dc.contributor.author
Hühn, Stephan
dc.date.accessioned
2018-06-07T22:50:39Z
dc.date.available
2010-04-29T13:50:48.439Z
dc.identifier.uri
https://refubium.fu-berlin.de/handle/fub188/9708
dc.identifier.uri
http://dx.doi.org/10.17169/refubium-13906
dc.description
Abstract Zusammenfassung Table of Contents I List of Abbreviations V Index of
Figures and Tables IX 1\. Introduction 1 1.1 Salmonella taxonomy and
serological classification 1 1.2 Epidemiology 3 1.2.1 Epidemiology of serovars
commonly found in broilers and biological properties 5 1.3 Disease and
Pathogenesis 7 1.4 Molecular typing and characterisation methods 8 1.4.1
Molecular characterisation of the genomic setting 9 1.5 Molecular
characteristics of the Salmonella genome 10 1.5.1 Salmonella Pathogenicity
Islands and Islets 10 1.5.2 Prophages 14 1.5.3 Salmonella virulence plasmid 15
1.5.4 Fimbrial clusters 15 1.5.5 Antimicrobial resistance and resistance
determinants 16 1.5.6 Elements associated with DNA-mobility 17 1.5.7
Salmonella Genomic Island 1 (SGI-1) 18 1.5.8 Genes involved in serovar
identification according to White- Kauffmann-Le Minor scheme 19 1.6 Aim of the
thesis 20 2\. Material and Methods 21 2.1 Materials 21 2.1.1 Instruments 21
2.1.2 Disposables 22 2.1.3 Bacterial strains 22 2.1.4 Culture media 24 2.1.5
Reagents and buffers 27 2.1.6 Reaction kits 30 2.1.7 Primers and
Oligonucleotides 30 2.1.8 Software 31 2.2 Methods 31 2.2.1 DNA microarray
production 31 2.2.1.1 Oligonucleotide design 31 2.2.1.2 Preparation of the
source plate 32 2.2.1.3 Print process 32 2.2.1.4 Post-coupling processing 34
2.2.2 Salmonella DNA purification 34 2.2.3 Fluorescence-labelling of genomic
DNA 35 2.2.3.1 DNA labelling 36 2.2.3.2 Purification of labelled DNA 36 2.2.4
Construction of an internal hybridisation control 37 2.2.5 Microarray
hybridisation of the labelled DNA 37 2.2.5.1 Hybridisation 37 2.2.5.2 Post
hybridisation washing 37 2.2.6 Data handling and DNA microarray analysis 38
2.2.6.1 DNA microarray slide scanning 38 2.2.6.2 Normalisation of signal
intensities 38 2.2.6.3 Analysis of microarray results 39 2.2.7 Validation of
the DNA microarray 39 2.2.7.1 Validation of the microarray signals by PCR 40
2.2.7.2 Evaluation of probe specificity 41 2.2.8 Pulsed-field gel
electrophoresis 42 2.2.8.1 Preparation of DNA-agarose blocks 43 2.2.8.2 Cell-
lyses in DNA-agarose blocks 44 2.2.8.3 Washing of DNA-agarose blocks 44
2.2.8.4 Digestion with XbaI 44 2.2.8.5 Preparation of the PFGE gel 44 2.2.8.6
Performance of electrophoresis and staining 44 2.2.8.7 Cluster analysis 45
2.2.9 Antimicrobial susceptibility testing by minimal inhibitory concentration
(MIC) 45 3\. Results 47 3.1. Salmonella DNA microarray development 47 3.1.1
Construction of microarray and controls 47 3.1.2 Validation of the DNA
microarray 48 3.1.3 Evaluation of the specificity of oligonucleotide probes 49
3.1.4 Internal hybridisation control 50 3.1.5 Experimental workflow 50 3.2.
Investigation of S. enterica subsp. enterica serovar 4,12:d:- 52 3.2.1
Analyses of serovar 4,12:d:- using PFGE 52 3.2.2 Virulence determinants
characterisation of the serovar 4,12:d:- 53 3.2.3 Comparison of serovar
4,12:d:- virulence determinants to other serovars 55 3.2.4 Other
chracteristics of serovar 4,12:d:- 57 3.3 Investigation of S. enterica subsp.
