Determining the fitness and competitive advantage of the epidemic ExPEC clone E. coli ST131 Abdulaziz Saad Alqasim A thesis submitted in partial fulfilment of the requirements of Nottingham Trent University for the degree of Doctor of Philosophy February 2015 Abstract Extraintestinal pathogenic E. coli (ExPEC) is the major aetiological agent of urinary tract infections (UTIs) in humans. The emergence of the CTX-M producing E. coli ST131 clone represents a major challenge to public health worldwide because of its ability to cause a wide range of difficult-to-treat infections in the healthcare and community settings. The key aim of this study was to characterise the traits that give E. coli ST131 a competitive fitness advantage over other potential ExPEC clones. Comparative phenotypic characterisation of a collection of ExPEC strains showed that there was no difference between ST131 and non-ST131 strains in terms of their growth rates in different culture media, their capacity to associate with, invade and form intracellular bacterial communities within T24 human bladder epithelial cells and their ability to persist within U937 human macrophages. Afterwards, this study tested and compared the metabolic activity of a collection of ST131 and non-ST131 strains using two different testing methodologies: API strips and phenotypic microarray (PM) technology. Our API data showed that ST131 strains had a lower metabolic activity for 5 substrates. Further testing of the metabolic activity of E. coli using phenotypic microarray demonstrated the absence of a specific metabolic profile for ST131 strains suggesting that ST131 is not a metabolically distinct lineage of ExPEC and thus altered metabolism might not contribute to the fitness of this clone. The gene content of a group of E. coli including ST131 and non-ST131 strains was investigated to identify the presence of other loci that are uniquely associated with ST131 H30Rx clade, which involves ST131 isolates belonging to the fimH30 lineage and I associated with fluoroquinolones resistance and CTX-M-15 production. Our data identified the presence of 150 loci unique to ST131 H30Rx strains, and the most striking finding at a genomic level was the identification of the secondary flagellar locus Flag 2 as a region uniquely associated with ST131 H30Rx strains. The ability of a collection of ST131 and non-ST131 strains to resist human serum was tested and compared. Our data showed that all ST131 and ST73 strains were associated with high serum resistance phenotype, and this might suggest serum resistance as an important factor in driving the current success of this ST131 as a major cause of bloodstream infections worldwide. Given many reports showing that polysaccharide capsules might be a major factor allowing E. coli to resist the human serum, and based on many studies demonstrating the genetic and biochemical diversity in the capsule region of ST131 strains, the capsule region of a collection of ExPEC belonging to ST131 H30Rx clade and non-ST131 was tested in more detail at a genomic and biochemical level. Our capsule genetics data showed a surprising level of diversity within the capsule locus of the H30Rx clade with a phylogenetic distribution highly suggestive of frequent recombination at the locus. Subsequent analysis demonstrated that this recombination had no obvious detectable effect on virulence- associated phenotypes in-vitro. Given the level of diversity observed at the capsule locus of ST131 H30Rx strains, it is tempting to speculate that there is significant selective pressure occurring at this site during the life cycle of the H30Rx clade, and that frequent recombination allows the clade to subvert that pressure and might provide a fitness advantage to ST131. This study provided detailed insights into the phenotypic, metabolic and genetic traits of ST131 and highlighted the factors that might drive its success. II Declaration I hereby declare that the work presented herein is the result of my original research work, except where references have been made to acknowledge the literature. This work is an intellectual property of the author. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests of any other use, or if a more substantial copy is required, should be directed in the first instance to the owner(s) of the intellectual property rights. Experiments were performed in the Pathogen Research Group at Nottingham Trent University and in the Microbiology Research Laboratory at the University of Surrey. Comparative metabolic studies were carried out in collaboration with Profs Roberto La Ragione and Richard Emes. The phenotypic microarray (PM) assay was carried out with the help of Dr Jane Newcombe, University of Surrey. Classical capsule typing assays were performed at the Statens Serum Institute, Denmark. Finally, writing the Post Assembly Genome Improvement Toolkit (PAGIT) script, pan-genome analysis using the Large- Scale Blast Score Ratio (LS-BSR) software and whole-genome phylogeny work were carried out by my director of studies Dr Alan McNally. Abdulaziz Alqasim III Acknowledgments I would like to thank Almighty Allah for giving me the strength and courage to lead this project to completion. There are many people to whom I owe my gratitude for helping me with the completion of this PhD project. Firstly, my very special thanks to my director of studies Dr Alan McNally for giving me the opportunity to carry out this PhD research project and for his support and encouragement throughout this project. This work would have not been possible without his invaluable advice and guidance. I would like to thank our collaborators Prof. Roberto La Ragione and Dr. Richard Emes for their efforts in our published work on comparative phenotypic microarray analysis of ExPEC strains. My deep thanks are also extended to Mr Gordon Arnott for his assistance with using the confocal microscope. I would like to extend my sincerest thanks and appreciation to my lovely father, mother, brother and sisters for their prayers and support throughout all my studies. Special thanks go to my beloved fiancée Mai, who has provided me with her never-ending emotional and practical support over the past year. I also owe my thanks to all members in the Pathogen Research Group, Nottingham Trent University for their help and kindness in the past four years. I would like also to thank my colleagues Fahad, Dhahi, Alya and Miquette for providing a co-operative, comfortable and most enjoyable environment for work. The administrative and financial support of the Saudi Arabian Cultural Bureau is gratefully acknowledged. IV Publications 1. Research articles Alqasim, A., Emes, R., Clark, G., Newcombe, J., La Ragione, R. & McNally, A. (2014). Phenotypic microarrays suggest Escherichia coli ST131 is not a metabolically distinct lineage of extra-Intestinal pathogenic E. coli. PloS One, 9, e88374. Alqasim, A., Scheutz, F., Zong, Z. & McNally, A. (2014). Comparative genome analysis identifies few traits unique to the Escherichia coli ST131 H30Rx clade and extensive mosaicism at the capsule locus. BMC Genomics, 15, 830. 