new sequence variants of staphylococcal enterotoxins ... · new sequence variants of staphylococcal...

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

[email protected]

@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




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 levels

environmental stress encountered in the food matrix



1. Introduction




1. Introduction


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



1. Introduction


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


SEB and SED


1. Introduction experimental design


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


7 SaPIs




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





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.


human hosts


2. Aim of the study




3. Materials and Methods

Bacterial Strains


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)


12 SEB

10 SEC

15 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



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


4. Results


sec Promoter and Gene Sequences (10 strains)



4. Results




4. Results



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 Acid (sed v1–v4)


4. Results



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

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. 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?




?

#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


Guerrino Macori, PhD

Post-Doctoral Researcher

Teagasc APC - Microbiome Institute

[email protected]@guerrinomacori

new sequence variants of staphylococcal enterotoxins ... · new sequence variants of staphylococcal...

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