lab food microbiology · lab food microbiology 1.3 control by hurdle technology b. cereus spore...

Lab Food Microbiology

Resistente micro-organismen:Voorkomen en controle met

hordentechnologie

Prof. Dr. Ir. Chris MichielsLabo LevensmiddelenmicrobiologieFaculteit Bio-ingenieurswetenschappenKU [email protected]

16.05.2017, Antwerpen

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mailto:[email protected]

Resistant foodborne microorganisms

1.Heat resistance

1.High pressure resistance

2.Acid tolerance

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Occurrence / relevance in foods

Cellular mechanisms

Control by hurdle technology

1. Heat resistant bacteria

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Am. J. Pub. Hlth 36:451 (1946)

1.1 Natural occurrence

Salmonella Senftenberg 775W

• Isolated from egg powder in 1946!

• More heat resistant than any otherSalmonella strain

• Can survive USDA minimum pasteurisation requirements:

• Whole egg: 3.5 min @ 60°C• Egg white: 3.5 min @ 56.7°C


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Cronobacter sakazakii

• Some strains are much more heat resistant than others

• May survive production of powdered infant formula

• May survive heating duringreconstitution of PIF

J. Food Safety 29:287 (2009)


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E. coli

• Isolates from environment where carcasses are steam-treated in 2011

• More heat resistant than otherE. coli strains

• Survive in beef patties heated tointernal T = 71°C

J. Appl. Microbiol. 110:840 (2001)


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1.2 Resistance mechanism

Cronobacter sakazakii

• Specific protein present in heat-resistant strains but not in heat-sensitive strains (2005)

• Function unknown

Proteomics 5:4161 (2005)


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• Heat-resistant strains of several Enterobacteriaceae share a ‘locus of heat resistance’• Heat resistance requires several genes of this locus• Locus also called: ‘transmissible locus for protein quality control (TLPQC)• Occurs in 2% of all sequenced E. coli genomes

Nguyen et al., mSystems 2:e00190-16 (2017)


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• E. coli strains without locus of heat resistance can also develop heat resistance• Overexpression of rpoH and/or rpoS

Appl. Environ. Microbiol. 82:6656 (2016)


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1.1 Heat resistance variation in bacterial spores

• Bacillus subtilis group: B. subtilis, B. licheniformis, B. amyloliquefaciens• Strains with heat resistant spores have spoVA genes on a Tn1546 transposon

Front. Microbiol. doi: 10.3389/fmicb.2016.01912

Food. Microbiol. 45:18 (2015)


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1.3 Control by hurdle technology

• Heat + natural antimicrobials• Essential oil compounds• Synergistic effect at very low concentration

Survival curves of S. Senftenberg in TSB broth at 55°C (B) with 0 (closed squares), 0.1 (circles), 0.5 (open squares) and 1 (triangles) mM D-limonene nanoemulsified

Log10 of times for 5 log reduction of E. coli O157:H7 at different temperatures in orange juice, with no Eos (filledcircles), orange EO (open circles) or limonene (open squares)

Food Bioprocess. Technol. 7:471 (2014)

Nanomaterials 7:65 (2017)


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B. cereus spore inactivation at 100°C (control, closed circles) and with 0.5 mM thymol in heating medium (closed squares) or in recovery medium (open squares)

Log D of C. sporogenes spores inactivation at 90C withdifferent concentrations of fatty acids in recovery medium. Palmitic (closed circles), stearic (closed triangles), palmitoleic(open circles), and oleic (open triangles)

= Exploitation of injury of spores

Food Microbiol. 48:35 (2015)

Int J Food Microbiol 141:242 (2010)

2. High pressure resistant bacteria

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> 250 million L HP juices sold in 2015

L. monocytogenes

E. coli


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Compilation of literature data on inactivation of E. coli and L. monocytogenes in neutral pH buffer, 20-25°C, 10 min treatment


• Very large strain variability (more than forheat)

• At low p, L. mono is on average more resistant than E. coli

• At high p, E. coli is on average more resistant than L. mono

• Some natural strains of E. coli are extremely HP resistant

• E. coli and some (but not all!) bacteriacan acquire extreme HP resistance


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Inactivation of wild-type E. coli strain MG1655 and its HP resistant mutant LMM1010 by 15 min treatment at different pressure-temperature combinations