2. Poster presentations Alqasim, A., Diggle, M., Weston, V., Cheetham P. & McNally, A. (2011). Investigation of clinical E. coli ST131 isolates. Society of General Microbiology Autumn Conference, University of York. Alqasim, A., Diggle, M., Weston, V., Cheetham P. & McNally, A. (2012). Determining the fitness and competitive advantage of the epidemic ExPEC clone E. coli ST131. School of Science & Technology Research Conference, Nottingham Trent University. Alqasim, A., Emes, R., Newcombe, J., La Ragione, R. & McNally, A. (2013). Phenotypic microarray analysis of ExPEC strains of different sequence types shows the absence of ST specific metabolism. Federation of Microbiological Societies in Europe Conference, Leipzig, Germany. V Table of Contents Abstract ................................................................................................ I Declaration ........................................................................................ III Acknowledgments ............................................................................... IV Publications ..........................................................................................V Table of Contents ................................................................................ VI List of Tables ........................................................................................X List of Figures ................................................................................... XII Abbreviations ..................................................................................... XV Chapter one: Introduction ........................................................................ 1 1. Introduction ...................................................................................... 2 1.1 The species Escherichia coli .............................................................. 2 1.2 The classification of E. coli strains ..................................................... 2 1.3 The genetic structure and phylogenetic history of E. coli ...................... 5 1.4.1 Overview of the medical and economic impact of major infections due to ExPEC .................................................................................. 10 1.4.2 Virulence factors of ExPEC ....................................................... 13 1.4.3 Antimicrobial resistance of ExPEC.............................................. 23 1.5 Microbial typing methods for the identification of pathogenic E. coli clones ............................................................................................... 24 1.5.1 Phenotypic methods ................................................................ 24 1.5.2 Genotypic methods ................................................................. 25 1.6 E. coli ST131 ................................................................................ 26 1.6.1 Bacterial characteristics of E. coli ST131 .................................... 26 1.6.1.1 Serotyping and phylogenetic group ..................................... 26 1.6.1.2 fimH subtyping of E. coli ST131 .......................................... 26 1.6.1.3 Antimicrobial resistance of E. coli ST131 .............................. 27 1.6.1.4 Phylogeny of E. coli ST131 clinical isolates ........................... 28 1.6.2 Pathogenic characteristics of E. coli ST131 ................................. 32 1.6.2.1 Scale of infection .............................................................. 32 1.6.2.2 Transmissibility ................................................................ 32 1.6.2.3 Virulence potential of E. coli ST131 ..................................... 33 1.6.2.4 Metabolic potential of ST131 .............................................. 35 1.6.2.5 Adhesion and invasion capacity of ST131 ............................. 36 1.7 Introduction to the project ............................................................. 37 1.8 Aims of the project ........................................................................ 38 Chapter two: General Materials and Methods ......................................... 40 2. General Materials and Methods ....................................................... 41 2.1 Bacterial strains ............................................................................ 41 2.2 Antibiotics .................................................................................... 46 2.3 Culture media ............................................................................... 46 2.3.1 Luria-Bertani (LB) medium ....................................................... 46 2.3.2 LB broth (LBB) ....................................................................... 46 2.3.3 Cysteine Lactose Electrolyte Deficient (CLED) agar ...................... 46 2.3.4 McCoy’s 5A modified medium ................................................... 46 2.3.5 SOC medium .......................................................................... 47 2.4 Bacterial culture maintenance and growth conditions ......................... 47 2.5 General media, buffers and reagents ............................................... 47 2.5.1 1X Tris-acetate EDTA (TAE) buffer ............................................ 47 2.5.2 Dulbecco’s phosphate buffered saline (PBS) ............................... 47 2.5.3 Saline solution ........................................................................ 47 2.5.4 80% glycerol solution .............................................................. 48 2.5.5 10% glycerol solution .............................................................. 48 VI 2.5.6 3% α-D-mannose solution ....................................................... 48 2.5.7 4% paraformaldehyde solution ................................................. 