Ready-to-eat Vegetables as new sources of contamination-infection of Pseudomonas aeruginosa - Alessandra Scano
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Alessandra Scano presenta alcuni aspetti del progetto RealTime Check IV Gamma, il progetto finanziato da Sardegna Ricerche con i fondi del POR FESR Sardegna 2014-2020 e gestito dall'Istituto di Scienze delle Produzioni Alimentari (ISPA) del CNR, in collaborazione con il Dipartimento di Scienze chirurgiche dell'Università degli Studi di Cagliari. Il progetto ha l’obiettivo di fornire alle imprese sarde che operano nel settore della IV Gamma due dispositivi per il monitoraggio rapido della qualità e dell'idoneità igienico-sanitaria di prodotti ortofrutticoli minimamente trasformati.
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Ready-to-eat Vegetables as new sources of contamination-infection of Pseudomonas aeruginosa - Alessandra Scano
- 1. Ready-to-eat vegetables as new sources of contamination-infection of Pseudomonas aeruginosa PhD student Alessandra Scano Laboratorio di Biologia Molecolare AOU Cagliari
- 2. Pseudomonas aeruginosa a multi-task pathogen P. aeruginosa Alginate Antibioticresistance -Gram negative -Motile -Rod-shaped -Facultative aerobe Biofilm
- 3. Quorum sensing Biofilm is the condicio sine qua non for Pseudomonas pathogenicity ! AHL
- 4. Alginate ✓ algD gene codes the guanosine of phospho-D-mannose dehydrogenase, a key process in the biosynthesis of alginate. ✓ The PalgD is a promoter that regulated the expression of alginate. ✓ Mutations of the negative regulator mucA, lead to an overproduction of alginate. Resistant Sensible M NM
- 5. The mucA mucB, mucC and mucD gene control the gene expression of the alginate operon mucA 585pb PAO1 Wild type Alg+ mucA acts as a negative regulator of the production of alginate because it can bind and sequester the factor σ 22 through the N-terminal cytoplasmic domain. mucA gene
- 6. Incidence of P. aeruginosa infections in nosocomial area http://www.cdc.gov/hai/organisms/Pseudomonas.html http://www.who.int Resistant to more than one antimicrobial agent. Extensively Drug-Resistant strains Resistant to all antimicrobial agents in all antimicrobial categories MDR Antibiotic Resistance XDR PDR - Intrinsic or natural mechanism - Acquired mechanism
- 7. Workflow CLINICAL ISOLATES INFECTIONS HOT SPOT Water contaminations Well Medical device Food Nosocomial infections Dental Units Ocular devices Breeding farms ovine Fresh ready-to- eat vegetables HUMAN FIELD VETERINARY FIELD AGRICULTURAL FIELD ➢ Pathogen detections ➢ mucA/Alginate profile ➢ Evaluation of potential new antimicrobials ➢ New cultural systems: design/use of bioreactors
- 8. Ready-to-eat vegetables as new sources of contamination-infection of Pseudomonas spp. CDC - Centers for disease Control and Prevention Human/animal Ready-to-eat vegetable (RTE) Hot points for food contaminations: • Human manipolation • Uncontrolled water • Peeling and cutting
- 9. BIOREACTOR SIMULATING CONTAMINATION IN VEGETABLES The strains of reference are: mucA gene GAC65GGC mutated mucA gene wild type Lactuca sativa 30 % of H2O The temperature at 4° test RTE product
- 10. MATERIALS AND METHODS • 1 cm2 of leaf has been cut with a precise aluminum ring. • DNA extraction by CTAB • PCR has been considered on a region of the 16S rRNA gene • The primers for the PCR (OG347 and OG348) were designed to a flanking of 177 bp (Genbank accession NC_AF104671) • Fisher’s test (All P values < 0.05 were considered significant)
- 11. Results The difference between muc+ and muc- strains in biofilm amount was assessed approximately to 1 log (P < 0.05). ✓ The risk of water contamination with muc+ P. aeruginosa strains ✓ The importance of sequencing method to evaluate the mucA gene mutations responsible for alginate production in this bacteria genus. ✓ Alginate overproduction greatly affects the physical properties of P. aeruginosa biofilms as well as the physiological properties (cell death and growth) of the bacterial cells inside the biofilms 0,E+00 2,E+08 4,E+08 6,E+08 8,E+08 1,E+09 1,E+09 1,E+09 1 Pagenomes/cm2 muc- muc+
- 12. Development of a molecular method to detect Pseudomonas spp. DUAL - FRET PROBE Bacillus Escherichia Staphylococcus Streptococcus P. aeruginosa P. putida P. fluorescens VS
- 13. The primers and the bi-functional probe were developed using the Oligo program version 4.0 .The universal primers for the 16S rRNA gene were designed using the 16S rRNA sequence (GenBank accession NC_X73965), in a fragment common to all Schizomycetes. Dual fret probes
- 14. . 35 cycles The thermal cycles were set as follow: - initial denaturation for 30 seconds at 95° - denaturation at 95° C for 0 seconds - alignment 53° C for 10 seconds - extension at 72° C for 12 seconds DNA extraction - 710 nm (infrared) emission is related to the total bacteria - 650 nm (red) indicates the presence of the P. spp. PCR CONDITIONS The universal primers for the 16S rRNA gene were designed using the 16S rRNA sequence (GenBank accession NC_X73965), in a fragment common to all Schizomycetes.
- 15. RESULTS Bacteria strain Real Time PCR results P. fluorescens P. putida P. aeruginosa A. actinomycetemcomitans E. coli S. mitis S. oralis S. gordonii S. mutans B. cereus B. subtilis S. aureus M. micros 650 nm + + + - - - - - - - - - - 710 nm + + + + + + + + + + + + + F2 F3 F2 650 nm Fluorescence signal obtained with different bacteria species. F3 710 nm Bacteria strain RealTime PCR results By implementing serial dilutions in P. aeruginosa and E. coli, quantification standards have been set up. Both lines show an acceptable correlation coefficient R2 (from 0.95 to 0.98). The sensitivity limit was around 50 CFU / PCR for Pseudomonas spp. and 150 CFU / PCR for E. coli . Similar results were obtained using as representative of the total P. aeruginosa, S. aureus and B. subtilis bacteria, where the sensitivity range oscillated between 300 and 50 CFU / PCR.
- 16. CONCLUSIONS ✓ The ubiquity of these microorganisms imposes a precise evaluation of the mass of the bacterium in the sample, namely the verification if the pathogen has reached excessive titers, close to the infective dose. The difficulty is control the sample temperature during transport, this to avoid artifacts due to unwanted bacterial growth. ✓ This methodology can represent in the laboratory diagnosis a helpful tool for clinician as well as to the laboratory technician, presenting some peculiarities with respect to the current microbiological diagnostics: fast procedure (average 30 minutes for 30 samples) and consequently rapidity in prophylaxis responses Salmonella spp. ✓ Other pathogens ? Listeria spp. E. coli