New sequence variants of Staphylococcal Enterotoxins detected: host specificity and characterization study

New sequence variants of Staphylococcal Enterotoxins detected: host specificity and

characterization studyMacori 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 2016Pacengo di Lazise (VR)

NRL for Coagulase Positive Staphylococci including Staphylococcus aureus

XVII Congresso Nazionale S.I.Di.LV.

Guerrino Macori, PhDPost-Doctoral ResearcherTeagascAPC - Microbiome [email protected]@guerrinomacori


Outline

1. Introduction2. Aim of the study3. Material and Methods4. Results 5. Discussion6. Conclusion

New Sequence Variants in S.aureus

NRL for Coagulase Positive Staphylococci including Staphylococcus aureusIZSPLV - Turin

Background




Background




Background

EFSA Journal 2015;13(12):4329




1. Introduction



Five major SEs SEA, SEB, SEC, SED, SEE


1. Introduction



Strain-specific variation of SE mRNA and protein levelsenvironmental 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).


1. Introduction



• 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.


1. Introduction



Accessory elements introExpression 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


SEA

1. Introduction



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


SEB and SED

1. Introduction



Accessory elements SEBThe 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 SEB


7 SaPIs

1. Introduction



Accessory elements SECsec 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


1. Introduction



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)


human hosts

2. Aim of the study



• 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.


BIOINFORMATIC CHALLENGE

3. Materials and MethodsBacterial Strains



350 isolatesDNA 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)


12 SEB10 SEC15 SED

3. Materials and Methods



• 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


4. Results



seb Promoter and Gene Sequences (12 strains)Five variants of the seb promoter (sevp v1–v5)


4. Results




V1 Multiple positions: Four variants of the seb gene (seb v1–v4) Firstly described

4. Results





4. Results





4. Results





4. Results



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)


4. Results




4. Results



Three variants of the sed promoter (sedp v1–v3)


Sed Promoter and Gene Sequences (12 strains)

4. Results




Sed Promoter and Gene Sequences (12 strains)

Four variants of the sed gene (sed v1–v4)

4. Results




Four variants of the sed Amino Avid (sed v1–v4)

Rabbit carcasses

4. Results




Four variants of the sed Amino Acid (sed v1–v4)

Rabbit carcassesSTOP CODON

4. Results




Four variants of the sed Amino Acid (sed v1–v4)

Rabbit carcassesSTOP CODONPOSITIVE RESULTS – ENTEROTOXIN DETECTED

SET-RPLA

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


5. Discussion



6. Conclusion1. 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?




• 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, PhDPost-Doctoral Researcher

TeagascAPC - Microbiome Institute

[email protected]@guerrinomacori

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New sequence variants of Staphylococcal Enterotoxins detected: host specificity and characterization study