enterica serovar Paratyphi B dT+ and serovar 4,5,12:b:- 57 3.3.1 Antimicrobial
resistance characteristics of serovar Paratyphi B dT+ 57 3.3.2 Analysis of
population structure 60 3.3.3 Virulence determinant characterisation of
serovar Paratyphi B dT+ 62 3.3.4 Other chracteristics of serovar Paratyphi B
dT+ 65 4\. Discussion 66 4.1 Development of the Salmonella DNA microarray 67
4.1.1 Development of the Salmonella DNA microarray 67 4.1.2 Internal
hybridisation and print control (IHC) 70 4.1.3 Validation of the DNA
microarray 70 4.2 Application of the DNA microarray to poultry associated
Salmonella serovars 72 4.2.1 Application of the Salmonella DNA microarray to
study the gene repertoire of serovar 4,12:d:- 72 4.2.2 Application of the
Salmonella DNA microarray to study the gene repertoire of serovar Paratyphi B
dT+ 76 4.3 Outlook 79 5\. References 81 6\. Eidesstattliche Erklärung 103 7\.
List of publications 104 8\. Acknowledgement 107 9\. Curriculum vitae 108 10\.
Appendix I 111
dc.description.abstract
Poultry is recognised as an important source for human infections caused by
Salmonella enterica subsp. enterica. A European baseline survey during the
years 2005 and 2006 has revealed that the monophasic Salmonella enterica
subsp. enterica serovar 4,12:d:- and the serovar Paratyphi B d-tartrate
positive (dT+) were one of the most frequently isolated serovars in German
broiler flocks. This study focuses on the genotypic characterisation of these
both serovars using pulsed- field gel electrophoresis and DNA microarray to
determine the clonality, the pathogenic gene repertoire, and resistance
determinants. For that purpose, a prototype of a Salmonella DNA microarray
comprising 276 60-mer and 5 40-mer oligonucleotide probes was developed and
in-house validated. Nearly identical PFGE profiles and a highly similar gene
repertoire were found among serovar 4,12:d:- isolates of feed, animal and
human sources. All strains were susceptible to 16 antimicrobial agents tested
and did not encode any resistance genes. The serovar lacked genes with known
contributions to pathogenicity in comparison to serovars highly prevalent in
humans. The comparison of the virulence gene repertoire to other serovars
indicated the highest relationship to serovar Derby (4,12:f,g:[1,2]). Among
serovar Paratyphi B dT+ isolates two major clonal lines, which could be
phenotypically differentiated by the expression of the O:5-antigen, were
identified. All O:5 antigen negative (O:5-) strains were multi-drug resistant
and originated from Western Europe (Belgium, Netherlands and Germany) poultry.
Strains exhibiting the O:5-antigen (O:5+) encoded by the oafA gene revealed a
more heterogeneous group including multi-drug resistant and susceptible
strains. Compared to O:5- isolates, serovar Paratyphi B dT+ O:5+ strains
possessed additional virulence determinants. The Salmonella Genomic Island 1
was only found in O:5+ strains. Five monophasic serovar 4,5,12:b:- isolates
lacking the phase-2 flagellar antigen were highly similar to serovar Paratyphi
B dT+ isolates of the O:5+ group. Currently it can not be estimated that
serovar 4,12:d:- exposes a high risk to humans caused by consumption of
poultry. In concern of serovar Paratyphi B dT+ a multi-drug resistant clone
still persists in chickens across Western Europe. Moreover, the existence of a
second, more heterogeneous serovar Paratyphi B dT+ O:5+ group was shown
encoding additional fimbrial and virulence genes suggesting a more diverse
origin and an ubiquitous spreading.
