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MOLECULAR and ANTIGENIC CHARACTERIZATION of Streptococcus Suis Serotype 2 ISOLATES in CENTRAL VIETNAM

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MOLECULAR and ANTIGENIC CHARACTERIZATION of Streptococcus Suis Serotype 2 ISOLATES in CENTRAL VIETNAM University of Sassari Department of Biomedical Sciences INTERNATIONAL PHD SCHOOL IN BIOMOLECULAR AND BIOTECHNOLOGICAL SCIENCES XXVIII Cycle MOLECULAR AND ANTIGENIC CHARACTERIZATION OF Streptococcus suis serotype 2 ISOLATES IN CENTRAL VIETNAM Director: Prof. LEONARDO A. SECHI Tutor: Prof. ALBERTO ALBERTI Co-tutor: VAN AN LE MD. PhD. PhD thesis of Dr. HOANG BACH NGUYEN ACADEMIC YEAR 2012-2015 ABBREVIATION ITS : 16S–23S ribosomal (r) DNA intergenic spacer region 6-PGD: : 6-phosphogluconate-dehydrogenase ABC : Ammonium bicarbonate ACN : Acetonitrile AmyA : Amylopullulanase BBB : blood-brain barrier BHI : Brain Heart Infusion BMEC : Brain microvascular endothelial cell BMEC : brain microvascular endothelial cells CNS : central nervous system CPEC : Choroid plexus epithelial cell CSF : Cerebrospinal fluid GAPDH : Glyceraldehyde-3-phosphate dehydrogenase LPXTG : Leu-Pro-any-Thr-Gly LTA : Lipoteichoic acid NET : Neutrophil-extracellular trap PCR : Polymerase chain reaction PSF : peptide-spectrum match SS2 : Streptococcus suis serotype 2 TFA : Trifluoroacetic acid WPE : Whole protein extracts TABLE OF CONTENTS ACKNOWLEDGEMENT ABBREVIATION TABLE OF CONTENTS I. ABSTRACT .......................................................................................................... 1 II. INTRODUCTION ................................................................................................ 2 2.1. Streptococcus suis: taxonomy and main biological features ........................... 2 2.2. Streptococcus suis serotype 2 and meningitis .................................................. 4 2.3. Invasion of Streptococcus suis ......................................................................... 7 2.3.1. Colonization: adherence and invasion of epithelial surfaces..................... 7 2.3.2. S. suis survival in blood and dissemination ............................................... 9 2.3.3. Inflammatory activation and septicemia .................................................. 11 2.3.4. Central nervous system invasion and meningitis .................................... 12 2.4. Epidemiology ................................................................................................. 15 2.5. Diagnosis and treatment of Streptococcus suis meningitis ............................ 16 III. RESEARCH OBJECTIVES .............................................................................. 19 VI. MATERIALS AND METHODOLOGIES ........................................................ 20 4.1. Ethical approval ............................................................................................. 20 4.2. Media and buffers .......................................................................................... 20 4.3. Sampling ........................................................................................................ 21 4.4. Bacterial strain, culture and isolation ............................................................. 22 4.5. DNA extraction .............................................................................................. 23 4.6. Capsular serotyping confirmation .................................................................. 23 4.7. 16S-23S rRNA intergenic spacer fragment amplication ............................... 24 4.8. Gene profile analysis ...................................................................................... 25 4.9. Whole protein extracts ................................................................................... 26 4.10. SDS-PAGE ................................................................................................... 26 4.11. Western immunoblotting .............................................................................. 26 4.12. Pooled WPE againts pooled patient sera by western immunoblotting ........ 27 4.13. 2-D PAGE .................................................................................................... 28 4.14. Band and spot excision and Coomassie-stained destaining ......................... 29 4.15. In-gel tryptic digestion and extraction of peptides digestion products ........ 29 4.16. LC-MS/MS ANALYSIS ............................................................................. 30 4.17. Mass spectrophotometry data analyses ........................................................ 31 V. RESULTS ........................................................................................................... 32 5.1. Species identification ..................................................................................... 32 5.2. Capsular serotype identification ..................................................................... 