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New sequence variants of Staphylococcal
Enterotoxins detected:
host specificity and characterization study
20th- 21st October 2016
Torino - Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d’Aosta
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
IX Workshop of the National Reference Laboratory for Coagulase Positive Staphylococci, including S.aureus
Guerrino Macori, PhD
Post-Doctoral Researcher
Teagasc
APC - Microbiome Institute
guerrino.macori@teagasc.ie
@guerrinomacori
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
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 aureusIZSPLV - Turin
Background
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
Background
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
Background
EFSA Journal 2015;13(12):4329
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
1. Introduction
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
Five major SEs
SEA, SEB, SEC, SED, SEE
New Sequence Variants in S.aureus
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
1. Introduction
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
Strain-specific variation of SE mRNA and protein levels
environmental stress encountered in the food matrix
New Sequence Variants in S.aureus
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
1. Introduction
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
1. Introduction
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
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
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
1. Introduction
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
Accessory elements and variation in SEB, SEC, and SED intro
•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
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
1. Introduction experimental design
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
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
New Sequence Variants in S.aureus
7 SaPIs
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
1. Introduction experimental design
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
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
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
1. Introduction experimental design
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
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.
New Sequence Variants in S.aureus
human hosts
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
2. Aim of the study
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
3. Materials and Methods
Bacterial Strains
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
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
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
3. Materials and Methods
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
• 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
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
4. Results
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
seb Promoter and Gene Sequences (12 strains)
Five variants of the seb promoter (sevp v1–v5)
New Sequence Variants in S.aureus
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
4. Results
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
Multiple positions: Four variants of the seb gene (seb v1–v4) Firstly described
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
4. Results
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
Multiple positions: Four variants of the seb gene (seb v1–v4) Firstly described
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
4. Results
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
Multiple positions: Four variants of the seb gene (seb v1–v4) Firstly described
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
4. Results
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
Multiple positions: Four variants of the seb gene (seb v1–v4) Firstly described
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
4. Results
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
sec Promoter and Gene Sequences (10 strains)
New Sequence Variants in S.aureus
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
4. Results
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
4. Results
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
Sed Promoter and Gene Sequences (12 strains)
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
4. Results
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
Sed Promoter and Gene Sequences (12 strains)
Four variants of the sed gene (sed v1–v4)
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
4. Results
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
Four variants of the sed Amino Acid (sed v1–v4)
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
4. Results
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
Four variants of the sed Amino Acid (sed v1–v4)
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
4. Results
NRL for Coagulase Positive Staphylococci including Staphylococcus aureus
New Sequence Variants in S.aureus
Four variants of the sed Amino Acid (sed v1–v4)
SET-RPLA
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
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
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
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
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
?
#FoodSeqInvestigating microbial communities in artisanal fermented foods to decrypt complex interactions and harness novel strains
This research project focuses on characterizing and optimizing the microbiology relating to a number of fermented
food products. In fact, food fermentation plays a major role in combating food spoilage and foodborne diseases,
while also improving the availability of some trace minerals, vitamins and anti-oxidants, and conferring enhanced
organoleptic, nutritional, and health promoting attributes.
harnessing of specific microbes as starter and adjunct strains, and accompanying novel flavour, metabolic and
health-promoting functionalities.
Methods:
Bioinformatics, Next Generation Sequencing, Next Generation Sequencing Data Analysis, Microbiological Analysis,
16s RNA sequencing, fermented foods
Thanks
IX Workshop IT-NRL for Coagulase Positive Staphylococci, including S.aureus
Guerrino Macori, PhD
Post-Doctoral Researcher
Teagasc APC - Microbiome Institute
guerrino.macori@teagasc.ie@guerrinomacori
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