmission Electron Microscopy

(TEM). All data revealed the formation of pure nano-sized Al2O3, CoAl2O4, and

Zn0.9Al0.1O NPs. Among the tested synthesized nanoparticles, aluminum doped zinc

oxide show powerful antimicrobial action on the tested bacteria and fungi except

Aspergillus sp.. The effectiveness of the synthesized nanoparticles (Al2O3, CoAl2O4,

and Zn0.9Al0.1O NPs) showed that the action of CoAl2O4 and Zn0.9Al0.1O NPs against

tested bacteria was determined to be bactericidal. Zn0.9Al0.1O NPs exhibited a

privileged ability to suppress the growth of nosocomial pathogens in culture media

and paints as an application. Transmission electron images clearly illustrated a

remarkable damage in E. coli cell’s structure upon exposure to Zn0.9Al0.1O NPs, the

observations suggest that Zn0.9Al0.1O NPs distorted and damaged bacterial cell wall

and cytoplasmic membrane resulting in leakage of protoplasmic content.

v

Table of Contents Acknowledgments .............................................................................................................. v

Abstract .............................................................................................................................. iv

Table of Contents ............................................................................................................... v

List of Tables ................................................................................................................... viii

List of Figures .................................................................................................................... ix

List of Abbreviations…………………………………………………………………….xi

1. Introduction ................................................................................................................ 1

1.1. Nanoparticles ............................................................................................................ 1

1.2. Metal Oxide Nanoparticles ....................................................................................... 1

1.2.1. An Overview of metal oxide nanoparticles ....................................................... 1

1.2.2. Physical-Chemical Characterization of Metal Oxide Nanoparticles ................. 2

1.2.3. Antimicrobial Activity of Metal Oxide Nanoparticles ...................................... 2

1.2.4. Mechanisms of Metal Nanoparticles on Microbes ............................................ 3

1.2.4.1. Interactions with Phospholipid Bilayer ........................................................... 4

1.2.4.2. Production of Reactive Oxygen Species (ROS) ............................................ 5

1.2.4.3. Binding to Cytosolic Proteins .......................................................................... 5

1.3. Aluminum Oxide, Cobalt Aluminum Oxide and Aluminum Doped Zinc Oxide Nanoparticles ....................................................................................................................... 5

1.3.1. Aluminum Oxide Nanoparticles ........................................................................ 5

1.3.2. Applications of Aluminum Oxide Nanoparticles .............................................. 6

1.3.3. Cobalt Aluminum Oxide Nanoparticles ........................................................... 7

1.3.4. Applications of Cobalt Aluminum Oxide Nanoparticles .................................. 8

1.3.5. Aluminum Doped Zinc Oxide Nanoparticles .................................................... 8

1.3.6. Applications of Aluminum Doped Zinc Oxide Nanoparticles .......................... 9

1.4. Toxicity of Nanoparticles ......................................................................................... 9

1.5. Hospital Acquired Infection .................................................................................... 10

1.5.1. Overview of Health Care Environment ........................................................... 10

1.5.2. Spread of Nosocomial Infections .................................................................... 11

1.5.3. Routes of Nosocomial Infection Transmission ............................................... 12

1.5.4. Types of Nosocomial Infection ....................................................................... 13

1.6. Control of Nosocomial Infection ............................................................................ 14

1.6.1. Infection Control Programs ............................................................................. 14

1.6.2. Antimicrobial Use and Resistance ................................................................... 15

vi

1.6.2.1 . Appropriate Antimicrobial Use.................................................................... 15

1.6.2.2. Antibiotic Resistance ....................................................................................... 15

1.6.3. Use of Biocides and Bacterial Resistance ....................................................... 16

1.6.3.1. Use of Biocides ................................................................................................ 16

1.6.3.2. Bacterial Resistance to Biocides ...................................................................... 17

1.7. Application of Nanoparticles in Hospitals and Health Care Facilities ................... 17

1.8. Aim of Study ........................................................................................................... 18