33 5.3. 16S-23S Intergenic spacer phylogenetic analysis .......................................... 33 5.4. SDS-PAGE, western immunoblotting and immunogenic protein identification ............................................................................................................................... 36 5.5. 2-D electrophoresis, western immunoblotting and immunogenic protein identification ......................................................................................................... 40 VI. DISCUSSION ................................................................................................ 44 VII. APPENDIX .................................................................................................... 51 VIII. REFERENCES ............................................................................................... 52 I. ABSTRACT Streptococcus suis (S. suis) is a Gram-positive bacterium with clinical relevance in pigs and is emerging in human. Based on differences in antigenic properties of the polysaccharide capsule, 35 S. suis serotypes have been distinguished to date, of which serotype 2 is most commonly associated with disease in human and pigs, worldwide. Recently, S. suis emerged as a zoonotic agent in human in contact with infected pigs or with their products(Wertheim et al. 2009)(Wertheim et al. 2009)(Wertheim et al. 2009)(Wertheim et al. 2009). In 2006 a major S. suis outbreak resulted in more than 200 human cases with a fatality rate of nearly 20% in China. More recently, several studies from Thailand, Hong Kong, Taiwan, Singapore and Viet Nam demonstrated that S. suis can cause adult endocarditis, septicemia, arthritis and especially meningitis with high fatality rate and severe neurological sequelae. Multilocus enzyme electrophoresis, restriction endonuclease analysis with HaeIII, ribotyping, repetitive extragenic palindromic (REP) or enterobacterial repetitive intergenic consensus (ERIC), arbitrarily primed PCR, and pulsed-field gel electrophoresis (PFGE) have been used to determine S. suis strains epidemiological relationships. Characterization of the 16S-23S ribosomal (r) DNA intergenic spacer region (ITS) has also been used to compare bacterial strains and to identify species within the genus Streptococcus, including S. suis. In this study, a specific and sensitive PCR assay for fast detection of S. suis serotype 2 is crucial to meningitis treatment based on the S. suis serotype 2 cps2J gene was developed and evaluated for diagnosis in meningitis patients hospitalized in Thua Thien Hue, Viet Nam. The intergenic spacer region was amplified by traditional PCR from strains obtained in this study and sequenced. ITS - based phylogenetic analysis is also presented and discussed. We also identified and characterized the main immunodominant antigens of Streptococcus suis serotype 2 based on the immunoproteomics approach. Nguyen Hoang Bach - Molecular and antigenic characterization of Streptococcus suis serotype 2 isolates in central Vietnam - Doctorate Thesis of International PhD School In Biomolecular and Biotechnological Sciences, University of Sassari 1 II. INTRODUCTION 2.1. Streptococcus suis: taxonomy and main biological features In the early 1950s, Jensen et al. (1951) and Field et al. (1954) reported streptococcal septicemia as cause of meningitis and arthritis in pigs and piglets in the United Kingdom and in the Netherlands. Later, de Moor el al. (1963) described similar hemolytic streptococci that had been isolated from septicemic pigs and could be differentiated biochemically and serologically from the known streptococcal species. These streptococci were placed into two Lancefield groups. Strains isolated in newborn pigs were designated “group S”, whereas strains from older pigs were designated “group R” (Staats et al. 1997). There were reports indicating that some strains of this species contained streptococcal group D antigens. However, later reports indicate that group R and group D antigens are similar but not identical, and observed reaction with group D antisera was due to cross-reaction. S. suis is a very heterogeneous species. So far, 35 capsular serotypes (types 1 to 34 and type 1/2) have been identified based on the difference of antigenic carbohydrate types of their capsule (Staats et al. 1997). The discovery of additional serotypes of this species led to a change in nomenclature used to indicate capsular types. Group R, the most common strain identified, is type 2, and group S is type 1. The important change to clinical microbiologists is that the only serotype identified from human has been type 2 (group R). There are difficulties when using the original method for serological grouping of S. suis with Lancefield procedure. Therefore, it is recommended that a capsular reaction should be used for identification of the various serotypes. This reaction is similar to
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