Canada Research Chair in Food Microbiology and Probiotics

Department of Agricultural, Food and Nutritional ScienceEdmonton, Alberta, Canadawww.ualberta.ca

Tales of an amateur thermophile:

Resistance of Escherichia coli to pathogen intervention

treatments in beef processing M. Gänzle, E. Dlusskaya, D. Pierce, L. Ruan, and L. McMullen

Enterohaemorraghic Escherichia coli(EHEC) on beef

Habitat: Colon of ruminants (up to 107 cfu / g)

Frequency: 4 – 7 cases per year and 100,000 population (USA and Canada) – estimated cost of > $60 million annually in Canada

Regulation: U.S.A., „zero tolerance“ for E. coli O157:H7 Canada, microbiological criteria for E. coli.

Virotype of E. coli (Genes for adhesion to intestinal mucosa and shiga-toxin)

Frequent serotypes of EHEC

E. coli O157:H7 (O26:H11, O111:H8, O157:NM)

Disease:

Bloody diarrhea, hemolytic-uraemic syndrome

Infectious dose: 10 cells

George Morris Centre. 2007. Cost of E. coli O157:H7 illness in Canada

Elimination of EHEC on beef: a formidable challenge!

EHEC

>1010 cells

none

E. coli

>1012 cells

< 103 cells / g

No pasteurisation or other physical treatment,no preservatives or other additives

Hide washing

Removal of hide

Evisceration

Lactic acid spray, neck only

Trimming, vacuuming

Steam pasteurization

Lactic acid spray

Cattle receiving and holding

Stunning

Chilling

Pathogen intervention in beef processing

Photos courtesy of Dell Allen, Cargill Meats

Current intervention reduce E. coli counts by less than 99% (2 log)

=> Determine effects of meat matrix on resistance of E. coli to interventions (Dana Pierce)

=> Determine resistance of > 50 strains of E. coli to intervention treatments (Lena Dlusskaya)

=> Use of functional genomics to elucidate determinants of heat resistance (Lifang Ruan)

EHEC on beef: an unsolved problem in food processing

Lab scale intervention treatments

Surface treatment with steam (8’’ or 15’’) and lactic

acid (4.5%)

Meat cylinders (lean or adipose tissue) with 4.9 cm2 surface area

Inoculation with ~ 107 E. coli

(single strain or strain cocktail)

surface temperature:>80°C after 2’’>90°C after 8’’pH: 4.3 (lean)

3.1 (fat)

Bactericidal effect of intervention on lean and adipose tissue

meat-fat

+/- lactic acid

=> Treatment effects in keeping with bactericidal effect on carcass=> Transformation with fluorescent marker does not influence sensitivity

AW 1.7 DM18.3 DM18.3 ATCC 25922 GFP

Distribution of cells on lean and adipose tissue

E. coli pRFP on fat E. coli pRFP on meat

Use of Green Fluorescent Protein and Red Fluorescent Protein as fluorescent probes

eGFP: derivative of GFP (denaturation at 75 - 80°C)RFP: red fluorescent protein, denaturation at 85 - 90°C

Z-Scan to determine survival beneath meat surface

0 µm30 µm90 µm240 µm475 µm

E. coli DM18.3 with RFP expression plasmidFluorescence microscopy of untreated lean tissueFocus 0 – 500 µm below surface, 1000 x magnification

● untreated cells, lean tissue (n= 551 cells) ○ untreated cells on fat tissue (n= 611 cells)▲ steam-treated cells on lean tissue (n= 89) steam-treated cells on fat tissue (n= 236)

Distribution of cells of E. coli DM18.3 RFP before and after steam treatment meat and fat

Bactericidal effect of intervention treatments- interim summary -

Penetration of E. coli

Penetration of steam and lactic acid treatments

Thermal insulating properties of fat mitigate effect of steam treatment

Buffering capacity of meat mitigates effect of lactic acid treatment

Are all E. coli the same? – strain variation in resistance

55 strains of E. coli isolated from beef processing plant„historic“ isolate – isolated prior to industrial use of steam and

lactic interventionsreference isolates from strain collection and live animals

Use of strain cocktails instead of single strains

# 1 # 2 # 5 # 6

Resistance of strain cocktails to steam and lactic acid intervention treatments

Treatment time (min)

0 6 12 18 24 30

Cel

l cou

nts

(log

CF

U/m

l)

3

4

5

6

7

8

9

0 6 12 18 24 30

3

4

5

6

7

8

9

70°C65°C60°C

Resistance of E. coli strains to heat

DM18.3 AW1.7

FUA 1041

GGG10FUA 1044K-12

Survival at 60°C Survival at 60, 65 and 70°C

D60 (min) Strain # (serotype) Reference:

0.11 - 0.32 K-12 this study, J. Food Prot. 70:851

0.4 - 1.5 LTH 5807 (O157:H-; Stx- ) Eur. Food Res. Technol.

