Development of genotypic and phenotypic methods for the identification and differentiation of hazardous Bacillus cereus group strains [Elektronische Ressource] / Martina Fricker

LEHRSTUHL FÜR MIKROBIELLE ÖKOLOGIE
DEPARTMENT FÜR GRUNDLAGEN DER BIOWISSENSCHAFTEN
TECHNISCHE UNIVERSITÄT MÜNCHEN



Development of genotypic and phenotypic methods for the
identification and differentiation of hazardous
Bacillus cereus group strains


MARTINA FRICKER


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines Doktors der Haushalts- und Ernährungswissenschaften
(Dr.oec.troph.)

genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr.-Ing. Dr.-Ing.habil. Ulrich M. Kulozik
Prüfer der Dissertation: 1. Univ.-Prof. Dr.rer.nat.habil. Siegfried Scherer
2. Univ.-Prof. Dr.med.vet. Dr.med.vet.habil. Dr.h.c. Johann Bauer
3. Univ.-Prof. Dr.rer.nat.habil. Rudi F. Vogel



Die Dissertation wurde am 19.07.2007 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 27.09.2007 angenommen.
























Nothing shocks me. I’m a scientist.

Harrison Ford as Indiana Jones Table of contents

TABLE OF CONTENTS

Table of contents……………………………………………………………..……………………………………………I
List of figures……………………………………………………………………………………………….……….…...IV
List of tables…………………………………………………………………………………..……………….…………..V
Summary…………………………………………………………………..………………………..……………………...VI
Zusammenfassung………………………………………………………………..…………………………………...VII
1 Introduction.................................................................................................................1
1.1 The Bacillus cereus group ..........................................................................................1
1.2 Pathogenicity of B. cereus2
1.3 Available methods for the detection of B. cereus.......................................................3
1.4 Aims of this work .......................................................................................................5
2 Material and methods..................................................................................................6
2.1 Bacterial strains ..........................................................................................................6
2.2 Cultivation methods....................................................................................................6
2.3 DNA isolation methods ..............................................................................................8
2.4 Molecular characterization .........................................................................................9
2.4.1 Standard PCR .............................................................................................................9
2.4.2 Inverse PCR and module jumping..............................................................................9
2.4.3 Cloning of PCR amplicons, DNA sequencing and sequence analysis.....................10
2.4.4 Sequence typing........................................................................................................10
2.4.5 Amplification and sequencing of the plcR regulator gene .......................................11
2.4.6 Standard PCR assay for detection of emetic B. cereus ............................................11
2.4.7 SYBR green Real-time PCR for detection of emetic B. cereus ...............................12
2.4.8 TaqMan Real-time PCR including an internal amplification control.......................13
2.4.9 Application of the novel Real-time assays in two recent food poisonings...............14
2.4.10 Multiplex PCR Assay for toxin gene profiling of toxigenic B. cereus.....................14
2.4.11 Southern analysis......................................................................................................15
2.5 Phenotypic characterization......................................................................................16
2.5.1 Test of growth limits ................................................................................................16
2.5.2 Influence of temperature and pH on growth.............................................................16
2.5.3 Gamma phage lysis assay and antibiotic resistance .................................................17
2.5.4 Analysis of phosphatidylcholine-phospholipase C (PL-PLC) activity ....................17
2.5.5 Fourier transform infrared (FTIR) spectroscopy......................................................17
I Table of contents

2.5.6 Artificial neural networks (ANN).............................................................................18
3 Results.......................................................................................................................19
3.1 Genotyping ...............................................................................................................19
3.2 Development of novel PCR assays...........................................................................21
3.2.1 Standard PCR assay for the detection of emetic B. cereus.......................................22
3.2.2 Real-time PCR assays for the detection of emetic B. cereus....................................23
3.2.3 Toxin gene profiling .................................................................................................29
3.3 Growth profiles of selected B. cereus isolates..........................................................31
3.3.1 Temperature correlated growth limits ......................................................................31
3.3.2 Influence of different temperatures and pH..............................................................33
3.4 Comparison of different selective plating media for the detection and identification
of B. cereus group strains ........................................................................................34
3.4.1 Plating media assessment .........................................................................................34
3.4.2 Gamma-phage sensitivity and antibiotic resistance of selected strains....................37
3.4.3 Detection of B. cereus from complex food samples on selective plating media......37
3.5 ANN assisted Fourier transform infrared spectroscopy ...........................................39
3.5.1 Analysis of FTIR spectra..........................................................................................39
3.5.2 Architecture and validation of the artificial neural networks (ANN).......................41
3.5.3 Wave numbers selected for the differentiation between the B. cereus group
members ...................................................................................................................42
3.5.4 Application of ANN assisted FTIR spectroscopy in population analysis in soil
samples and a rice sample from a food poisoning outbreak ....................................44
3.6 Application of the developed methods for the characterization of B. cereus strains
isolated from foods and in recent food poisonings..................................................46
3.6.1 Characterization of B. cereus isolates from naturally contaminated cream .............46
3.6.2 Identification of the etiological agent in recent food borne outbreaks.....................48
4 Discussion.................................................................................................................50
4.1 Genotypic and phenotypic characterization of B. cereus group isolates..................50
4.1.1 Sequence typing confirms the monomorphic structure of emetic B. cereus strains.50
4.1.2 Emetic B. cereus strains show distinct growth characteristics .................................51
4.1.3 Metabolic fingerprinting of B. cereus group organisms by Fourier transform
infrared spectroscopy ...............................................................................................52
4.2 Evaluation of selective plating media.......................................................................56
4.3 Development of genotypic methods for the detection of emetic B. cereus isolates.59
II Table of contents

4.3.1 PCR assays for the detection of emetic B. cereus isolates .......................................59
4.4 Application of the developed methods .....................................................................62
4.5 General conclusions and perspectives ......................................................................63
5 References.................................................................................................................65
6 Publications...............................................................................................................78
7 Appendix...................................................................................................................79
7.1 Bacterial strains ........................................................................................................79
7.2 Oligonucleotide primers and probes..............................................................