antimicrobial resistance in the food chain - … bsmt food... · 2014-05-28 · refrigeration cold...
TRANSCRIPT
David McDowell
Emeritus Professor of Food Microbiology
School of Health Sciences
University of Ulster
Copyright McDowell 2014
Epidemiology of Antimicrobial Resistance
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The genetic nature of bacteria
(and antibiotic resistances)
may
be changed
DURING
food production and processing
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Transport No genetic change
Transport
+gene transfer
+mutation
Food Chain
Transport
+ gene transfer
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terile” products
Considerable collateral damage
to other desirable food characteristics
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“New” food processing “More natural” Less collateral damage Bacteriostatic (inhibitory) Multiple hurdle treatments
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“Traditional” food processing Bacteriocidal = few survivors (permanent effects)
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“Traditional” food processing Bacteriocidal = few survivors (permanent effects) “New” food processing Bacteriostatic = many stressed survivors (inhibited)
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Sublethal (bacteriostatic)stress in the food chain
and the implications
for antimicrobial resistance
in the human food chain
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Stress sites/types
Food/process Stress Human body
Mayonnaise, fermented foods, pepperoni, salami
Acid (pH) Stomach, intestine/colon, phagosomes
Mild/rare/sous vide cooking Heat Temperature intracellular environment
Fish, brines, Na+, marinades Osmolarity Stomach
H2O2, in foods O2/oxidation Phagocytes
Refrigeration Cold Temperature excretion
Surfaces in food plants Starvation Nutrient dilution in water on defecation/macrophages
Sous vide/Vacuum packing Anaerobiosis Phagosomes
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What does not kill them,
makes them stronger
i.e. more
Persistent? Virulent?
& Antimicrobial Resistant?
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Stress responses
σB expression
persistence of replication errors
genomic promiscuity
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Stress responses
Short term adaptation (phenotypic + reversible)
Longer term change
(genomic + permanent)
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Does sub-lethal stress increase
[1] development of new ABR in foodborne pathogens?
and/or
[2] transfer of extant ABR genes among foodborne pathogens?
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E. coli, Staph aureus
Salmonella Typhimurium
“Stressed” = 75% reduction in growth rate
MICs determined [1] under stress,
[2] after removal of stress
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Organism
Stress applied
to achieve 75% reduction in growth rate)
Low temp High temp NaCl pH
E. coli 10 45 0 4.5
S. aureus 21 45 12 5.0
S. Typhimurium 10 45 4.5 4.5
Controls 37 37 0 7.4
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Changes in MICs in stressed cell suspensions
E. coli (n=4)
Antibiotic Control MIC
(μg/ml)
Applied stress
Low
temp
High
temp NaCl pH
Amikacin 15 --- --- ++ ++
Ceftriaxone 0.09 --- --- + +++
Naladixic acid 7.5 - 0 + +++
0 = no change in MIC
+/- = 1.5- 2 fold increase/decrease in MIC (p<0.05)
++/-- = 2.1- 4 fold increase/decrease in MIC (p<0.05)
+++/--- = greater than 4 fold increase/decrease in MIC (p<0.05)
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Changes in MICs in stressed cell suspensions
S. aureus (n=4)
Antibiotic Control MIC
(μg/ml)
Applied stress
Low
temp
High
temp NaCl pH
Gentamicin 1.9 -- -- +++ +++
Oxacillin 0.19 0 -- +++ -
Erythromycin 1 0 --- + +++
0 = no change in MIC
+/- = 1.5- 2 fold increase/decrease in MIC (p<0.05)
++/-- = 2.1- 4 fold increase/decrease in MIC (p<0.05)
+++/--- = greater than 4 fold increase/decrease in MIC (p<0.05)
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Changes in MICs in stressed cell suspensions
S. Typhimurium
Antibiotic Control MIC
(μg/ml)
Applied stress
Low
temp
High
temp NaCl pH
Amikacin 20 --- --- 0 ++
Ceftriaxone 1 -- -- + +++
Naladixic acid 3 --- --- ++ +++
0 = no change in MIC
+/- = 1.5- 2 fold increase/decrease in MIC (p<0.05)
++/-- = 2.1- 4 fold increase/decrease in MIC (p<0.05)
+++/--- = greater than 4 fold increase/decrease in MIC (p<0.05)
Copyright McDowell 2014
Persistent changes in MICs in post stressed cell suspensions
E. coli (n=4)
Antibiotic Control MIC
(μg/ml)
Applied stress
Low
temp
High
temp NaCl pH
Amikacin 15 + --- ++ ++
Ceftriaxone 0.09 0 -- +++ +++
Naladixic acid 7.5 - - ++ ++
0 = no change in MIC
+/- = 1.5- 2 fold increase/decrease in MIC (p<0.05)
++/-- = 2.1- 4 fold increase/decrease in MIC (p<0.05)
+++/--- = greater than 4 fold increase/decrease in MIC (p<0.05)
Copyright McDowell 2014
Persistent changes in MICs in post stressed cell suspensions
