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New sequence variants of Staphylococcal Enterotoxins detected: host specificity and characterization study
- 1. New sequence variants of Staphylococcal Enterotoxins detected: host specificity and characterization study Macori Guerrino1, Gallina Silvia1, Johler Sophia2, Sihto Henna-Maria2, Stephan Roger2 and Decastelli Lucia 1 1National Reference Laboratory for Coagulase-Positive Staphylococci including Staphylococcus aureus, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Torino, Italy 2Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zurich, Zurich, Switzerland. 28th- 30th September 2016 Pacengo di Lazise (VR) NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. Guerrino Macori, PhD Post-Doctoral Researcher Teagasc APC - Microbiome Institute guerrino.macori@teagasc.ie @guerrinomacori
- 2. XVII Congresso Nazionale S.I.Di.LV. Outline 1. Introduction 2. Aim of the study 3. Material and Methods 4. Results 5. Discussion 6. Conclusion New Sequence Variants in S.aureus NRL for Coagulase Positive Staphylococci including Staphylococcus aureus IZSPLV - Turin
- 3. Background NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. New Sequence Variants in S.aureus
- 4. Background NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. New Sequence Variants in S.aureus
- 5. Background EFSA Journal 2015;13(12):4329 NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. New Sequence Variants in S.aureus
- 6. 1. Introduction NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. Five major SEs SEA, SEB, SEC, SED, SEE New Sequence Variants in S.aureus
- 7. 1. Introduction NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. Strain-specific variation of SE mRNA and protein levels environmental stress encountered in the food matrix Glucose stress (30%) can lead to decreased sed expression in stationary phase. •Enterotoxin D production is decreased in the presence of nitrite (c = 150 mg/L). •Staphylococcus aureus growth is not significantly affected by nitrite (c = 150 mg/L). New Sequence Variants in S.aureus
- 8. 1. Introduction NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. • Gene expression regulated by mRNA turnover modulation, factors in transcript degradation are poorly understood. • In S.aureus, the accessory regulator, SarA, (RNA-binding protein) turnover modulator. • A sigma factor (σ factor) is a protein needed only for initiation of RNA synthesis. • Alternative sigma factor B has an essential role in enterotoxin D regulation. • Staphylococcal accessory regulator A positively regulates enterotoxin D production. • Loss of accessory gene regulator does not alter enterotoxin D production. New Sequence Variants in S.aureus
- 9. 1. Introduction NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. Accessory elements intro Expression of the phage-encoded: • life cycle of the phage • nucleotide sequence variation in the sea gene and upstream promoter region Ana Camacho, and Margarita Salas FEMS Microbiol Rev 2010;34:828-841 New Sequence Variants in S.aureus SEA
- 10. 1. Introduction NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. Accessory elements and variation in SEB, SEC, and SED • seb and sed promoters region from -98 to -59 is required for the expression and regulation of seb • region from -34 to +18 is required for sed promoter function -98 -59 -34 +18 New Sequence Variants in S.aureus SEB and SED
- 11. 1. Introduction NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. Accessory elements SEB The seb gene resides in one of seven different S.aureus pathogenicity islands Variation in SEB levels produced Five allelic variants of SEB have been described that vary in biological activity SEB SEB SEB SEB SEB SEB New Sequence Variants in S.aureus 7 SaPIs
- 12. 1. Introduction NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. Accessory elements SEC sec gene located in 4 different SaPIs. • Four variants of SEC (SEC1-4) associated with human S.aureus strains • Host-specific variants SEC-bovine and SEC-ovine SaPIn1 SaPIm1 SaPImw2 SaPIbov1 SEC New Sequence Variants in S.aureus
- 13. 1. Introduction NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. Accessory elements SED • sed gene highly conserved, located on a plasmid • strains harboring a single base deletion in various locations in the sed sequence have been reported in S. aureus isolates from human. Argudin et al., 2010) New Sequence Variants in S.aureus human hosts
- 14. 2. Aim of the study NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. • Analyse the promoter and gene sequences of seb, sec, and sed from S.aureus strains originating from different sources; • Strain-specific variation in SE expression; • Monitoring the evolution of S.aureus pathogenicity and host adaptation. New Sequence Variants in S.aureus BIOINFORMATIC CHALLENGE
- 15. 3. Materials and Methods Bacterial Strains NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. 350 isolates DNA microarray enterotoxin hybridization patterns, spa types and clonal complexes . Population studied (37 strains) published data and characteristics well described • SFP outbreaks (9) • symptomatic nasal colonization or cases of infections in humans (16) • rabbit carcasses (8) • bovine mastitis milk (4) New Sequence Variants in S.aureus 12 SEB 10 SEC 15 SED
