University College London Nicola D’Arcy Exploring the Nature and Diversity of Microorganisms in Healthcare and Educational Settings Eastman Dental Institute Doctor of Philosophy Supervisors: Dr David A. Spratt and Professor Nigel Klein 2014 I, Nicola D’Arcy confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Signed………………………. Date………………………… Abstract Many human populations spend approximately 90 % of their time indoors, yet relatively little is known about the microbial communities associated with indoor environments. This is despite knowledge that these microorganisms can contribute to adverse health effects, including the acquisition of healthcare-associated infections, which cause significant morbidity and mortality. The concept of the ‘indoor microbiome’ is relatively new and to date, few studies have been field-based, systematic and long-term. Hospitals in particular, are unique environments which have been shown to drive microbial evolutionary processes as they contain a different sub-set of the human population. The study of the hospital microbiome could have important implications for healthcare and infection control. This thesis explores a range of methods for investigating microorganisms in different indoor environments, including a classroom and outpatient’s waiting areas and wards in a hospital. Results show that the classroom is much more heavily contaminated in terms of total viable counts (TVCs) of bacteria recovered than the hospital environment. This was thought to be attributed to the absence of a strict cleaning regime in the classroom. High-touch items were less contaminated than other objects, likely due to them being obvious cleaning targets. Potential pathogens, including a number of Enterobacteriaceae were cultured from the classroom, outpatient’s waiting area and ward. Virus nucleic acid was recovered from an outpatient’s area, including norovirus and rotavirus RNA. Adenovirus DNA was frequently isolated throughout a 3 month screening protocol and there appeared to be evidence to suggest that a viral marker may be more appropriate than TVCs for identifying viral contamination. Human-associated bacteria were found to be dominant on a hospital ward over a 12 month longitudinal screening study and the presence of numerous bacterial taxa, which may be of concern in the context of paediatrics and immunodeficient patients, was also demonstrated. 4 Acknowledgements I would first like to thank my supervisors Dave Spratt and Nigel Klein for taking on my project and for their encouragement and support. Both were exceptionally calming during the stressful times. Thanks to Louise, Melisa, Paula and Jon at HIRC for helping to kick-start the project Lena for proof-reading and Dimitrios Margaritis for the maps of sampling locations displayed throughout this thesis. I would also like to thank Anna Tymon and those at the Eastman Dental Institute for their help. Thanks to all at the Camelia Botnar labs, especially Elaine Cloutman-Green for lab space, countless cups of tea, and advice. Thanks also to Kathryn Harris, John Hartley and Garth Dixon for providing practical help, reagents and knowledge and to Paul Locke and his team for their wisdom and support. Ronan Doyle provided barcode primers used in Chapter 6 and thanks also to him for bioinformatics support. Thanks to Infection Control for access to the wards and data and to all on Tiger and Koala who put up with me getting in the way taking my swabs; particularly Tom Kennedy. Samana, thanks for the advert for the PhD! Thanks also to her and the rest of my amazing friends; Taemi, Paul, Gron, Alex, Kylie, Chris, Kara, Quinton, Paul and Kate. Without their support and that of others too many to mention, I wouldn’t have been able to focus on the task at hand and complete the project. Thanks to Chris for being so wonderful and supportive. 5 Finally, I would like to thank my family, Mum, Will, Bob and my Grandparents. Without their encouragement, love and endless support, I would not have achieved anything in life. I hope you’re proud. 6 Table of Contents Declaration 2 Abstract 3 Acknowledgements 5 Table of Contents 7 List of Figures 12 List of Tables 15 1. Introduction…………………………………………………………….18 1.1 Overview 18 1.2 The indoor microbiome: current research 21 1.3 Indoor microbial source and distribution 26 1.3.1 Human microbial sources .......................................................................... 26 1.3.2 Other sources of microorganisms .............................................................. 29 1.3.3 Spatial distribution ..................................................................................... 31 1.4 Factors influencing the indoor microbiome 32 1.4.1 Physical parameters ................................................................................... 32 1.4.2 Temporal factors ........................................................................................ 33 1.4.3 Bacterial survival on surfaces .................................................................... 34 1.5 Environmental sampling methods 35 1.5.1 Air Sampling ............................................................................................. 35 1.5.2 Surface sampling ....................................................................................... 39 1.5.3 Post-sampling processing: culture methods ............................................... 41 1.5.4 Post-sampling processing: molecular methods .......................................... 44 1.5.5 16S ribosomal RNA................................................................................... 51 1.5.6 Alternatives to 16S rRNA gene PCR for culture-independent bacterial identification ............................................................................... 58 1.6 Hospitals, nosocomial infection and the role of the environmental microbiome 59 1.6.1 Infection control and the hospital environment ......................................... 60 1.7 Clinically important pathogens 63 1.7.1 Enterobacteriaceae ..................................................................................... 63 1.7.2 Clostridium difficile ................................................................................... 64 1.7.3 Acinetobacter species ................................................................................ 64 1.7.4 Pseudomonas aeruginosa .......................................................................... 65 1.7.5 Staphylococcus aureus .............................................................................. 66 1.7.6 Drug-resistant enterococci ......................................................................... 67 1.7.7 Viruses ....................................................................................................... 67 7 1.7.8 Fungi .......................................................................................................... 68 1.8 Infection and paediatric patients 68 1.9 Aims of the project 69 2. Materials and Methods………………………………………………...72 2.1 Safety, sterility and quality control 72 2.2 Environmental sampling 73 2.2.1 Contact plates ............................................................................................ 73 2.2.2 Swabbing ................................................................................................... 74 2.2.3 Air sampling .............................................................................................. 75 2.2.4 Recording of environmental parameters .................................................... 75 2.3 Culture methods 76 2.3.1 Culture for preparation of known numbers of bacteria ............................. 76 2.3.2 Miles and Misra method ............................................................................ 77 2.4 Post-culture analysis 78 2.4.1 Gram staining ............................................................................................ 78 2.4.2 Biochemical identification ......................................................................... 79 2.5 Nucleic acid extraction methods 81 2.5.1 DNA extraction from pure bacterial cultures ............................................ 81 2.5.2 DNA extraction from cotton swabs ........................................................... 81 2.5.3 DNA extraction from gelatine filters ......................................................... 83 2.6 PCR methods 83 2.6.1 16S rDNA end-point PCR ......................................................................... 83 2.6.2 Quantitative real-time PCR ....................................................................... 89 2.7 Gel electrophoresis 96 2.7.1 Standard agarose gels ................................................................................ 96 2.7.2 E-gels® ....................................................................................................... 96 2.7.3 Size SelectTM 2 % E-gels® ......................................................................... 97 2.8 PCR purification 98 2.8.1 Silica membrane column purification ........................................................ 98 2.8.2 SephadexTM column purification ............................................................... 98 2.9 DNA concentration 99 2.9.1 NanoDrop spectrophotometer .................................................................. 100 2.9.2 Qubit® 2.0 Fluorometer ........................................................................... 100 2.9.3 Agilent Bioanalyzer