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dc.description.abstract
Geflügel stellt eine wichtige Quelle für durch Salmonella enterica subsp.
enterica verursachte Lebensmittel assoziierte Humaninfektionen dar. Das in den
Jahren 2005 und 2006 durchgeführte Europäische Masthähnchen Monitoring zeigte,
dass der monophasische Salmonella enterica subsp. enterica Serovar 4,12:d:-
und der Serovar Paratyphi B, d-Tartrat fermentierend (dT+), die am häufigsten
isolierten Serovare aus Masthähnchen Herden waren. Die in dieser Studie
durchgeführte genotypische Charakterisierung untersucht diese beiden Serovare
hinsichtlich ihrer Klonalität, dem Pathogenitätsgenrepertoire und
Resistenzdeterminanten mittels Pulsfeld-Gelelektrophorese und DNA Microarray.
Zu diesem Zweck wurde ein neuer Prototyp eines Salmonella DNA Microarrays,
ausgestattet mit 276 60-mer und 5 40-mer Oligonucleotiden, entwickelt und
validiert. Nahezu identische PFGE Profile und ein sehr ähnliches Genrepertoire
wurden für Serovar 4,12:d:- Isolate aus Futtermittel, Tier und Mensch
gefunden. Alle Stämme waren sensibel gegenüber den 16 getesteten Antibiotika
und kodierten keine Resistenzgene. Dem Serovar fehlten Pathogenitätsgene, die
üblicherweise von häufig in Menschen vorkommenden Serovaren kodiert werden.
Der Vergleich des Virulenzgenrepertoires mit anderen untersuchten Serovaren
zeigte die höchste Verwandschaft zum Serovar Derby (4,12:f,g:[1,2]). Serovar
Paratyphi B dT+ Isolate unterteilten sich in zwei klonale Linien, die
phänotypisch durch die Expression des O:5 Antigens unterschieden werden
konnten. Alle aus Westeuropa (Belgien, Niederlande und Deutschland) stammenden
O:5 negativen (O:5-) Stämme waren multiresistent und aus Geflügel isoliert.
Stämme die über das O:5 Antigen verfügten (O:5+), kodiert durch oafA, zeigten
eine heterogene Gruppe die sowohl sensible, als auch resistente Stämme
einschloss. Im Vergleich zu den O:5- Isolaten, besaßen O:5+ Stämme zusätzliche
Virulenzdeterminanten. Die Salmonella Genomic Island 1 wurde nur in den O:5+
Stämmen gefunden. Fünf monophasische Serovar 4,5,12:b:- Stämme, denen das
Phase2-Antigen fehlte, waren den Serovar Paratyphi B dT+ Isolaten der O:5+
Gruppe sehr ähnlich. Zur Zeit kann nicht davon ausgegangen werden, dass
Serovar 4,12:d:- ein hohes Risiko beinhaltet, nach Verzehr von Geflügel
Humaninfektionen auszulösen. Für Paratyphi B dT+ O:5- konnte die Persistenz
eines multiresistenten Klones in Westeuropa in Geflügelbeständen nachgewiesen
werden. Weiterhin wurde die Existenz einer zweiten, heterogenen O:5+ Serovar
Paratyphi B dT+ Gruppe nachgewiesen, die zusätzliche Fimbrien- und
Virulenzgene kodiert und sehr wahrscheinlich ubiquitär verbreitet ist.
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dc.format.extent
IX, 136 S.
dc.rights.uri
http://www.fu-berlin.de/sites/refubium/rechtliches/Nutzungsbedingungen
dc.subject.ddc
600 Technik, Medizin, angewandte Wissenschaften
dc.title
Development and validation of a DNA microarray for characterisation and typing
of Salmonella isolates
dc.contributor.contact
huehn.stephan@vetmed.fu-berlin.de
dc.contributor.firstReferee
Prof. Dr. Appel
dc.contributor.furtherReferee
Prof. Dr. Multhaup
dc.date.accepted
2010-02-05
dc.identifier.urn
urn:nbn:de:kobv:188-fudissthesis000000017206-3
dc.title.translated
Entwicklung und Validierung eines DNA Mikroarrays zur Charakterisierung und
Typisierung von Salmonella Isolaten
de
refubium.affiliation
Biologie, Chemie, Pharmazie
de
refubium.mycore.fudocsId
FUDISS_thesis_000000017206
refubium.mycore.derivateId
FUDISS_derivate_000000007504
dcterms.accessRights.dnb
free
dcterms.accessRights.openaire
open access