E. coli - Dipartimento di Scienze Mediche Veterinarie

 DOTTORATO DI RICERCA IN SCIENZE VETERINARIE XXVI CICLO BORSA FONDO GIOVANI Valorizzazione dei prodo= >pici dell’agroalimentare e sicurezza alimentare aFraverso nuovi sistemi di caraFerizzazione e garanzia di qualità DoForando: Gian Marco Baranzoni Tutor: DoF.ssa Sabrina Albone= Milk clean up protocol: Quan&fica&on of Listeria monocytogenes using molecular techniques: Evalua&on of shelf-­‐life food products: v  Survival of Listeria monocytogenes in ready-­‐to-­‐
eat frozen meat broth -­‐ Paper under submission. v  Development and valida>on of an alterna>ve quan>fica>on method of Listeria monocytogenes combining MPN technique with real-­‐>me PCR -­‐ Work in progress. v  Evalua>on of Listeria monocytogenes growth in raw milk at refrigerator storage temperatures -­‐ Paper under revision. 10 ml
Sample
Contamina>on EDTA
5 min. at 40 °C
v  Development of methods for DNA extrac>on, detec>on, and quan>fica>on of Listeria monocytogenes in milk using quan>ta>ve polymerase chain reac>on (qPCR). At USDA ARS ERRC Philadelphia, USA -­‐ Work in progress. v  Partecipa>on at a mul>disciplinary project to evaluate rabbit meat shelf life (microbiological analysis) -­‐ Work in progress. Lysis buffer
SDS, NaCl, MgCl2
and DNase
Trypsin
Milk
Skim
206
0
100
193
0
100
273
0
100
412
4
99
176
8
96
200
1
100
18
119
13
17
213
7
32
159
17
Raw
These graphics show the fluorescence per cycle of real-­‐>me amplifica>ons using power SYBR green Master mix, Applied Biosystem (above) and TaqMan Environmental Master Mix 2.0, Applied Biostystem (on the side). Curves of standard made from pure culture of L. monocytogenes extracted with DNAeasy, QIAgen are in red and the blue ones correspond to DNA e x t r a c t e d f r o m c l e a n e d m i l k s u p p l e m e n t e d b y D N A o f L . monocytogenes standard. Results: v  Detec>on and isola>on techniques for Shiga toxin-­‐
producing Escherichia coli (STEC) O104 from sprouts. At USDA ARS ERRC Philadelphia, USA -­‐ Paper under composi;on. Experimental design: Conclusions: multiplex
PCR assay
real&me IMS and Latex real&me
PCR on colonies Agglu&na&on PCR on enrichment! recovery!
Test colonies!
2/2"
2/2"
2/2"
2.4"
1/1"
1/1"
1/1"
1/1"
Legend: 2/2"
2/2"
2/2"
2/2"
• 
stx1-­‐2 3.6"
1/1"
1/1"
1/1"
1/1"
• 
aggR 3.2"
1/1"
1/1"
1/1"
1/1"
• 
ehzA • 
2/2"
2/2"
2/2"
2/2"
wzx104 • 
IPC 2/2"
2/2"
2/2"
2/2"
1/1"
1/1"
1/1"
1/1"
4.4"
STEC O104"
6.4"
mRBA CHROMagar O104 STEC O104:H-­‐ Samples with low level of contamina>on Ar&ficial contamina&on cfu/g!
E. coli strain!
real&me IMS and Latex real&me
PCR on colonies Agglu&na&on PCR on enrichment! recovery!
Test colonies!
