ProNNaC Whole Genome Sequencing. Staphylococcus aureus

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1 ProNNaC Whole Genome Sequencing and Staphylococcus aureus NRL for Coagulase Positive Staphylococci including S.aureus Angelo Romano National Reference Laboratory for Coagulase Positive Staphylococci, including S.aureus S.C. Controllo Alimenti e Igiene delle Produzioni Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d Aosta

2 Project ProNaCC: Production of Natural Contaminated Cheeses GMI PT 2015 proficiency test I C o N S : Integrative and Collaborative Staphylococcus aureus Database, from classic biotyping to NGS and some backward analysis

3 Introduction Project ProNaCC Production of «naturally» contaminated cheeses (laboratory dairy plant) Initial purposes: CPS and SE homogeneity and stability in complex matrix growth/viability of CPS distribution of CPS/SE expression of se genes during the process 1st step (Anses): choice of CPS strains test in BHI / milk / cheese 2nd step (NRL IT / Anses): productions of cheeses and analysis

4 Material and Methods Project ProNaCC Choice of CPS strains Objectives: Verify the growth of CPS strains and SE production in 3 different culture media. Methods: Selection of CPS strains producing different types of SE. 3 experiments: in broth, milk and cheese Image F.Auvray EU-RL CPS

5 Material and Methods Objectives: Project ProNaCC 2nd step : productions of cheeses - mimic the natural CPS contamination of cheese - verify the CPS growth and SE production during the manufacturing of cheese. Methods: production of cheese in the dairy plant of IZSTO 4 selected CPS strains sent from Anses + 2 CPS strains carrying SE genes (seg/ sei and seh respectively) + DESC project strain (sea, sed, sej, ser) + mix of different strains

6 Results Project ProNaCC Strain Name 2nd step : productions of cheeses Milk after incubation with strain(s) (18 Fresh Cheeses after sweating (3 h) h) SEs qualit CPS Quantification SEs qualit CPS Quantification SE TV UFC/g SE (VIDAS) TV UFC/g (VIDAS) Batch 1 S.aureus sed pos 2,76 2,1*10^7 pos 2,64 2,8*10^8 Batch 2 S.aureus see pos 2,47 5,3*10^7 pos 1,91 2,5*10^8 Batch 3 S.aureus sec pos 1,38 3,0*10^8 / / / Batch 4 S.aureus sea pos 2,54 1,1*10^8 pos 2,58 2,9*10^8 Batch 5 S.aureus seg/sei neg 0 9,2*10^7 neg 0,01 5,6*10^8 Batch 6 S.aureus seh neg 0 9,2*10^7 neg 0,01 2,5*10^8 Batch 7 S.aureus sea, sed,sej, ser neg 0 1,0*10^8 neg 0,04 2,7*10^8 Batch 8 09CEB253 / 09CEB314 / 11CEB73 / 12CEB304 / 14SBCL84STA / 14SBCL881STA / DESC STRAIN pos 2,94 7,8*10^7 pos 2,57 5,0*10^8 6

7 Results Project ProNaCC 8 cheeses produced 2nd step : productions of cheeses Problem with batch 3 (sec 11CEB73): the milk was already curdled after 24 h of incubation. addition of rennet and starter, incubation and place in the forms but the curd doesn't formed. The cheesemaking was repeated the second day but the same result was obtained. Problem encountered with batch 7 (DESC strain from IZSTO): SE result was negative with Vidas SET2 => new production Mix of strains: reduction of the number of strains => new production 7

8 Conclusions Project ProNaCC For all batches Very high level of: - CPS growth in cheese (> 10 6 cfu/g) - Production of SE (2 ng/g for the lowest) - No difference between core and periphery Batch 7 (DESC) Results close to those obtained in DESC project: - Production of SEA and SED simultaneously - Concentration levels of [SEA] < [SED] Batch 8 (mix) Production of SEA, SED and SEE simultaneously ([SEA] < [SEE] < [SED]) growth of the 3 strains 8

9 Conclusions Project ProNaCC What s next? To continue - Choice of one or two strain(s) to produce a new batch less contaminated - Reduce the level of CPS / SE: control of growth / SE production: temperature? No pre-culture in milk? Lower [CPS] contamination of milk? - Larger production to study homogeneity and stability - Transcriptome response of CPS in different conditions: Product composition Storage conditions Competition between strains 9

10 Bioinformatic study Project ProNaCC Application of WGS typing to S.aureus with free online resources Center for Genomic Epidemiology (DTU, Denmark) Galaxy Public Server (ARIES - ORIONE) - ResFinder spa Typer MLST PathogenFinder PROKKA - CSI Phylogeny 1.4

11 Bioinformatic study Project ProNaCC ResFinder 2.1 ProNaCC 01 ProNaCC 02 ProNaCC 04

12 Bioinformatic study Project ProNaCC MLST 1.8 (MultiLocus Sequence Typing) ProNaCC 01 spatyper 1.0 ProNaCC 02 ProNaCC 04 ProNaCC 01 ProNaCC 02 ProNaCC 04

13 Bioinformatic study Project ProNaCC PathogenFinder 1.1

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15 IZSTO Whole Genome Sequencing MLST spa-type Phylogenetic analysis SNPs Outbreak Resolution Antibiotic Resistance Virulence Genes Risk analysis Treatment Intervention Prediction of pathogenicity

16 GMI PT proficiency test Global Microbial Identifier aim of applying novel genomic technologies and informatics tools to improve global patient diagnostics surveillance research by data exchange and analysis tools for characterization of all microbial organisms and microbial communities.

17 GMI PT 2015 proficiency test Focused on: Salmonella enterica Escherichia coli Staphylococcus aureus Item 1a) Bacterial cultures. DNA extraction, library-preparation and WGS: upload reads identify MLST/resistance genes Item 1b) WGS of pre-prepared DNA Item 2) Variant detection and phylogenetic analysis of three datasets: 20 genomes of S. enterica, E. coli and S. aureus

18 GMI PT 2015 proficiency test Item 2 Diversity of bioinformatics tools Three topologies clustered samples quite similarly (>93%) labs would reach similar conclusions when the methods are applied to trace back and source raking investigations Need of standardized approach

19 ICoNS Project: Integrative and Collaborative Staphylococcus aureus Database from classic biotyping to NGS - Increase the analysis capability - Development and evaluation of new methods for specific target detection WGS data To reach standardized approach Integrative and Collaborative S. aureus Database

20 and some backward analysis Research Project to investigate MRSA - Raw milk dispenser - Farms dairy cows screening of bulk milk

21 and some backward analysis ResFinder 2.1

22 and some backward analysis MLST 1.8 spatyper 1.0

23 and some backward analysis CSI Phylogeny 1.4

24 Thanks for the attention Angelo Romano National Reference Laboratory for Coagulase Positive Staphylococci, including S.aureus S.C. Controllo Alimenti e Igiene delle Produzioni Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d Aosta