mi bmicrobe hth ost fdf ood it tii nteractions · the future …… food extract dna/rna sequence...
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Mi b h t f d i t tiMicrobe : host : food interactionsimplications for food safetyimplications for food safety.
Colin Hill
Microbiology Department
University College Cork
Ireland
Many factors determine the outcome of bacterial host interactionsbacterial host interactions
HealthAge
Microbial NumbersVirotype [EHEC]
Host Microbe
WellSick
AgeMicrobiome
Virotype [EHEC]History
Dead
Food
Food type [RTE?]P iProcessingConstituents
Listeria monocytogenesy g• Causes a rare but often fatal illnessCauses a rare but often fatal illness,
listeriosis (Increasing in EU).
• Associated with dairy products,Associated with dairy products, delicatessan meats and other ready‐to‐eat foods
• Can grow in the fridge, survive high temperatures, resist high salt and low pH, withstand high pressureslow pH, withstand high pressures
• Certain strains more likely to cause epidemic outbreaks (serotype 4b)p ( yp )
• Regulatory confusion? 100cfu/g appropriate?pp p
EC regulation 2073 / 2005
Immediate historyy
100
10
ival
1
0.1
0.01
pH 7 pH 5(30 min)
pH 3.5
% surv
0.01
0.001
30 60 90 12030 60 90 120hours
pH 7 pH 3.5
The acid tolerance response
O' Driscoll et al. 1996. Appl. Environ. Microbiol. 62: 1693‐1698.
Immediate historyYoghurt (pH3.9) Cottage cheese (pH4.7)
y
100
10
1
0.1
100
10
1
0.1% survival
% survival
0.01
0.001
12 24 36 48
0.01
0.001
4 8 12 16
Cheddar (pH5.1) Low‐fat Cheddar (pH5.2)
hours days
100
10
1
Cheddar (pH5.1)
100
10
1
Low fat Cheddar (pH5.2)
vival
vival
1
0.1
0.01
0.001
1
0.1
0.01
0.001
% surv
% surv
18 36 54 72days
18 36 54 72days
Gahan et al. 1996. Appl. Environ. Microbiol. 62:3128‐3132.
Cross adaptation between stresses
Acid
Sub‐lethal (induction)
Acid
Lethal (challenge)
Acid
Salt
Acid
Salt
Heat Heat
AMP’s AMP’s
Pressure Pressure
3% NaCl increases pressure resistance >1000‐fold>1000 fold
10
10
treatm
ent 500 Mpa for 5 minutes
6
7
10
10
10
er pressure t
4
5
Requires uptake of
10
10
10
urvivors afte
2
3carnitine or betaine from
the food
10log su
1
Δgbu, opuC, betLLO28
3% NaCl 3% NaCl 0% NaCl 0% NaCl
LO28
Smiddy et al. 2005. Applied Environ. Microbiol. 70: 7555‐7557.
The ability to adapt affects virulence
5
Log cfu/spleen on day 3
41000‐foldThe immediate history of a bacterium can
3
000 o d
significantly influence it’s survival and virulence potential2
Adapted Un‐adapted
ATM56 ATR‐1
virulence potential
Unable to adapt
Permanently adapted
Mechanism of adaptation– GAD systemp yH+
H+
H+
H+
H+glu
GadT
GABA
H+
GABAglu
H+
H+
H+ CO2GadDH+
H+
H+H+
H+
H+
H+H
A proton is consumed(inside the cell)
Glutamate is exchanged for GABA(outside the cell) (inside the cell)
Cotter et al. 2001. Mol. Microbiol. 40:465‐75.