- 16. 3. Materials and Methods NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. • 350 strains DNA Extraction and PCR Amplification • Microarray and molecular typing • Selected strains PCR Purification and Sequencing • Sequence variants: Toxin detection by SET-RPLA • Amino acid identity, bioinformatic New Sequence Variants in S.aureus
- 17. 4. Results NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. seb Promoter and Gene Sequences (12 strains) Five variants of the seb promoter (sevp v1–v5) New Sequence Variants in S.aureus
- 18. 4. Results NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. New Sequence Variants in S.aureus V1 Multiple positions: Four variants of the seb gene (seb v1–v4) Firstly described
- 19. 4. Results NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. New Sequence Variants in S.aureus V2 Multiple positions: Four variants of the seb gene (seb v1–v4) Firstly described
- 20. 4. Results NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. New Sequence Variants in S.aureus V3 Multiple positions: Four variants of the seb gene (seb v1–v4) Firstly described
- 21. 4. Results NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. New Sequence Variants in S.aureus V4 Multiple positions: Four variants of the seb gene (seb v1–v4) Firstly described
- 22. 4. Results NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. Three variants of the sec promoter (secp v1–v3) Four variants of the sec gene (sec v1–v4) sec Promoter and Gene Sequences (10 strains) New Sequence Variants in S.aureus
- 23. 4. Results NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. New Sequence Variants in S.aureus
- 24. 4. Results NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. Three variants of the sed promoter (sedp v1–v3) New Sequence Variants in S.aureus Sed Promoter and Gene Sequences (12 strains)
- 25. 4. Results NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. New Sequence Variants in S.aureus Sed Promoter and Gene Sequences (12 strains) Four variants of the sed gene (sed v1–v4)
- 26. 4. Results NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. New Sequence Variants in S.aureus Four variants of the sed Amino Avid (sed v1–v4) Rabbit carcasses
- 27. 4. Results NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. New Sequence Variants in S.aureus Four variants of the sed Amino Acid (sed v1–v4) Rabbit carcasses STOP CODON
- 28. 4. Results NRL for Coagulase Positive Staphylococci including Staphylococcus aureus XVII Congresso Nazionale S.I.Di.LV. New Sequence Variants in S.aureus Four variants of the sed Amino Acid (sed v1–v4) Rabbit carcasses STOP CODON POSITIVE RESULTS – ENTEROTOXIN DETECTED SET-RPLA
- 29. One SED variant exhibited a deletion in sed premature stop codon and a truncated sed amino acid precursor. FBOs isolates, deletions at two nucleotide positions (150 and 514) premature stop codon A deletion in sed identical to the one seen in rabbit isolates in this study (nucleotide position 521) has been reported in S. aureus isolates originating from humans and from food but not success the detection by Western Blot and qPCR. In contrast, in this study, three of four rabbit strains tested with truncated sed variants yielded a weak, but positive result for SED in the SET-RPLA assay NRL for Coagulase Positive Staphylococci including Staphylococcus aureus 5. Discussion New Sequence Variants in S.aureus XVII Congresso Nazionale S.I.Di.LV.
- 30. 6. Conclusion 1. sequence variation in enterotoxin genes of S. aureus strains isolated from various sources. 2. All SED+ rabbit strains tested in this study harbored a sed variant that exhibited a deletion in sed leading to a premature stop codon. 3. The sequence data -> sequence variation in enterotoxin genes of S. aureus strains isolated from various sources, truncated proteins and specificity of host? Prospects: quantifying gene expression? mRNA, genomic profiles, Mass spectrometry? NRL for Coagulase Positive Staphylococci including Staphylococcus aureus New Sequence Variants in S.aureus XVII Congresso Nazionale S.I.Di.LV.
- 31. • Thanks to SIDiLV for the opportunity; • Thanks to the Institute for Food Safety and Hygiene (IFSH) of the University of Zurich and the Director Stephan Roger for guest me as a Visitor Scientist and the • Thanks to the researcher of the IFSH, Sophia Johler for the great experience, the competence and the quality of the researches she is conducting. It was very inspiring , I learnt a lot! • Thanks to Henna-Maria Sihto for the collaboration; • Thanks to the Italian NRL for Coagulase Positive Staphylococci, in particular the Head of the Laboratory Lucia Decastelli and Silvia Gallina for the chance to work with you! • The researchers of the Food Control lab in Turin; • Thanks to Paul D Cotter the head of the Vision I lab at Teagasc, Food Bioscience Dept. (APC Microbiome Institute Fermoy, Co. Cork, Ireland) where I’m working. Guerrino Macori, PhD Post-Doctoral Researcher Teagasc APC - Microbiome Institute guerrino.macori@teagasc.ie @guerrinomacori Keep in touch!