2. Materials and Methods ............................................................................................ 19

1.1. Materials ................................................................................................................. 19

2.1.1. Microbial Pathogens ........................................................................................ 19

2.1.2. Chemicals and Supplements ............................................................................ 19

2.1.3. Culture Media .................................................................................................. 19

2.1.4. Reagents........................................................................................................... 20

2.2. Methods................................................................................................................... 21

2.2.1. Preparation of Nanoparticles ............................................................................... 21

2.2.1.1. Preparation of Aluminum Oxide (Al2O3) NPs by Co-Precipitation Method ... 21

2.2.1.2. Preparation of Cobalt Aluminum Oxide (CoAl2O4) NPs by Co-Precipitation Method ………………………………………………………………………….…….22

2.2.1.3. Preparation of Aluminum Doped Zinc Oxide (Zn0.9Al0.1O) NPs by Co-Precipitation Method .......................................................................................................... 22

2.2.2. Nanoparticles Characterization ........................................................................... 23

2.2.2.1. X-ray Powder Diffraction (XRD) .................................................................... 23

2.2.2.2. Transmission Electron Microscope (TEM) ..................................................... 24

2.2.2.3. Ultraviolet-Visible Absorption Spectroscopy (UV-vis) .................................. 25

2.2.2.4. Fourier Transform Infrared- Spectroscopy (FTIR) ......................................... 26

2.2.3. Preparation of Culture Media .............................................................................. 27

2.2.4. Stock Culture of Microorganisms ....................................................................... 27

2.2.4.1. Agar Slants ...................................................................................................... 27

2.2.4.2. Glycerol Stock ................................................................................................. 27

2.2.5. Inoculum Preparation and Standardization ......................................................... 27

2.2.5.1. Preparation of Bacterial Inocula ...................................................................... 27

2.2.5.2. Preparation of Fungal Inocula ......................................................................... 28

2.2.6. Antimicrobial Susceptibility Tests ...................................................................... 28

2.2.6.1. Disk Diffusion method .................................................................................... 28

vii

2.2.6.2. Broth Micro Dilution Method.......................................................................... 29

2.2.6.2.1. Determination of Minimum Inhibitory Concentration (MIC) ................... 29

2.2.6.2.2. Determination of Minimum Bactericidal Concentration (MBC) .............. 29

2.2.6.3. Time Kill Study ............................................................................................... 30

2.2.6.4. Transmission Electron Microscope ................................................................. 30

2.2.7. Application of Aluminum Doped Zinc Oxide Nanoparticles in a Paint Sample 31

3. Results and Discussion ............................................................................................. 32

3.1. Characterization of Al2O3, CoAl2O4, and Zn0.9Al0.1O Nanoparticles ..................... 32

3.1.1. X-ray Diffraction (XRD) ................................................................................. 32

3.1.2. Transmission Electron Microscope (TEM) ..................................................... 35

3.1.3. Ultraviolet -Visible Absorption Spectroscopy (UV-vis) ................................. 36

3.1.4. Fourier Transform Infrared Spectroscopy (FTIR) ........................................... 38

3.2. Antimicrobial Susceptibility Tests .......................................................................... 40

3.2.1. Disc Diffusion Method .................................................................................... 40

2.2.2. Broth Micro Dilution Method.......................................................................... 43

3.2.2.1. Determination of Minimum Inhibitory Concentration (MIC) ................... 43

3.2.2.2. Determination of Minimum Bactericidal Concentration (MBC) .............. 45

3.2.3. Time-kill Study ................................................................................................ 45

3.2.3. Transmission Electron Microscope ................................................................. 60

3.3. Application of Aluminum Doped Zinc Oxide NPs in a Paint Sample ................... 62

4. Recommendation ...................................................................................................... 64

5. Summary ................................................................................................................... 65

6. References .......................................................................................................... 67

67

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