S. aureus (n=4)
Antibiotic Control MIC
(μg/ml)
Applied stress
Low
temp
High
temp NaCl pH
Gentamicin 1.9 0 -- ++ 0
Oxacillin 1.9 -- 0 - 0
Erythromycin 1.9 + -- ++ +
0 = no change in MIC
+/- = 1.5- 2 fold increase/decrease in MIC (p<0.05)
++/-- = 2.1- 4 fold increase/decrease in MIC (p<0.05)
+++/--- = greater than 4 fold increase/decrease in MIC (p<0.05)
Copyright McDowell 2014
Persistent changes in MICs in post stressed cell suspensions
S. Typhimurium (n=4)
Antibiotic Control MIC
(μg/ml)
Applied stress
Low
temp
High
temp NaCl pH
Amikacin 20 -- - -- -
Ceftriaxone 1 - - -- 0
Naladixic acid 3 -- 0 -- -
0 = no change in MIC
+/- = 1.5- 2 fold increase/decrease in MIC (p<0.05)
++/-- = 2.1- 4 fold increase/decrease in MIC (p<0.05)
+++/--- = greater than 4 fold increase/decrease in MIC (p<0.05)
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pH or NaCl stressed pathogens show increased ABR
Some changes persists during
and after stress
Bacteriostatic food preservation stress may contribute to development/expression of ABR in food related pathogens
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E. coli
Stress Antibiotic “Hyper resistant”
colonies
Control,
10°C or /45°C
Amikacin 0
Ceftriaxone 0
Nalidixic acid 0
pH
Amikacin 0
Ceftriaxone +
Nalidixic acid +
NaCl
Amikacin +
Ceftriaxone +
Nalidixic acid +
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S. aureus
Stress Antibiotic “Hyper resistant”
colonies
Control
10°C
45°C
Oxacillin 0
Erythromycin 0
Gentamycin 0
pH
Oxacillin +
Erythromycin +
Gentamycin 0
NaCl
Oxacillin +
Erythromycin 0
Gentamycin 0
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S. Typhimurium
Stress Antibiotic “Hyper resistant”
colonies
Control
10°C
45°C
Amikacin 0
Ceftriaxone 0
Trimethoprim 0
pH
Amikacin 0
Ceftriaxone 0
Trimethoprim +
NaCl
Amikacin +
Ceftriaxone +
Trimethoprim +
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Differences between MICs
of parent and stress associated “resistant” isolates
Organism Antibiotic Stress MIC increase (fold)
E. coli Ceftriaxone NaCl 833
Nalidixic acid pH 5
S Typhimurium Trimethoprim pH 4
S. aureus Erythromycin pH 2
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Multiple stresses
=
more hyper-resistance
Possible ABR impact of
Multiple hurdle technology?
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[1] stress increases expression of
ABR in foodborne pathogens
[2] stress spreads of extant ABR genes among foodborne
pathogens?
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Effects of the high/low temp,
osmotic and pH stress on
Rates of plasmid transfer during filter mating between
[1] E. coli/E. coli
[2] E. coli/S. Typhimurium
Donor and recipient each carried 1 known ABR
Transconjugants expressed both ABRs
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Strain/Species Role Resistance
E. coli NCTC 50021 donor Plasmid bearing, R386, Tetracycline
resistance
E. coli NCTC 50338 donor Plasmid bearing, TP 3 07
Gentamycin, Streptomycin,
Tobramycin & Apramycin resistance
E. coli ATCC 35695 recipient Streptomycin
E. coli ATCC 33694 recipient Streptomycin
S. Typhimurium DT104 st11 recipient Ampicillin
Conjugant Pairs
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Donor (plasmid) Recipient Stress (in LLB) Transfer rate (Power)
E. coli 50021
(plasmid R 386)
E. coli 35695
Control (37oC)
pH 4.3
4% NaCl
5oC
2.1 x 10-8
5.6 x 10-6
1.2 x 10-5
8.1 x 10-6
-8
-6
-5
-6
E. coli 33694
Control (37oC)
pH 4.3
4% NaCl
5oC
2.7 x 10-9
1.5 x 10-5
1.25 x 10-7
3.8 x 10-6
-9
-5
-7
-6
Transfer rate = N0/Tranconjugants/N0 recipients
Plasmid Transfer Rates
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Donor (plasmid) Recipient Stress (in LLB) Transfer
rate
(Power)
E. coli 50338
(plasmid TP307)
E. coli 35695
Control (37oC)
pH 4.3
4% NaCl
5oC
6.8 x 10-12
5.4 x 10-8
1.2 x 10-5
4.0 x 10-5
-12
-8
-5
-5
E. coli 33694
Control (37oC)
pH 4.3
4% NaCl
5oC
2.7 x 10-11
8.1 x 10-8
3.1 x 10-7
1.2 x 10-3
-11
-8
-7
-3
S. Typhimurium
DT104 st11
Control (37oC)
pH 4.3
4% NaCl
5oC
4.5 x 10-12
7.3 x 10-10
1.4 x 10-7
1.7 x 10-5
-12
-10
-7
-5
Transfer rate = N0/Tranconjugants/N0 recipients
Plasmid Transfer Rates (2)
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E. coli/E. coli (R386 2-4 fold increase)
&
E. coli/E. coli
& E.coli/S. Typhimurium
(TP307 5-7 fold increase)
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[1] stress increases expression of
ABR in foodborne pathogens
[2] stress spreads of extant ABR genes among foodborne
pathogens
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Need to
understand how sublethal stress
stimulates ABR in foods
Review elements of multiple hurdle technologies/develop safer
alternative combination treatments
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Acknowledgements
Dr Ann McMahon
Funding from
Research and Development Office,
Department of Health,
Northern Ireland
Infectious Diseases RRG
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