48 2.5.8 1% triton X-100 ..................................................................... 48 2.5.9 Vectashield mounting medium with 4’, 6-diamidino-2-phenylindole (DAPI) ........................................................................................... 48 Chapter three: Comparative phenotypic characterisation of strains belonging to different ExPEC STs ........................................................... 50 3.1 Introduction ................................................................................. 51 3.1.1 The role of studying bacterial growth in pathogenesis .................. 51 3.1.2 UPEC attachment, invasion and intracellular survival in host bladder epithelial cells ................................................................................. 52 3.1.3 UPEC intracellular survival in host macrophages ......................... 53 3.1.4 Aims of the study .................................................................... 54 3.2 Materials and Methods ................................................................... 56 3.2.1 Bacterial strains ...................................................................... 56 3.2.2 Plasmid and primer sets........................................................... 59 3.2.3 Comparative bacterial growth assays......................................... 59 3.2.3.1 Turbidity measurement assay ............................................. 59 3.2.3.2 Viable cell count assay ...................................................... 60 3.2.4 Genomic DNA extraction .......................................................... 61 3.2.5 Polymerase chain reaction (PCR) screening of the fimB insertion ... 61 3.2.6 DNA analysis by agarose gel electrophoresis .............................. 62 3.2.7 Preparation of Saccharomyces cerevisiae (S. cerevisiae) yeast suspension ..................................................................................... 62 3.2.8 Yeast cell agglutination assay ................................................... 62 3.2.9 Mini prep Plasmid purification ................................................... 63 3.2.10 Preparation of electro-competent cells ..................................... 63 3.2.11 Transformation of electro-competent cells by electroporation ...... 64 3.2.12 Confirmation of pMN402 plasmid transformation by PCR ............ 64 3.2.13 Cell culture methods .............................................................. 65 3.2.13.1 Cell lines ........................................................................ 65 3.2.13.2 Cell culture media ........................................................... 65 3.2.13.3 Cell line growth and maintenance ...................................... 66 3.2.13.4 Preparation of bacterial inocula ......................................... 67 3.2.13.5 Comparative T24 cell infection assays ................................ 67 3.2.13.6 Confocal fluorescent microscopy ....................................... 68 3.2.13.7 Statistical analysis for T24 cell culture data ........................ 68 3.2.13.8 Persistence of E. coli strains within U937 cell line ................ 69 3.2.13.9 Statistical analysis for U937 cell culture data ...................... 69 3.3 Results ........................................................................................ 70 3.3.1 Growth curves of E. coli strains determined by OD measurement .. 70 3.3.2 Growth curves of E. coli strains determined by viable cell count measurement ................................................................................. 74 3.3.3 Type 1 fimbriae expression results ............................................ 78 3.3.3.1 Screening of the fimB transposon insertion in E. coli ST131 strains ........................................................................................ 78 3.3.3.2 Testing the ability of E. coli ST131 to express functional type 1 fimbriae ...................................................................................... 79 3.3.4 Confirmation of plasmid transformation by PCR .......................... 82 3.3.5 T24 cell culture results ............................................................ 83 3.3.5.1 Association profiles of GFP-tagged E. coli ST131 and non-ST131 strains ........................................................................................ 83 3.3.5.2 Invasion profiles of GFP-tagged E. coli ST131 and non-ST131 strains ........................................................................................ 87 3.3.6 Persistence of E. coli strains within U937 cell line results.............. 96 3.4 Discussion .................................................................................... 99 VII 3.5 Conclusion ................................................................................. 106 Chapter four: Comparative studies on the metabolic potential and gene content of a group of E. coli ST131 and non-ST131 strains .................. 108 4.1 Introduction ............................................................................... 109 4.1.1 The role of metabolism in bacterial colonisation and pathogenesis109 4.1.2 The proposed role of E. coli ST131 metabolic potential in enhancing its fitness ..................................................................................... 110 4.1.3 Overview of the phenotypic methods used for testing the bacterial metabolic potential ........................................................................ 111 4.1.3.1 Biotyping ....................................................................... 111 4.1.3.2 Phenotypic microarray (PM) technology ............................. 112 4.1.4 Gene content analysis for the identification of unique ST131 loci . 113 4.1.4.1 Bacterial “pan-genome” approach as a tool for gene content analysis .................................................................................... 113 4.1.4.2 The genetic architecture of E. coli ST131 H30Rx clade ......... 114 4.1.5 Aims of the study .................................................................. 116 4.2 Material and Methods .................................................................. 117 4.2.1 Bacterial strains .................................................................... 117 4.2.2 Comparative metabolic profiling assays ................................... 120 4.2.2.1 Metabolic profiling assay using API test reagents ................ 