(outside the cell)
History and constituents combine
non‐adapted
140
adapted
GAD *** 10
100
Porcine stomach juice (pH 2.5 x 20 min)
12010080
GADactivity
0.1
1
survival
604020
0.001
0.01
%
0LO28 ΔgadD
ND
wt wt +glu
Δgad+/‐glu
GAD is a major player in the
g g
Can dramatically affect acid Acid Tolerance Response tolerance ‐ and gastric transit
Supporting Evidence pp gClinical strains have high GadBC activity
d i b tt i t i id
100
and survive better in gastric acid
DPC 4609
LO28
S ttA10
% survival in gastric juice (pH2.5) after 60 min
FH 1965 DPC 4593
ScottA10
LO28ΔgadAB
EGD
DPC 4591DPC 4594
1
LO28ΔgadABND
0 20 40 60 80 100 120
% GAD activity relative to LO28
Role of GAD in survival in food
• The immediate history of a bacterium i ifi tl i fl it’ i lcan significantly influence it’s survival
and virulence potential• Food constituents can significantly
influence growth and survival (andinfluence growth and survival (and infectious dose?)
Cotter et al. 2001. Mol. Microbiol. 40:465‐75.
Strain diversityGadT
Some strains have two GAD systems
GadD
two GAD systems
The second is encoded on aGadT GadT2 encoded on a Stress Survival I l d [SSI 1]GadD GadD2
Island [SSI‐1]GadD GadD2
SSI‐1
Ryan, et al. 2010. J. Appl. Microbiol. 109:984‐995.
Strain diversity
days0 1 2 3 4 5 6 8
106
105
CFU/gNo 4b strains (epidemic strains)
104
ΔSSI‐1contain SSI‐1 [of >20 tested]
403020100104
Time (d)
Ryan, et al. 2010. J. Appl. Microbiol. 109:984‐995.
Strain diversity and virulence
Lineage II, III‘food &Lineage I ‘food &
environmental strains’
Lineage I(Serotype 4b)
‘epidemic strains’strains’
Only 51 genes unique to 4b strainsOnly 51 genes unique to 4b strainsNelson et al, 2004
Listeriolysin S
Non‐4b(100%)
4b(63%)
LIPI‐3
LlsA is a haemolysin cfu/organ
(only expressed in vivo).
Knockouts are less virulent
Liver Spleen
107
108
* **
in murine models and in human PMN’s.
106
107
10F2365 lls‐ F2365 lls‐
Cotter et al. 2008. PLoS Pathogens 4:e1000144
• The immediate history of a bacterium ycan significantly influence it’s survival and virulence potentialand virulence potential
• Food constituents can significantly influence growth and survival (and infectious dose?)infectious dose?)
• Strain diversity (genetic complement) may significantly affect survival and virulence potentialvirulence potential
‘Food’ v ‘Disease’ strains?GadT2
GadD2
‘Food’ strains ‘Disease’ strains
Lineage 1 (non‐4b), lineage II and IIISSI‐1 +ve LIPI‐3 ‐ve
Lineage 1 (esp. 4b)SSI‐1 ‐ve LIPI‐3 +ve
Grow well in food, no 2nd
hemolysin less virulent?Grow poorly in food, 2nd
hemolysin more virulent?hemolysin – less virulent? hemolysin – more virulent?
35 strain surveyGadT2
GadD2
FSL R2‐503FS R 503
CD878
H7858
FSL J1‐194
FSL J1 208 CD147
F2365
F6854FSL J2 071
CD1078
FSL N1 017
CD1066
CD1198
CD1742
CD1038 C241
CD243
CD246
CD83
CD1032 CD1121
CD749
CD748 CD1028
CD1061
In order to predict outcomes more accurately …
HealthAge
Microbial NumbersSpecies or Strain
Host Microbe
WellSick
AgeMicrobiota
Species or StrainHistory
Dead
FoodWe need to assess risk based on:risk based on:
• Genetic complement
Food typeP i
• Stress history• Food constituents• Host parameters
ProcessingConstituents
p
the future ……
FoodExtract DNA/RNA Sequence
16S: Complete quantitative microbial profileB t i l i l f lBacterial, viral, fungal
[spoilage, commensal, pathogenic]
BioinformaticsTotal DNA: gene‐based risk assessment[pathogenicity islands, survival islands,
metabolic potential] Output: Quantitative risk assessment
RNA: Viability assessment, strain
Output: Quantitative risk assessmentEpidemiological certaintyTherapeutic optionsE i th
y ,history, stress adaptation status Emerging pathogens
Rapidity (minutes/hours)
Metagenomics of IMFculture dependent culture independent
Infant milk formula
Strain number
Closest homologue (accession number)
IMF1 Bacillus licheniformis strain LQ98 (Ef472268)
IMF2 Bacillus cereus strain APB2 (Hm046583)
Strain number
Closest homologue (accession number)
PIF1 Uncultured firmicutes.
