New antimicrobials to target gut and food pathogens Enriqueta Garcia-Gutierrez A thesis submitted for the degree of Doctor of Philosophy to the University of East Anglia Quadram Institute Bioscience Teagasc September 2019 ©This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived therefrom must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. PhD Thesis 2019 Enriqueta Garcia-Gutierrez New Antimicrobials to Target Gut and Food Pathogens ABSTRACT There is a pressing need for the discovery of new antimicrobials to fight antibiotic resistant bacteria. The aim of this thesis was the discovery and characterisation of new bacteriocins from two sources, fermented foods and human faeces, testing the hypothesis that bacteria from the same niche will produce antimicrobials uniquely suited to act in this niche. Isolates from culture collections and new isolates from food and faecal samples were screened against a panel of pathogens responsible for food spoilage and human disease. Promising candidates were selected for genome sequencing, antimicrobial characterisation and biological study. The genome of Lactobacillus gasseri LM19 showed the presence of antimicrobial genes encoding, among others, a new bacteriocin, gassericin M. L. gasseri LM19 could survive and express its bacteriocin genes under colonic conditions. Its administration modulated the effects of Clostridium perfringens on the gut microbiota composition. Streptococcus agalactiae DPC7040 was previously shown to produce the natural variant nisin P. MALDI-ToF analysis confirmed that nisin P is three amino acids shorter than nisin A and that two lanthionine rings were absent in 50% of molecules. This structure impacted on its antimicrobial activity, which was weaker than that of nisin A and nisin H in faecal fermentations. Staphylococcus epidermidis strains isolated from faecal samples were compared with skin isolates with respect to genomic and phenotypic traits. It was concluded that S. epidermidis has no specific genomic features to colonise different body sites but is likely to adapt its metabolism to the different conditions. Potential novel antimicrobials were found in Lactobacillus amylovorus and Lactobacillus crispatus isolates, which showed probiotic properties and interesting phenotypic differences between strains. Together, this work further demonstrates that fermented food and gut environments are valuable sources of new isolates, the study of which can yield new antimicrobials and give insights into bacterial ecology and evolution. II Table of Contents Abstract .................................................................................................................................. II Table of contents .................................................................................................................. III List of figures...................................................................................................................... VIII List of tables ..........................................................................................................................XI Outputs of this project ...................................................................................................... XIII Abbreviations .................................................................................................................... XIV Symbols .............................................................................................................................. XVI Acknowledgements .......................................................................................................... XVII CHAPTER I. General introduction .......................................................................................... 1 1.1. The antimicrobial resistance problem ......................................................................... 2 1.2. Microbial ecology ........................................................................................................ 4 1.3. Bioactive potential of the human gastrointestinal tract (GIT) .................................... 4 1.3.1. Conditions and microbiota composition in the human GIT ................................. 5 1.3.2. Antimicrobial activity in the gut ........................................................................... 8 1.4. Bacteriocins ............................................................................................................... 12 1.4.1. Classification ....................................................................................................... 13 1.4.2. Regulation of bacteriocin biosynthesis ............................................................... 27 1.4.3. Identification, production and characterisation of bacteriocins ........................ 28 1.4.4. Bioengineering of bacteriocins ........................................................................... 29 1.4.5. Prospective applications of bacteriocins ............................................................ 30 1.4.6. Challenges and future developments in bacteriocin research ........................... 33 1.5. Aims and objectives of the thesis .............................................................................. 35 CHAPTER II. General material and methods ....................................................................... 37 2.1. Microbiology .............................................................................................................. 38 2.1.1. Culture media ..................................................................................................... 38 2.1.2. Bacterial strains and their growth conditions .................................................... 39 2.1.3. Measurement of cell growth .............................................................................. 51 2.1.4. Microscopy .......................................................................................................... 51 2.1.5. Bioassays to measure antimicrobial activity ...................................................... 52 2.1.6. Assessment of characteristics of probiotic activity ............................................ 55 2.1.7. Preparation of a faecal inoculum for faecal fermentations ............................... 55 III 2.1.8. Biofilm formation ............................................................................................... 56 2.1.9. Phenotypic studies using BIOLOG system .......................................................... 58 2.2. Molecular biology ..................................................................................................... 58 2.2.1. PCR primers ........................................................................................................ 59 2.2.2. General PCR methods ......................................................................................... 60 2.2.3. Agarose gel electrophoresis ............................................................................... 62 2.2.4. DNA purification ................................................................................................. 62 2.2.5. 16S rDNA sequencing ......................................................................................... 62 2.2.6. Extraction of genomic DNA (gDNA) .................................................................... 63 2.2.7. RNA work ............................................................................................................ 63 2.2.8. qPCR and RT-qPCR .............................................................................................. 64 2.2.9. Bioinformatics analysis ....................................................................................... 66 2.2.10. Transformation of Lactobacilli ......................................................................... 67 2.3. Protein work .............................................................................................................. 68 2.3.1. Assays to assess the nature of the antimicrobial ............................................... 68 2.4. General chemistry ..................................................................................................... 72 2.4.1. Organic acid quantification ................................................................................ 72 2.5. Statistical analysis ..................................................................................................... 73 CHAPTER III. Faecal and food screening for antimicrobial activity ................................... 74 3.1. Introduction .............................................................................................................. 75 3.2 Methods ..................................................................................................................... 76 3.2.1. Food and faecal screening .................................................................................. 76 3.2.2. Isolates obtained from culture collections ......................................................... 77 3.3. Results ....................................................................................................................... 77 3.3.1. Isolation and screening ...................................................................................... 77 3.3.2. Isolate testing ....................................................................................................