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Dissemination, Antibiotic Susceptibility, Proteomic And Dissemination, antibiotic susceptibility, proteomic and genomic characterization of antibiotic-resistant staphylococci recovered from general public settings Zhen Xu School of Biological and Chemical Sciences Queen Mary University of London A thesis submitted for the degree of Doctor of Philosophy May 2016 Signed declaration I, Zhen Xu, confirm that the research included within this thesis is my own work or that where it has been carried out in collaboration with, or supported by others, that this is duly acknowledged below and my contribution indicated. Previously published material is also acknowledged below. I attest that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual Property Right, or contain any confidential material. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. Signed (Candidate):............. ……….......................................Date...................... Full name:................................................................................................. Signed (Supervisor)…………………………………………..Date…………….. Full name:………………………………………………… …………… Collaborations and publications Collaborations Whole genome sequence Sanger Institute Gavin K Paterson Public Health England Raju Misra, Saheer Gharbia BioNumerics analysis Applied Math, Belgium Katleen Vranckx, Bruno Pot Public Health England Haroun Shah Conferences 25th ECCMID 2016 Comparative genomics of an environmental multiple Poster antibiotic-resistant Staphylococcus epidermidis Presentation 25th ECCMID 2016 Mass spectra based systematics analysis of antibiotic Poster resistant environmental staphylococci Presentation 18th PHE 2015 The spread of antibiotic resistant staphylococci in the Poster public libraries presentation 6th FEMS 2015 Widespread distribution of multidrug resistance in Poster community associated staphylococci presentation 17th PHE 2014 Identification of environmental isolates using Poster MALDI-TOF/MS presentation 24th ECCMID 2014 Molecular characterization of methicillin-resistant Poster coagulase negative staphylococci isolated from presentation environment Publications Xu Z, Mkrtchyan H V, Cutler RR. Antibiotic resistance and mecA characterization of coagulase-negative staphylococci isolated from three hotels in London, UK. Frontiers in microbiology. 2015, 6: 947. Acknowledgements I would like to thank Dr Hermine V Mkrtchyan, who gave me the instructions necessary to complete this project, and without her overwhelming support I would not have been able to produce this thesis. I want to thank Dr Ronald R Cutler and Prof Conrad Mullineaux for their guidance and support. I would also like to thank Prof Haroun Shah, Dr Min Fang, Dr Raju Misra, Dr Renata Culak, Dr Katleen Vranckx, Dr Bruno Pichon, Prof Saheer Gharbia and Dr Gavin K Paterson for all their support, for making both equipments and facilities available. I am very grateful to all members, past and present, of School of Biological and Chemical Sciences. I am also grateful to Nan Wang and all final year project students who carried out their projects in our labaratory. I particularly thank the China Scholarship Council and Queen Mary University of London for providing funds for 4 years and giving me this great opportunity to complete my Ph D studies. I am also deeply grateful to my boyfriend Dr Yongfei Cui, who encouraged me to overcome difficulties. Finally, I would like to give my most heartfelt gratitude to my parents: Mr Weidong Xu and Mrs Yan Zhao, for all their support throughout my PhD studies. Without their unquestioning love and support, this would have been impossible to complete. This is for both of you. Abstract Staphylococci are opportunistic pathogens responsible for a range of infections. Many staphylococcal species are frequently found to be resistant to antibiotics. The environment is considered a potential reservoir of genes conferring antibiotic resistance, which known as the ‘resistomes’. Monitoring the dissemination of antibiotic resistant staphylococci is instrumental to mitigating this global health risk. The overall aim of this study was to generate informative data regarding dissemination of antibiotic resistance in environmental and public settings. This included looking into the distribution, epidemiology characteristic and transfer of oxacillin resistant determinant mecA; gaining an insight into genomic features that contribute to multiple antibiotic resistance and pathogenicity of one S. epidermidis isolate; and understanding the stress responses in mediating oxacillin resistance in S. aureus. The use of MALDI-TOF MS allowed identification of staphylococci to species level. MALDI-TOF MS data were used for taxonomic analysis of staphylococci, and taxonomic data were then combined with isolation sites and antimicrobial susceptibility profiles to aid the understanding of dissemination of environmental resistant staphylococci. The widespread dissemination of antibiotic resistant staphylococci in the environment was demonstrated. 12% of staphylococci harboured mecA gene. Community associated SCCmec types IV and V were more prevalent than nosocomial associated SCCmec types I, II, and III in the environment. 52% of SCCmec were non-typable. In addition, 14 new environmental S. epidermidis MLST types were reported. 9 antibiotic resistant determinants that were responsible for the resistant to 7 antimicrobial classes have been 1 identified in environmental S. epidermidis 118 (G6_2). Proteomic analysis revealed that stress responses, including SOS response, stringent response and heat shock response, mediate oxacillin resistance in S. aureus. These results demonstrate widespread multiple drug resistance in different staphylococcal species isolated from non-healthcare environments. This uncontrolled dissemination of multidrug resistant bacteria poses a potential public health threats. 2 Contents Abstract ............................................................................................................................. 1 Contents ............................................................................................................................ 3 List of tables ...................................................................................................................... 8 List of figures .................................................................................................................. 10 List of abbreviations ........................................................................................................ 12 Chapter 1 Introduction ................................................................................................... 14 1.1 Microbiology of staphylococci .............................................................................. 14 1.1.1 Morphology .................................................................................................... 14 1.1.2 Biochemical properties ................................................................................... 14 1.1.3 Taxonomy of Staphylococcus spp. ................................................................. 15 1.1.4 Lab identification ............................................................................................ 19 1.1.5 Molecular characterization ............................................................................. 25 1.2 Epidemiology of staphylococci ............................................................................. 30 1.2.1 Ecological niches of staphylococci ................................................................. 30 1.2.2 Hospital associated staphylococci .................................................................. 33 1.2.3 Community associated staphylococci ............................................................. 38 1.2.4 Environmental staphylococci .......................................................................... 38 1.2.5 Transmission of staphylococci........................................................................ 39 1.3 Pathogenicity of staphylococci .............................................................................. 41 1.3.1 Extracellular toxins ......................................................................................... 43 1.3.2 Surface proteins .............................................................................................. 45 1.3.3 Pathogenicity determination approaches ........................................................ 47 1.4 Antibiotic resistance .............................................................................................. 51 1.4.1 History of antibiotic resistance ....................................................................... 51 1.4.2 Limitation in tackling with antibiotic resistance............................................. 53 1.4.3 Antibiotic resistance mechanisms ................................................................... 54 1.4.4 In vitro susceptibility testing overview ........................................................... 61 1.5 Resistome .............................................................................................................
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