PIF2 Streptococcus thermophiles strain T1
A b ill fl ith l LK4formula IMF3 Bacillus thuringiensis strain BL40 (Hq180398)
IMF4 Bacillus sp. JM4 (Ef062991)
IMF5 Bacillus anthracis strain (HM854238)
IMF6 Bacillus sp. ITO01(Fr667164)
IMF7 U lt d B ill l Filt 93 (H 152681)
PIF3 Anoxybacillus flavithermus clone LK4
PIF4 Streptococcus thermophiles strain T1 1
PIF5 Flavobacterium frigidarium strain A2i
PIF6 Uncultured bacterium clone gir_aah96b03 16S
PIF7 Enterococcus faecium strain: ML4IMF7 Uncultured Bacillus sp. clone Filt.93 (Hm152681)
IMF8 Lysinibacillus fusiformis strain CBII‐2 (Hm068891)
IMF9 Brevibacillus parabrevis strain M3 (Ab215101)
IMF10 Brevibacillus brevis strain P‐30 (Hm185814)
IMF11 Paenibacillus sp JAM‐FM32 (Ab526335)
PIF7 Enterococcus faecium strain: ML4
PIF8 Uncultured bacterium clone 3‐1B‐G12
PIF9 Leuconostoc lactis YKLAB10
PIF10 Streptococcus uberis 0140J
PIF11 Lactococcus piscium strain: NJ119IMF11 Paenibacillus sp. JAM FM32 (Ab526335)
IMF12 Aeromicrobium erthreum (NR_024846)
IMF13 Staphyloccus hominis (Fj380978)
IMF14 Enterococcus faecium (Ab481104)
IMF15 Enterococcus durans (Eu419600)
PIF11
PIF12 Lactococcus lactis subsp. Cremoris strain: YIT 2007
PIF13 Uncultured Streptococcus sp.clone ChR‐II‐cI12
PIF14 Lactococcus sp.YM05004
PIF15 Cronobacter sakazakii strain 05CHPL95
IMF16 Cronobacter sakazakii ATCC BAA‐894
IMF17 Enterobacter cloacae (Fp929040)
IMF18 Citrobacter koseri (CP000822)
IMF19 Escherichia vulneris (Af530476)
PIF16 Uncultured bacterium W05
PIF17 Uncultured bacterium W05
PIF18 Uncultured Trichococcus sp. TCE‐134
PIF19 Uncultured firmicutes bacterium clone PLF34 16S
IMF20 Uncultured bacterium (Hm309271) PIF20 Streptococcus thermophilus strain T1
Conclusions (1)To predict the outcome of pathogen host interactions (or to derive safe exposure levels), (do) we need to know?
• Host factors (age, health, immune status)• Bacterial numbers
• Genetic complement of strain• Growth potential, survival potential, virulence
i lpotential• History (exposure to stress)
• Impact gastric transit virulence potential• Impact gastric transit, virulence potential• Food constituents
• What about co‐consumed foods?What about co consumed foods?
Conclusions (2)( )
• Should inform policy, but how?• Specific regulations for specific strainsSpecific regulations for specific strains
(EHEC v E. coli)?
• Specific regulations for specific foods?• Zero tolerance in high glutamate foods?Zero tolerance in high glutamate foods?
• Specific advice for high‐risk groups?• Certain combinations of foods to be avoided (e.g. intensive care)
Acknowledgementsg
Listeria research Cormac Gahan
Paul Cotter
group, UCCPaul Cotter
(GAD and LlPI‐3)
Sheila Ryan, Maire Begley(SSI‐1)
Science Foundation Ireland, ,Department of Agriculture, Enterprise Ireland