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Mechanisms of Resistance to Β-Lactam Antibiotics in Streptomycetes Wedad

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Mechanisms of Resistance to Β-Lactam Antibiotics in Streptomycetes Wedad Mechanisms of resistance to β-lactam antibiotics in streptomycetes Wedad Mohamed Omran Alkut A thesis submitted in partial fulfilment of the requirements of Liverpool John Moores University for the degree of Doctor of Philosophy April 2016 Contents DECLARATION ............................................................................................................... ix ACKNOWLEDGEMENTS ................................................................................................ x ABSTRACT ................................................................................................................... xi ABBREVIATIONS ............................................................................................................ 1 Literature review ............................................................................................................. 2 1. Streptomycetes Biology and Life cycle ...................................................................... 2 1.1 Characteristics of Streptomycetes ......................................................................... 2 1.2 The life cycle of Streptomyces .............................................................................. 3 1.3 Streptomyces coelicolor A3 (2) ............................................................................. 4 1.3.1 Actinorhodin (ACT) ........................................................................................... 5 1.3.2 Undecylprodigiosin (RED) ................................................................................ 6 1.3.3 Calcium-dependent antibiotic (CDA) ................................................................ 6 1.3.4 Methylenomycin ................................................................................................ 7 1.4 Morphological differentiation in Streptomyces coelicolor ................................... 7 1.5 Genes specifically required for formation of aerial hyphe.................................... 8 1.6 Genes specifically required for sporulation .......................................................... 8 1.7 Antibiotics produced by Streptomyces .................................................................. 9 1.8 Streptomyces are a potential source of novel antibiotics ...................................... 9 1.9 Antibiotics ........................................................................................................... 10 1.10 Common enzymatic aspect of antibiotic resistance in bacteria ........................ 11 1.11 Beta-lactam antibiotics ...................................................................................... 12 1.12 β-Lactam antibiotics and their role in resistance as inhibitors of cell wall synthesis .................................................................................................................... 13 1.13 Beta-lactamases and their role in resistance...................................................... 15 1.14 Structure of peptidoglycan in Gram-positive bacteria ...................................... 16 1.15 Peptidoglycan synthesis in Gram-positive bacteria .......................................... 18 1.16 Penicillin binding proteins (PBPs) .................................................................... 19 1.17 Penicillin binding proteins in S. coelicolor ....................................................... 21 1.18 Resistance to β-Lactam antibiotics in Streptomyces ......................................... 22 1.19 Relationship between β-Lactam Biosynthetic Gene, β-Lactamase and PBP .... 23 1.20 Measuring antibiotic resistance in Streptomyces sp .......................................... 24 1.21 Difficulties in growing Streptomyces in continuous culture ............................. 27 1.22 Project aims and objectives ............................................................................... 28 Chapter Two ...................................................................................................................... 30 Materials and methods .................................................................................................. 30 2. Sample collection and isolation of actinomycetes .................................................... 30 ii 2.1 Soil samples ............................................................................................................ 30 2.2 Isolation of pure culture of actinomycetes .............................................................. 30 2.3 Maintenance of bacteria .......................................................................................... 31 2.3.1 Streptomyces coelicolor A3 (2) and mutants ................................................... 31 2.4 DNA extraction ....................................................................................................... 31 2.4.1 Polymerase Chain Reaction amplifications (PCR) of DNA ............................ 32 2.5 The PCR product purification ................................................................................. 33 2.6 DNA purity was verified by agarose gel electrophoresis........................................ 33 2.7 Scanning Electron Microscopy (SEM) ................................................................... 33 2.8 Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of penicillin G to Streptomyces strains .................. 34 2. 8. 1 Calculation of oxygen concentration ............................................................. 35 2.8.2 Determination of Bacterial Growth rate ........................................................... 36 2.9 Screening for β-lactamase activity .......................................................................... 37 2.10 BOCILLIN FL ...................................................................................................... 37 2.11 Chemostat culture experiments ............................................................................. 38 2.11.1 Preparation of inoculum of Streptomyces coelicolor 1147 ............................ 38 2.11.2 Chemostat culture conditions ......................................................................... 38 2.11.3 Inoculation and fermentation ......................................................................... 39 2.11.4 Analytical methods ........................................................................................ 42 Chapter Three ................................................................................................................ 43 Results ........................................................................................................................... 43 3.1 Isolation and morphological characteristics of actinomycetes ................................ 43 3.1.1 Growth of Streptomyces sp on Solid Media ..................................................... 43 3.1.2 Growth of Streptomyces in Liquid Media ........................................................ 55 3.2 Identification of the actinomycetes ......................................................................... 58 3.3. The minimal inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of penicillin G for isolated Streptomyces strains ....................... 64 3.4. β-lactamase activity of isolated Streptomyces strains ............................................ 67 3.5 16S rRNA gene and phylogenetic analysis ............................................................ 71 3.6. Investigate the interaction between penicillin G and PBPs in isolated Streptomyces strains ............................................................................................................................ 77 Chapter Four ..................................................................................................................... 85 4. Continuous culture (Fermentation) studies of S. coelicolor 1147............................. 85 4.1 Fermentation Conditions ......................................................................................... 85 4.2 Culture Purity and Stability ..................................................................................... 87 4.3 Batch culture of Streptomyces coelicolor 1147 ...................................................... 88 4.4 Growth of S. coelicolor 1147 in continuous culture ............................................... 94 iii 4.4.1 Dilution rate 0.04 h-1 ........................................................................................ 95 4.4.2Dilution rate 0.06 h-1 ....................................................................................... 102 4.4.3 Dilution rate 0.08 h-1 ...................................................................................... 108 4.4.4. Dilution rate 0.1 h-1 ....................................................................................... 113 Chapter Five .................................................................................................................... 119 5. DISCUSSION ......................................................................................................... 119 5.1 Isolation and morphological characteristics of Streptomyces ............................... 119 5.3 Visualization of penicillin binding proteins of Streptomyces ............................... 121 5.4 Fermentations studies of Streptomyces coelicolor 1147 ....................................... 122 Chapter six .....................................................................................................................
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