Dissemination, antibiotic susceptibility, proteomic and genomic characterization of antibiotic ...

316 Pages·2017·3.81 MB·English

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
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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.

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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 .............................................................................................................. 69
1.6 Genomics related to antibiotic resistance .............................................................. 69
1.6.1 Lateral transfer of antibiotic resistance ........................................................... 70
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1.6.2 Vertical transfer of antibiotic resistance ......................................................... 71
1.7 Research Aims ....................................................................................................... 74
Chapter 2 Materials and Methods ................................................................................. 75
2.1 Sample collection .................................................................................................. 75
2.2 Isolation of staphylococci ...................................................................................... 76
2.3 Identification ......................................................................................................... 78
2.3.1 MALDI-TOF MS identification ..................................................................... 78
2.3.2 Reproducibility of MALDI-TOF MS ............................................................. 79
2.3.3 16S rRNA gene sequencing identification...................................................... 80
2.4 Antibiotic susceptibility test .................................................................................. 82
2.4.1 Disc diffusion test ........................................................................................... 82
2.4.2 MIC test .......................................................................................................... 82
2.4.3 Interpretation ................................................................................................... 83
2.5 Molecular characterization of staphylococci ......................................................... 84
2.5.1 mecA gene detection ....................................................................................... 84
2.5.2 mecC gene detection ....................................................................................... 88
2.5.3 SCCmec typing ............................................................................................... 89
2.5.4 Multi-locus sequence typing of S. epidermidis ............................................... 94
2.6 Whole genomic sequence ...................................................................................... 96
2.6.1 Identification of S. epidermidis 118 (G6_2) ................................................... 96
2.6.2 General test of S. epidermidis 118 (G6_2) ..................................................... 97
2.6.3 Whole genomic sequence assembly and comparative genomics .................... 97
2.6.4 Reference strains ............................................................................................. 98
2.6.5 Nucleotide sequence accession numbers ........................................................ 99
2.6.6 Antibiotic resistance gene determination ........................................................ 99
2.7 mecA gene transfer via broth mating experiment .................................................. 99
2.7.1 Cell stock ........................................................................................................ 99
2.7.2 Mating experiment .......................................................................................... 99
2.7.3 ProlexTM staph XTRA latex tests .................................................................. 100
2.7.4 MIC oxacillin tests ........................................................................................ 100
2.7.5 Pulse-field gel electrophoresis ...................................................................... 100
2.8 Proteomic analysis ............................................................................................... 101
2.8.1 Protein extraction .......................................................................................... 102
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2.8.2 Protein concentration .................................................................................... 102
2.8.3 One-dimensional SDS-PAGE ....................................................................... 103
2.8.4 In-gel tryptic digestion of protein for LC-MS/MS ....................................... 103
2.8.5 LC-MS/MS analysis of tryptic peptides ....................................................... 103
2.9 Bioinformatic analysis ......................................................................................... 105
2.9.1 mecA and mecC gene .................................................................................... 105
2.9.2 SCCmec typing ............................................................................................. 105
2.9.3 BioNumerics analysis ................................................................................... 105
2.9.4 Multi-locus sequence typing ......................................................................... 107
Chapter 3 Isolation, purification of environmental Staphylococci ............................... 108
3.1 Sample collection ................................................................................................ 108
3.2 Isolation ............................................................................................................... 110
3.3 Purification .......................................................................................................... 112
3.4 Chapter summary ................................................................................................ 112
Chapter 4 Identification of environmental staphylococci ............................................. 113
4.1 Bacterial identification by MALDI-TOF MS ..................................................... 113
4.2 Reproducibility of MALDI-TOF MS .................................................................. 118
4.3 16S rRNA gene sequencing ................................................................................ 120
4.4 MALDI-TOF MS data analysis ........................................................................... 124
4.4.1 Cluster analysis of selected staphylococci .................................................... 124
4.4.2 Cluster analysis of Staphylococcus spp. recovered from each site ............... 125
4.5 Chapter summary ................................................................................................ 138
Chapter 5 Phenotypic analysis of environmental staphylococci ................................... 140
5.1 Antibiotic susceptibility test ................................................................................ 140
5.2 MIC (oxacillin) test ............................................................................................. 146
5.3 Antibiotic susceptibility of taxonomically closely related staphylococci ........... 146
5.3.1 Antibiotic susceptibility of staphylococci..................................................... 146
5.3.2 Antibiotic resistance patterns of taxonomically closely related staphylococci
............................................................................................................................... 146
5.3.3 Cluster analysis of each staphylococcal species combined with antibiotic
susceptibility profile .............................................................................................. 148
5.3.4 Variations of antibiotic susceptibility profile of taxonomically closely related
staphylococci ......................................................................................................... 152
5.3.5 Percentage of multiple resistant staphylococci recovered from each site .... 154
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5.4 Chapter summary ................................................................................................ 156
Chapter 6 Genotypic analysis of selected staphylococci .............................................. 157
6.1 mecA gene ............................................................................................................ 158
6.1.1 Detection of mecA gene for all staphylococci .............................................. 158
6.1.2 mecA gene expression in selected staphylococci .......................................... 165
6.2 SCCmec typing .................................................................................................... 166
6.3 Multi-locus sequence typing ............................................................................... 169
6.4 Chapter summary ................................................................................................ 172
Chapter 7 Genomic analysis of S. epidermidis 118 (G6_2) .......................................... 173
7.1 Bacteria ................................................................................................................ 173
7.2 Phylogenetic relationship with clinical reference S. epidermidis ........................ 174
7.3 General genomic features and plasmids .............................................................. 176
7.4 Genotypic prediction of antibiotic resistance ...................................................... 179
7.5 Functional genes uniquely found in S. epidermidis 118 (G6_2) ......................... 183
7.6 Comparative virulence genes .............................................................................. 183
7.7 Chapter summary ................................................................................................ 189
Chapter 8 mecA gene transfer via mating experiment .................................................. 190
8.1 mecA gene amplification in conjugants ............................................................... 190
8.2 16S rRNA gene sequencing of conjugant ........................................................... 191
8.3 Prolex staph XTRA latex tests and MIC of conjugant ........................................ 191
8.4 Southern blotting ................................................................................................. 192
8.5 PFGE results ........................................................................................................ 192
8.6 Comparative proteomic analysis ......................................................................... 193
8.6.1 Protein extraction .......................................................................................... 194
8.6.2 In-gel trypsin digestion ................................................................................. 194
8.6.3 Comparative analysis of protein expression ................................................. 195
8.7 Chapter summary ................................................................................................ 201
Chapter 9 Discussion .................................................................................................... 203
Staphylococcus spp. ................................................................................................... 204
MALDI-TOF MS ...................................................................................................... 206
Antibiotic resistance .................................................................................................. 209
SCCmec and MLST ................................................................................................... 216
Antibiotic resistant and virulence genes in S. epidermidis 118 (G6_2) .................... 220
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