221:187 0.47 204P (O157:H7) J. Food Sci. 60:606

1.9 EDL-931, A 9218-C1, 45753-35,

933 (all O157:H7) Food Res. Int. 32:23

3.2 204P, 505B, 933, 932, F 501, F

585 J. Food Sci. 69:FMS97

4.2 ATCC 25922 Chem. Papers. 61:121 6.5 LMM1030 Appl. Envir. Microbiol. 63:945 6.7 380-94 Appl. Environ. Microbiol. 66:1726

14.6 DM 18.3 this study

71.4 AW 1.7 this study

Resistance of E. coli strains to heat:Comparison of D60 values with literature data

Resistance of E. coli strains to lactic acid

AW1.7

GGG10

Survival of E. coli in LB with 4% lactic acid (pH 2.3)

Survival of E. coli in hamburger patties grilled to internal temperature of 63 or 71°C

untreated 63°C 71°C

cell

cout

ns [l

og (

cfu/

g) ]

2

4

6

8

10

< 1.3 < 1.3

GGG10, AW 1.7

Heat resistance of cattle and beef isolates of E. coli - interim summary -

>1012 cells – a vast majority are heat sensitive

< 103 cells / g – some (most?) are heat resistant

Steam and lactic interventions

E. coli isolates from beef processing plants exhibit exceptional heat resistance!

Gene name

Fold expression (AW1.7/GGG10)

Gene function

Genes overexpressed in E. coli AW 1.7

nmpC 21 outer membrane porin protein C precursor; locus of qsr prophage

trs5-5 11 IS5 transposase and trans-activator trs5_9 8.4 IS5 transposase

potH 4.4 putrescine ABC transporter

permease protein PotH

putative 3.8 putative UDP-galactose 4-epimerase

yicM 3.6 predicted transporter

hisM 3.5 amino acid transporter subunit

Determination of gene expression in E. coli AW1.7 and E. coli GGG10

- Use of genome-wide microarray covering three E. coli genomes- Comparison of E. coli AW1.7 and E. coli GGG10 grown at 37°C, and after

heat shock at 50°C

Red, bold genes are related to transport functions

Validation of microarray data by quantitative reverse-transcription PCR

Validation of microarray data by Proteomics

40kD

AW1.7 GGG10 Ladder

OmpC

NmpC and OmpC

Separation of membrane proteins by SDS-PAGE and protein identification by LC/MS/MS

time at 60°C (min)

0 4 8 12 16

log

(cf

u m

L-1

)

2

4

6

8

10

AW 1.7 yfpAW 1.7 yafQ-dinJ GGG10 yfpGGG10 nmpC

Validation of microarray data by expression of NmpC from E. coli AW1.7 in E. coli GGG10

Expression of nmpC in E. coli GGG10 – YFP served as controlExpression of yafQ-dinJ in E. coli AW1.7 – YFP served as control

Determination of heat resistance at 60°C

Genetic determinants of heat resistance – interim summary

- Many genes overexpressed in heat resistant E. coli related to the transport of amino acids or related compounds

- Expression of nmpC increases heat resistance in sensitive strains

- Heat resistance is maintained at the expense of acid resistance

• Prevalence of heat resistant strains• design interventions that are more effective

– Non-thermal technologies– Biocontrol with nonpathogenic organisms

Where to go from here?

Further work needed to findthe Achilles’ heel of heat resistant E. coli!

Acknowledgements

Collaborators• Aaron Pleitner and Mandi Socholotuik

Financial support• ALIDF

…..for your attention

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

TMW 2.497 DM 18.3 RFP EC Top 10 RFP

Strains

Tre

atm

en

t Eff

ect

[Log

(N

0/N

1)

CFU

/cm

2]

Meat - Steam

Meat Steam & Lactic

Fat - Steam

Fat - Steam & LacticX X

*

*

Reduction in the numbers of transgenic Escherichia coli inoculated on the surface of lean or fat tissue by treatments with steam (8 s) or treatments with steam, followed by application of 4.5% v/v lactic acid spray. Strains EC Top 10 and DM 18.3 were transformed with a plasmid coding for the expression of RFP. Strain TMW 2.497 caries a plasmid coding for expression of rsGFP. An asterix (*) indicates a significant difference (p < 0.1) between treatments with steam and treatments with steam and lactic acid for the same strain and tissue; an X indicates a significant difference (p < 0.03) between steam treatments on lean and fat tissue for the same strain.

Photograph of E. coli DM 18.3 eGFP inoculated onto lean tissue and counterstained with Syto 17 before (a) and after steam and lactic acid

treatment (b). Photographed using the double exposure program with the Rhodamine and GFP filter sets.

