Antibacterial activity testing of cotton medical textiles sonochemically impregnated with metal oxide

257 Pages·2015·3.95 MB·English

by  Gagandeep Singh

Antibacterial activity testing of cotton
medical textiles sonochemically
impregnated with metal oxide
nanoparticles.

Singh, G.

Submitted version deposited in CURVE March 2016

Original citation:
Singh, G. (2014) Antibacterial activity testing of cotton medical textiles sonochemically
impregnated with metal oxide nanoparticles. Unpublished PhD Thesis. Coventry: Coventry
University

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ANTIBACTERIAL ACTIVITY TESTING OF
COTTON MEDICAL TEXTILES
SONOCHEMICALLY IMPREGNATED WITH
METAL OXIDE NANOPARTICLES

By
GAGANDEEP SINGH

PhD

December 2014
ANTIBACTERIAL ACTIVITY TESTING OF
COTTON MEDICAL TEXTILES
SONOCHEMICALLY IMPREGNATED WITH
METAL OXIDE NANOPARTICLES

By
GAGANDEEP SINGH

December 2014

A thesis submitted in partial fulfilment of the University’s
requirements for the Doctorate of Philosophy
Table of Contents
Acknowledgements ..................................................................................................... i
Abstract…………………………………………………………………………………... …..ii
Published work………………………………………………………………………………iv
1 Introduction and Literature Review...................................................................... 1
1.1 Bacteria ......................................................................................................... 1
1.2 Description of bacterial ultrastructure ............................................................ 2
1.2.1 Cell wall .................................................................................................. 3
1.2.2 Cytoplasmic membrane .......................................................................... 5
1.2.3 Capsule .................................................................................................. 5
1.3 Control of microbial growth............................................................................ 6
1.3.1 Antiseptics and Antibiotics ...................................................................... 7
1.3.2 Antibiotic resistance ................................................................................ 8
1.4 Hospital acquired infections (HAIs) ............................................................. 10
1.4.1 Staphylococcus aureus......................................................................... 13
1.4.2 Pseudomonas aeruginosa .................................................................... 14
1.4.3 Escherichia coli ..................................................................................... 15
1.4.4 Acinetobacter baumannii ...................................................................... 15
1.4.5 Klebsiella pneumoniae ......................................................................... 16
1.4.6 Biofilms and their role in nosocomial infections .................................... 17
1.5 Infection prevention ..................................................................................... 19
1.5.1 Medical textiles and infections .............................................................. 20
1.5.2 Survival of pathogens on textile surfaces ............................................. 24
1.5.3 The role of antimicrobial textiles ........................................................... 25
1.5.4 Antimicrobial agents for textiles ............................................................ 26
1.5.5 Organic antimicrobial agents ................................................................ 26
1.5.6 Triclosan ............................................................................................... 27
1.5.7 Quaternary ammonium compounds (QACs) ......................................... 28
1.5.8 Chitosan ............................................................................................... 29
1.5.9 Polyhexamethylene biguanide hydrochloride (PHMB) .......................... 30
1.5.10 Inorganic antimicrobial agents .............................................................. 30
1.5.11 Silver..................................................................................................... 31
1.5.11.1 Research into silver antimicrobial textiles ...................................... 32
1.5.12 Zinc oxide (ZnO) ................................................................................... 36
1.5.12.1 Research into ZnO antimicrobial textiles ........................................ 37
1.5.13 Copper oxide (CuO) ............................................................................. 40
1.5.13.1 Research into CuO antimicrobial textiles ....................................... 41
1.6 Textile application methods ......................................................................... 43
1.6.1 Confinement of antibacterial agents in resins ....................................... 44
1.6.2 Pad-dry-cure method ............................................................................ 44
1.6.3 Sonochemical method of impregnating textile fabrics ........................... 45
1.6.4 Ultrasound ............................................................................................. 46
1.6.5 Principles of ultrasonics sonochemistry ................................................ 47
1.6.5.1 Cavitation in a liquid medium............................................................. 47
1.6.5.2 Cavitation at or near to a solid surface .............................................. 48
1.6.6 Sonochemical synthesis and impregnation of textile fabrics with metal
oxide nanoparticles ........................................................................................... 49
1.6.7 The SONO project ................................................................................ 50
2 Research aims and objectives .......................................................................... 54
2.1 Objectives ................................................................................................... 55
3 Methodology ..................................................................................................... 57
3.1 Quality control and safety ............................................................................ 57
3.2 Equipment and apparatus ........................................................................... 58
3.3 Bacterial strains ........................................................................................... 59
3.4 Materials used ............................................................................................. 60
3.4.1 Reagents and culture media ................................................................. 60
3.4.1.1 Nutrient broth (NB) ............................................................................ 60
3.4.1.2 Soyabean-Casein Digest broth with Lecithin and Polysorbate 80
medium. .......................................................................................................... 60
3.4.1.3 Plate count agar (PCA) ..................................................................... 61
3.4.1.4 Nutrient agar (NA) ............................................................................. 62
3.4.1.5 Sterile saline ...................................................................................... 63
3.4.1.6 Phosphate buffered saline ................................................................. 63
3.4.1.7 Staining buffer (Flow cytometery) ...................................................... 63
3.5 Preparation of inoculum (ISO 20743:2007) ................................................. 63
3.5.1 Inoculum for growth on solid media ...................................................... 63
3.5.2 Preparation and incubation of test inoculums ....................................... 64
3.5.3 Calibration of bacterial suspensions ..................................................... 65
3.6 Preparation of fabric samples ...................................................................... 65
