antimicrobial resistance in the food chain - … bsmt food... · 2014-05-28 · refrigeration cold...

David McDowell

Emeritus Professor of Food Microbiology

School of Health Sciences

University of Ulster

Copyright McDowell 2014

Medicine Agriculture


Epidemiology of Antimicrobial Resistance


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The genetic nature of bacteria

(and antibiotic resistances)

may

be changed

DURING

food production and processing


Transport No genetic change

Transport

+gene transfer

+mutation

Food Chain

Transport

+ gene transfer


terile” products

Considerable collateral damage

to other desirable food characteristics


“New” food processing “More natural” Less collateral damage Bacteriostatic (inhibitory) Multiple hurdle treatments


“Traditional” food processing Bacteriocidal = few survivors (permanent effects)


“Traditional” food processing Bacteriocidal = few survivors (permanent effects) “New” food processing Bacteriostatic = many stressed survivors (inhibited)


Sublethal (bacteriostatic)stress in the food chain

and the implications

for antimicrobial resistance

in the human food chain


first bacteria” first “bacteria”

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


“No growth”

“No activity”


“No growth” no activity”

“slowed” metabolism is

undesirable metabolism


Sublethal stress

= Phenotypic/genomic changes


What does not kill them,

makes them stronger

i.e. more

Persistent? Virulent?

& Antimicrobial Resistant?


Stress responses

σB expression

persistence of replication errors

genomic promiscuity


Stress responses

Short term adaptation (phenotypic + reversible)

Longer term change

(genomic + permanent)


Does sub-lethal stress increase

[1] development of new ABR in foodborne pathogens?

and/or

[2] transfer of extant ABR genes among foodborne pathogens?


E. coli, Staph aureus

Salmonella Typhimurium

“Stressed” = 75% reduction in growth rate

MICs determined [1] under stress,

[2] after removal of stress


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


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)



S. aureus (n=4)


(μg/ml)

Applied stress

Low

temp

High

temp NaCl pH

Gentamicin 1.9 -- -- +++ +++

Oxacillin 0.19 0 -- +++ -

Erythromycin 1 0 --- + +++







S. Typhimurium


(μg/ml)

Applied stress

Low

temp

High

temp NaCl pH

Amikacin 20 --- --- 0 ++

Ceftriaxone 1 -- -- + +++

Naladixic acid 3 --- --- ++ +++






Persistent changes in MICs in post stressed cell suspensions

E. coli (n=4)


(μg/ml)

Applied stress

Low

temp

High

temp NaCl pH

Amikacin 15 + --- ++ ++

Ceftriaxone 0.09 0 -- +++ +++

Naladixic acid 7.5 - - ++ ++







S. aureus (n=4)


(μg/ml)

Applied stress

Low

temp

High

temp NaCl pH

Gentamicin 1.9 0 -- ++ 0

Oxacillin 1.9 -- 0 - 0

Erythromycin 1.9 + -- ++ +







S. Typhimurium (n=4)


(μg/ml)

Applied stress

Low

temp

High

temp NaCl pH

Amikacin 20 -- - -- -

Ceftriaxone 1 - - -- 0

Naladixic acid 3 -- 0 -- -






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


12% NaCl

stress Control

S. aureus AB

disc


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 +


S. aureus


colonies

Control

10°C

45°C

Oxacillin 0

Erythromycin 0

Gentamycin 0

pH

Oxacillin +

Erythromycin +

Gentamycin 0

NaCl

Oxacillin +

Erythromycin 0

Gentamycin 0


S. Typhimurium


colonies

Control

10°C

45°C

Amikacin 0

Ceftriaxone 0

Trimethoprim 0

pH

Amikacin 0

Ceftriaxone 0

Trimethoprim +

NaCl

Amikacin +

Ceftriaxone +

Trimethoprim +


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


12%

NaCl

stress Control

S. aureus AB

disc


Multiple stresses

=

more hyper-resistance

Possible ABR impact of

Multiple hurdle technology?


[1] stress increases expression of

ABR in foodborne pathogens

[2] stress spreads of extant ABR genes among foodborne

pathogens?


Plasmid transfer during conjugation


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


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


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


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)


E. coli/E. coli (R386 2-4 fold increase)

&

E. coli/E. coli

& E.coli/S. Typhimurium

(TP307 5-7 fold increase)


[1] stress increases expression of

ABR in foodborne pathogens

[2] stress spreads of extant ABR genes among foodborne

pathogens


Need to

understand how sublethal stress

stimulates ABR in foods

Review elements of multiple hurdle technologies/develop safer

alternative combination treatments


Acknowledgements

Dr Ann McMahon

Funding from

Research and Development Office,

Department of Health,

Northern Ireland

Infectious Diseases RRG


Questions

&

Comments?


antimicrobial resistance in the food chain - … bsmt food... · 2014-05-28 · refrigeration cold...

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