(a) GFP filter. Exposure time = 171 ms. 1000x magnification.(b) GFP filter. Exposure time = 1027 ms. 1000x magnification.

Treatment: 8'' steam and 2% lactic

acid

Treatment: 15'' steam and 4.5%

lactic acid

Cocktail # and strains in the

cocktail LB agar Endo agar

Cocktail # and strains in the

cocktail LB agar Endo agar

#1 MB 3.4 2 2 #1 MB 3.4 3 1 AW 1.3 2 3 AW 1.3 3 5 GM 11.9 4 5 GM 11.9 2 1 GM 16.7.2 2 1 GM 16.7.2 1 2 GM 18.4 3 1 GM 18.4 4 4

#2 MB 2.1 2 1 #2 MB 2.1 1 2 AW 1.7 1 4 AW 1.7 1 5 GM 11.5 1 3 GM 11.5 1 1 GM 18.6 4 2 GM 18.6 6 4 GM 18.3 5 2 GM 18.3 4 1

#3 GM 9.5 3 1 #5b GM 11.3 5 4

ATCC 25922

0 0

GM 12.6 1 2 GGG10 2 3 GM 16.2 2 1 FUA 1050 6 5 GM 16.6 2 5 FUA 1051 3 3

#4 MB 1.3 1 4 #6 FUA 1040 3 1 MB 3.3 2 2 FUA 1041 3 4 MB 10.1 3 4 FUA 1044 1 1 GM 9.8 3 2 FUA 1045 2 4

AW 12.2 4 1 FUA 1048 4 3

Genes overexpressed in E. coli GGG10

putative 0.34 head-tail preconnector gp5 phage portal protein lambda family

putative 0.31 putative ATP-dependent DNA helicase (together with adjacent 3 orfs)

putative 0.30 putative minor tail protein

sgcA 0.27 putative PTS system enzyme II A component

putative 0.22 partial putative tail component of prophage CP-933R

putative 0.20 putative antitermination protein Q for prophage CP-933V

ynjC 0.20 putative transport system permease protein

dinJ 0.18 YafQ-DinJ toxin-antitoxin system, damage-inducible protein J

ymcD 0.15 formation of the O-antigen

hokD 0.15 small toxin polypeptide destructive to membrane potential

putative 0.12 Putative Transposase within prophage

fimA 0.088 major type 1 subunit fimbrin (pilin)

Escherichia coli DM 18.3 eGFP on lean tissue, counterstained with propidium iodide.

Use of Green Fluorescent Protein and Red Fluorescent Protein as fluorescent probes

E. coli

nuclei of meat cells

Cost of E. coli O157:H7 infections in Canada

• Cost to health care: $29.6 million• Market demand loss: $30 million• Recalls: ???

• Total estimate: $60 million + ???

~ 25% of EHEC infections attributed to meat consumption

George Morris Centre. 2007. Cost of E. coli O157:H7 illness in Canada

Genes overexpressed in E. coli GGG10 compared to AW1.7 at 50ºC

Gene Ratio (GGG10/AW1.7)

Gene function

dnaK 0.29 chaperone Hsp70, co -chaperone with DnaJ

ydfI 0.29 predicted mannonate dehydrogenase

Nfi 0.27 endonuclease V

rpsK 0.25 30S ribosomal subunit protein S11

yfiD 0.20 pyruvate formate lyase subunit

rpsC 0.1 9 30S ribosomal subunit protein S3

fimA 0.18 major type 1 subunit fimbrin

hokD 0.18 small toxin polypeptide

cpxP 0.16 periplasmic protein combats stress

yccV 0.16 DNA -binding protein, hemimethylated

rplD 0.15 50S ribosomal subunit protein L4

yfiA 0.1 4 cold shock protein associated with 30S ribosomal subunit

rpmC 0.13 50S ribosomal subunit protein L29

rpsG 0.13 30S ribosomal subunit protein S7

Plasmid Strains Transformed Expression

pGFP EC Top 10, GM 18.6 Yes/No

pRFP DM 18.3, EC Top 10, K12 Yes/Yes/Yes

pEGFP DM 18.3, EC Top 10, K12 Yes/Yes/Yes

pYFP DM 18.3, EC Top 10, K12, AW 1.7 Yes/Yes/Yes/Yes

Use of Green Fluorescent Protein and Red Fluorescent Protein as fluorescent probes

GFP, YFP, eGFP: derivatives of GFP (denaturation at 75 - 80°C).RFP: red fluorescent protein, denaturation at 85 - 90°C

Transformation did not influence survival after steam / lactic acid treatments

E. coli DM 18.3 on lean meat stained with fluorescein and propidium iodide

Visualisation of treatment effects by fluorescence microscopy

before after treatment