0.16"
3/3"
3/3"
3/3"
3/3"
0.24"
2/3"
2/3"
2/2"
2/2"
4/4"
4/4"
4/4"
4/4"
0/2"
0/2"
0/0"
0/0"
0.56"
4/5"
4/5"
4/4"
4/4"
0.76"
3/3"
3/3"
3/3"
3/3"
0.24"
2/3"
2/3"
2/2"
2/2"
0.36"
2/3"
2/3"
2/2"
2/2"
0.52"
2/2"
2/2"
2/2"
2/2"
1/4"
1/4"
1/1"
1/1"
1.72"
3/3"
3/3"
3/3"
3/3"
0.96"
5/5"
5/5"
5/5"
5/5"
0.36"
0.52"
0.88"
O104:H4 "
STEC O104"
In the two tables above the results of all the steps of the protocol for detec>on and isola>on of E. coli O104 in sprouts are listed. Samples are grouped by the amount of ar>ficial contamina>on. Every >me the mul>plex real>me PCR was posi>ve the colonies of E. coli O104 were isolated and resulted posi>ve for both latex agglu>na>on and the second real>me PCR. Unfortunately not all the samples inoculated with 101 bacteria were posi>ve, but 4 and 5 samples, respec>vely for E.coli O104:H4 and STEC O104, on 20 resulted nega>ve. Conclusions: An>bodies an>-­‐O104 work really well both in the IMS and in the latex agglu>na>on kit. As a maFer of fact, plates were full of target colonies (mauve/purple) regarding the high level of coliforms present in sprouts ad excep>on of STEC O104:H-­‐ in CHROMagar STEC. Moreover, the presence of the gene wxz104 which codifies for the an>gen O104 was always detected in every colony posi>ve to latex agglu>na>on. The technique resulted to be very strong and able to detect less than 1 cfu/g and nega>ve samples can not be considered as false-­‐nega>ve because the level of inocula>on was very low and the contaminated sprouts were stressed before the analysis. The PCR assay with aggR can be used as a screening method for E. coli O104:H4 responsible of the German outbreak. Latex
agglutination
(anti-O104)
mBPWp + ACV
37→42 °C for 18h
2/2"
3.8"
CHROMagar and
modified Rainbow agar
Enrichment
Aker primers concentra>on and mel>ng temperature op>miza>ons, milk clean up, DNA extrac>on and real>me PCR protocols work on pasteurized milk samples. However, further experiments are needed to reduce the high variability among the samples. Then, it will be possible to start new trials with raw milk. 1.8 O104:H4"
Novel antibody
provided by
Samples with high level of contamina>on 2.7"
IMS +
Isolation
4 °C for 48 h
E. coli O104:H4 E. coli strain!
Artificial
contamination
The table shows the number of cfu collected by plate count in oxford plates from ar>ficially contaminated milk. From these results it has been decided to set up the protocol first in pasteurized milk and then switch to the raw one. The graph above display a quan>ta>ve real>me PCR of DNA extracted from cleaned milk ar>ficially contaminated with 100, 101 and 102 cfu/ml of L. monocytogenes. Ar&ficial contamina&on cfu/g!
HotShot
+
Neutralizer
Shiga toxin-­‐producing Escherichia coli (STEC) O104 Recovery
Supernate
in pellet
(cfu)
(%)
Pellet
(cfu)
Whole
DNA extrac>on NaCl, EDTA
+
NaCl, Tween
Results: mRBA CHROMagar STEC Aker having carefully selected five couple of primers and probes, two mul>plex real>me PCR were op>mized detec>ng the genes stx1-­‐2, ehzA and wzx104 (graphics above) and stx1-­‐2, aggR and wzx104 (right side graphic) respec>vely. TaqMan®Exogenous Internal Posi>ve Control Reagents, VIC Probe (Life Technologies, USA) was used in both assays. Mul>plex real>me PCRs were performed on DNA extracted aker the enrichment through PrepSEQ Rapid Spin Sample Prepara>on kit (Applied Biosystem, USA) in order to screen all the samples and on the same colony tested for latex agglu>na>on as a double check. The two pictures on the lek show the isola>on of E. coli O104:H4 (responsible of the German outbreak in 2011) and STEC O104:H-­‐ on CHROMagar and modified Rainbow agar (mRBA) aker Immuno-­‐Magne>c Separa>on. Both strains are able to grow on mRBA, but CHROMagar STEC is too selec>ve for STEC O104H-­‐. v  Isola>on of genomic and plasmid DNA from Escherichia coli, making DNA libraries and sequencing of the E. coli genomes and plasmids using the Ion Torrent Personal Genome Machine next genera>on sequencer. At USDA ARS ERRC Philadelphia, USA -­‐ work in progress. 12,216 bp E. coli O104:H-­‐ O104:H21 Before 2011, the serogroup O104 was considered a minor issue. It was associated with a small outbreak of 11 cases in USA (E. coli O104:H21) and 16 sporadic human cases in Germany, United Kingdom, Korea, France, Finland, Norway, Denmark, Belgium, Sweden and Austria. Among these, 4 presented Hemoly>c Uremic Syndrome (ECDC and EFSA, 2011). The concern for this serogrup increased in May 2011 when a big outbreak of Escherichia coli (E. coli) O104:H4 located mainly in north Germany occurred. Thanks to Ion Torrent Personal Genome Machine the genomic and plasmid DNA of E. coli O104:H-­‐, a Shiga toxin producer isolated from caFle, and E. coli O104:H21 were sequenced. At the moment a comparison between the obtained sequences, E. coli O104:H4 and other STEC genomes is in progress. On the right are shown a gel electrophoresis of two plasmid extrac>ons, a picture of the Ion Torrent Personal Genome Machine and an output example of a plasmids sequenced with barcode.