3.7 Antibacterial assessment of test fabrics ...................................................... 67
3.7.1 Absorption method (ISO 20743) – plate count enumeration ................. 68
3.7.2 Serial dilutions ...................................................................................... 69
3.7.3 Agar plating .......................................................................................... 70
3.7.3.1 Pour plating ....................................................................................... 70
3.7.3.2 Spread plating ................................................................................... 71
3.7.4 Test results interpretation ..................................................................... 72
3.7.4.1 Judgment of test effectiveness .......................................................... 72
3.7.5 Calculation of antibacterial activity value .............................................. 72
3.7.6 Calculation of percentage reduction ..................................................... 74
3.7.7 Calculation of growth reduction ............................................................ 74
3.8 Disc diffusion method (Semi-quantitative test) ............................................ 75
3.9 Shake flask tests ......................................................................................... 76
3.9.1 Shake flask in nutrient broth (Semi-quantitative test) ........................... 76
3.9.2 Shake flask test in saline (Quantitative method) ................................... 77
3.10 Saline leaching tests ................................................................................... 78
3.11 Inductively coupled plasma - optical emission spectrometer (ICP – OES) .. 78
3.11.1 Measurement of the total concentration of metals on the fabrics .......... 80
3.11.2 Measurement of the concentration of metals in the leachates .............. 80
3.12 Shake flask tests with different sized fabric pieces ..................................... 80
3.13 Effect of sample preparation on viable cell numbers ................................... 82
3.14 Preparation and incubation of test inoculums.............................................. 82
3.15 Flow cytometry (FC) .................................................................................... 83
3.15.1 Storage and handling of staining solutions ........................................... 83
3.15.2 Preparation of live and dead bacterial suspensions ............................. 83
3.16 Bacterial suspension and staining ............................................................... 84
3.17 Data acquisition and analysis ...................................................................... 86
3.18 Determination of sensitivity of detection by flow cytometry ......................... 87
3.19 Fluorescence microscopy............................................................................ 87
3.20 Cytotoxicity studies ..................................................................................... 88
3.20.1 Cell culture ............................................................................................ 88
3.20.2 Cytotoxicity by indirect contact ............................................................. 88
3.20.3 MTT assay ............................................................................................ 89
3.21 Statistics ...................................................................................................... 90
4 Results and Discussion ..................................................................................... 91
4.1 Testing of lab scale ZnO and CuO impregnated cotton fabrics ................... 91
4.1.1 Antibacterial efficacy testing of lab scale ZnO and CuO cotton fabrics
using the absorption method from ISO 20743:2007 .......................................... 92
4.1.2 Antibacterial activity of PEC fabrics coated with silver and triclosan .. 101
4.1.3 Assessment of the wash durability of ZnO, CuO, silver and triclosan
fabrics in terms of antibacterial activity ............................................................ 105
4.1.4 Enzymatic pre-treatment of cotton prior to ZnO impregnation ............ 107
4.1.5 Antibacterial activity testing of lab scale ZnO and CuO fabrics by a disc
diffusion method .............................................................................................. 109
4.1.6 Shake flask testing to assess the antibacterial activity of lab scale ZnO
and CuO fabrics .............................................................................................. 113
4.1.6.1 Shake flask test in nutrient broth ..................................................... 114
4.1.6.2 Shake flask test in saline ................................................................. 117
4.1.7 Determination of minimum inhibitory concentration of lab scale fabrics in
terms of sample size ....................................................................................... 120
4.1.8 ICP analysis of lab scale fabrics to measure the amount of zinc and
copper on fabrics and in leachates ................................................................. 122
4.1.8.1 Antibacterial activity of Zn and Cu ions by shake flask method ....... 125
4.1.8.2 Antibacterial activity of metal salts against E. coli ........................... 128
4.2 Testing of CuO fabrics from the pilot scale machines ............................... 130
4.2.1 Antibacterial efficacy testing of CuO impregnated PEC fabrics from the
prototype machines using the absorption method ........................................... 132
4.2.2 Antibacterial activity testing of pilot scale CuO PEC fabrics by disc
diffusion method .............................................................................................. 137
4.2.3 Antibacterial activity of pilot scale CuO PEC fabrics by shake flask
method in saline .............................................................................................. 139
4.2.4 Determination of minimum inhibitory concentration of pilot scale PEC
fabrics in terms of sample size ........................................................................ 140
4.2.5 Measurement of the amount of copper on fabrics and in leachates by
ICP…………………………………………………………………………………… 144
4.2.5.1 Antibacterial activity of saline leachates from pilot scale CuO
samples.. ...................................................................................................... 146
4.2.5.2 Leaching of copper in to nutrient broth ............................................ 150
4.2.6 Differences in the homogeneity of the impregnation (Klopman vs
Davo)………………………………………………………………………………… 152
4.2.6.1 Antibacterial activity of fabric E from pale and dark area of coating 153
4.3 Flow cytometry (FC) .................................................................................. 153
4.3.1 Effect of sample preparation on bacterial viable count (FC
experiments).. ................................................................................................. 154
4.3.2 Measurement of the sensitivity of cell detection by FC on the BD FACS
system.. ........................................................................................................... 160
4.3.3 Comparing the enumeration of viable cells from lab scale ZnO and CuO
fabrics by FC and agar plate counting ............................................................. 164
4.3.4 Comparing the enumeration of viable cells from 4 pilot scale CuO PEC
fabrics by FC and agar plate counting ............................................................. 173
4.4 Cytotoxicity studies on pilot scale ZnO and CuO fabrics ........................... 182
5 Conclusion ...................................................................................................... 187
6 Future work ..................................................................................................... 200
7 References ...................................................................................................... 203
Appendices See attached disc