Characterization of novel extended spectrum beta- lactamase producing bacteria and their bacteriophages from wastewater by Nanpon Miri The thesis is submitted in partial fulfilment of the requirements for the award of the degree of DOCTOR OF PHILOSOPHY of the University of Portsmouth School of Pharmacy and Biomedical Sciences September 2020 i Abstract Multidrug-resistant bacteria strains possessing extended-spectrum β-lactamases (ESBLs) has become an increasing problem worldwide. Bacteria resistant to antibiotics are mostly enteric, they can contaminate the environment and, through ingestion, enter new hosts to cause infections. Therefore, emphasis was put on isolating ESBL-producing bacteria from treated wastewater. The overall aim of this thesis was to isolate, identify and characterise ESBL-producing bacteria from wastewater followed by isolation and characterisation of bacteriophages specific for these bacteria. Bacterial isolates were recovered after growth on selective media and multiplex PCR was used to amplify SHV, TEM, CTX-M and OXA genes. Biochemical test and whole genome sequencing were applied to identify and characterise the isolated strains. The cell-free supernatants were then used to isolate bacteriophages. A high titer lysate was then used to test the specificity of bacteriophages for different bacterial strains. The nature of phage genetic material was established and morphology of isolated phages was determined by the transmission electron microscopy imaging. Two cold-tolerant bacteria isolated harbouring CTX-M gene were studied by 16S rRNA gene sequence analysis and housekeeping gene sequences analysis which revealed that these isolates showed no close similarity to any known member of the Enterobacteriaceae but are related to the members of Rahnella, Rouxiella and Ewingella genera. The phenotypic characteristics of the two isolates were, however, discrete from these 3 genera. Furthermore, two different bacteriophages infected the two newly identified cold tolerant bacterial strains. These bacteriophages were found to be novel viruses, most likely belonging to the Siphoviridae family, based on their characteristics, morphology, and genome size. In conclusion, all the analysis showed that they two isolates belong to a novel genus in the Enterobacteriaceae family. Given the menacing impact of disease outbreaks caused by contaminated water resources, work presented here identifies novel bacteria with harmful capabilities and potentially offers new tools for environmentally safer treatment to overcome this threat. ii Declaration Whilst registered as a candidate for the above degree, I have not been registered for any other research award. The results and conclusions embodied in this thesis are the work of the named candidate and have not been submitted for any other academic award. Word Count: 30534 iii Table of Contents Abstract ............................................................................................................................................... ii Declaration ......................................................................................................................................... iii List of Figures .................................................................................................................................... vii List of Tables .................................................................................................................................... viii Acknowledgement .............................................................................................................................. ix List of Abbreviations ............................................................................................................................ x 1.0 Introduction ........................................................................................................................... 1 1.1 Wastewater treatment ....................................................................................................... 1 1.2 Enteric microorganisms present in wastewater ............................................................ 3 1.2.1 The Enterobacterales ................................................................................................ 3 1.2.2 Pseudomonadales .................................................................................................... 7 1.3 Antibiotics, β-lactam antibiotics, and mode of resistance ........................................... 7 1.3.1 Antibiotic resistance ................................................................................................. 9 1.3.2 β-lactamases ............................................................................................................ 11 1.3.3 Extended-spectrum β-lactamases ......................................................................... 12 1.3.4 Extended-spectrum β-lactamases in Rahnella and Ewingella ........................... 13 1.3.5 Virulence factors of extended-spectrum β-lactamases ...................................... 14 1.3.6 The rise of antibiotic resistance and importance of finding new treatments ... 15 1.4 Phage Biology and classification .................................................................................. 16 1.5 Phage Infection................................................................................................................ 17 1.6 Phage evolution............................................................................................................... 19 1.7 Bacterial defence system against phages .................................................................... 19 1.7.1 Adsorption inhibition .............................................................................................. 19 1.7.2 Restriction modification systems .......................................................................... 20 1.7.3 CRISPR-Cas ............................................................................................................. 21 1.7.4 Abortive infection .................................................................................................... 21 1.8 History of phages and phage therapy ........................................................................... 23 1.9 Mechanism of Phage Therapy ....................................................................................... 24 1.10 Statement of aims and objectives ................................................................................. 25 2.0 Materials and Methods ....................................................................................................... 26 2.1 Media preparation ........................................................................................................... 26 2.1.1 LB medium ............................................................................................................... 26 2.1.2 LB agar ..................................................................................................................... 26 2.1.3 Mueller-Hinton Agar ................................................................................................ 26 2.1.4 Tryptone soy broth ................................................................................................. 26 2.1.5 Tryptone soy agar ................................................................................................... 26 2.1.6 Soft agar ................................................................................................................... 27 2.1.7 Bacteriophage buffer ............................................................................................. 27 2.2 Strains used in this study .............................................................................................. 27 iv 2.3 Collection of water samples and bacterial enrichment ............................................... 27 2.4 Isolation of extended spectrum beta lactamase producing bacteria ........................ 28 2.5 Identification and characterization of ESBL producing bacteria using microbiological techniques ................................................................................................................................... 29 2.5.1 Antimicrobial susceptibility test ............................................................................ 29 2.5.2 ESBL and AmpC production .................................................................................. 30 2.5.3 Identification of isolated ESBL producing bacteria using API 20 E strip test .. 31 2.5.4 Biolog GEN III extended Biochemical test ............................................................ 32 2.5.5 Growth of isolated bacteria at different temperatures ........................................ 32 2.6 Identification and characterization of ESBL producing bacteria using molecular biology techniques ..................................................................................................................... 32 2.6.1 Purification of Genomic DNA ................................................................................. 32 2.6.2 Purification of Plasmid DNA .................................................................................. 33 2.6.3 Multiplex PCR Amplification of TEM, CTX and SHV genes ................................ 33 2.6.4 Multiplex PCR Amplification of OXA group genes .............................................. 34 2.6.5 PCR amplification of the 16S rRNA gene ............................................................