120 4.2.2.2 Biolog phenotypic microarray (PM) assay ........................... 120 4.2.2.3 Phenotypic Microarray data analysis .................................. 121 4.2.2.4 Statistical analysis .......................................................... 122 4.2.2.5 Comparative genomics for the identification of the presence or absence of metabolic-associated loci ............................................ 122 4.2.3 Gene content analysis of a group of ST131 and non-ST131 strains ................................................................................................... 123 4.2.3.1 Bacterial genome data ..................................................... 123 4.2.3.2 E. coli ST131 genome re-assembly.................................... 123 4.2.3.3 Comparative gene content analysis of a group of E. coli ST131 and non-ST131 strains using Gegenees software ........................... 124 4.2.3.4 Core and pan-genome analysis of a group of E. coli ST131 and non-ST131 genomes using LS-BSR software ................................. 125 4.3 Results ...................................................................................... 126 4.3.1 Metabolic activity of E. coli strains obtained from API test reagents ................................................................................................... 126 4.3.2 Phenotypic Microarray data .................................................... 130 4.3.2.1 Choosing the signal value calculation approach for the PM data analysis .................................................................................... 130 4.3.2.2 Metabolic activity of E. coli strains obtained from PM ........... 132 4.3.2.3 Principal component analysis of PM data ............................ 137 4.3.2.4 Differences in the carbon source utilisation by ST131 strains obtained from using two measurement methods ............................ 138 4.3.2.5 Relating the metabolism observations to the presence/absence of ST131 associated metabolic loci ............................................... 140 4.3.3 Gene content analysis of E. coli strains .................................... 143 4.3.3.1 E. coli ST131 genome re-assembly data ............................ 143 4.3.3.2 Comparative genome analysis of E. coli strains using Gegenees software ................................................................................... 144 4.3.3.3 Identification of genetic loci unique to the E. coli ST131 H30Rx clade as shown by pan-genome analysis ....................................... 149 4.3.3.4 Distribution of functional categories of genes unique to E. coli ST131 H30Rx strains .................................................................. 155 4.4 Discussion .................................................................................. 158 4.5 Conclusion ................................................................................. 165 VIII Chapter five: Comparative studies on the serum resistance and capsule genetics of a group of E. coli ST131 and non-ST131 strains ................. 168 5.1 Introduction ............................................................................... 169 5.1.1 E. coli resistance to human serum ........................................... 169 5.1.2 The proposed role of E. coli cell surface polysaccharides in serum resistance .................................................................................... 170 5.1.3 E. coli ST131 resistance to human serum ................................. 172 5.1.4 Genetic and biochemical diversity of E. coli ST131 capsule locus . 173 5.1.5 Aims of the study .................................................................. 174 5.2 Material and Methods .................................................................. 175 5.2.1 Bacterial strains .................................................................... 175 5.2.2 Bacterial genome data ........................................................... 178 5.2.3 Identification of capsule loci in E. coli ST131 genomes ............... 179 5.2.4 Whole genome phylogeny ...................................................... 179 5.2.5 Phenotypic assays ................................................................. 179 5.2.5.1 Classical capsule typing ................................................... 179 5.2.5.2 Comparative serum resistance assay ................................. 180 5.2.5.3 Capsule production assay ................................................ 180 5.2.5.4 Biofilm formation assay ................................................... 181 5.2.6 Statistical analysis for serum resistance data ............................ 181 5.3 Results ...................................................................................... 182 5.3.1 Serum resistance profiles of E. coli strains ............................... 182 5.3.2 Comparative statistical analysis of the serum resistance profiles of E. coli strains.................................................................................... 184 5.3.3 Identification and comparison of genetic loci present in the capsule region of a collection of ExPEC strains .............................................. 187 5.3.4 Genetic and biochemical diversity of capsule locus in the E. coli ST131 H30Rx clade ....................................................................... 191 5.3.5 Whole genome phylogeny of E. coli ST131 H30Rx strains ........... 195 5.3.6 The effect of E. coli ST131 capsule locus diversity on other virulence- associated phenotypes ................................................................... 196 5.4 Discussion .................................................................................. 197 5.5 Conclusion ................................................................................. 204 Chapter six: Final conclusions and future directions ............................ 206 6. Final conclusions and future directions ......................................... 207 Chapter seven: Appendix ..................................................................... 214 7. Appendix ....................................................................................... 215 7.1 Metabolism work supplementary information .................................. 215 7.2 Bioinformatics software scripts and command lines ......................... 224 References ........................................................................................... 226 IX