• E. coli can acquire extreme HP resistanceby natural selection (1996)

• Strains resist HP up to 2 GPa

Appl Environ Microbiol 63:945 (1997)



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• Different mechanisms exist• Increased RpoS expression (cfr. heat resistance)• Loss of cya/CRP catabolite repression system

Inactivation of wild-type E. coli O157:H7 strain (left) and four selected HP resistant mutants by (A) pressure (300 MPa), (B) pressure (600 MPa) and (C) heat (56°C),

300 MPa

600 MPa

56°C


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• Combined treatment HP + different EO compounds• Limited number of compounds were synergistic

t-cinnamaldehyde

carvacrol

thymol

eugenol

linalool

linalool oxidecitral

geraniol

eucalyptol

α- terpineol

α-pinene

allyl

isothiocyanate

Front. Microbiol. doi: 10.3389/fmicb.2016.01912

Innov Food Sci Em Technol 27:26 (2015)


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No treatment

t-CIN alone

HP alone

HP + t-CIN

2.2 Control by hurdle technology• Strong synergy of HP + cinnamaldehyde at MIC level (4 mM)• Works on all tested bacteria, including HP resistant strains

trans-cinnamaldehyde


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α, β – unsaturated carbonyls Isothiocyanates

t-cinnamaldehyde

citral

allyl isothiocyanate


• Most synergistic compounds are electrophiles that react with thiol groups (proteins,

AcCoA, glutathion…)

• HP treatment is known to disrupt bacterial thiol-disulfide homeostasis

• Some synergistic compounds have other mode of action (e.g. linalool oxide)

• BUT: Compounds from plant essential oils have strong flavour…

Other thiol-reactive electrophiles with less side-effects, or with beneficial properties?


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The lactoperoxidase system (LPER)

• Milk enzyme: SCN- + H2O2 OSCN- + H2O

• OSCN- reacts with thiol groups

• LPER has a mostly bacteriostatic effect

• Applied in milk in remote areas of tropical countries

• Nontoxic in animals; no off-flavour

MG1655 - 300 MPa

LMM1010 - 300 MPa

LMM1010 - 400 MPa

LMM1010 - 500 MPa

Survival during storage at 20°C of E. coli strains

after HP treatment in milk (), milk with H2O2

(■), and milk with the LPER system (▲)

Combination HP + LPER

• HP sensitizes bacteria for LPER system

• HP-sensitized bacteria undergo rapid

inactivation after HP treatment

• Also HP resistant mutants are sensitized

• 6-D reduction of ALL vegetative cells can be

achieved at p < 600 MPa and T < 40°C in food




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Sulphoraphane

• Isothiocyanate (reactive with thiols)

• Moderate flavour

• Health-promoting properties

1 m

MSU

L

Un

trea

ted

HP

HP

+ S

UL

Reuterin

• Aldehyde that reacts with thiol groups

• Produced by Lactobacillus reuteri

• No taste or flavour

• L. reuteri is used as probiotic

• Can be converted to acrolein (cancerogen!)


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L. monocytogenes counts in smoked salmon after treatment with HP + LPER during storage at 5°C

Innov Food Sci Em Technol 16:26 (2012)

3. Acid tolerant bacteria

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3.1 Occurrence of acid resistance in E. coli

• Survival of extreme acid challenge has been well studied in E. coli and Salmonella• Growth at (moderately) low pH has been less studied

Acid resistance (AR) systems that support survival of E. coli at extremely low pH (pH 2.0 – 2.5)


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3.1 Occurrence of acid tolerance (growth) in E. coli

Predicted growth – no growth interfaces of 180 E. coli strains at low pH and different temperatures without or with lactic acid (25 mM)

Food Microbiol 45:222 (2015)


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3.2 Mechanisms of acid tolerance: role of butanediol fermentation

Fermentation pathways in Enterobacteriaceae

• Mixed acid fermenters• E. coli, Salmonella,…• MR+ VP-

• Butanediol fermenters• Enterobacter, Klebsiella,…• MR- VP+


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Growth of S. plymuthica control strain (black) and budAB knockout strain (grey) in tomato juice at 20°C



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VRBG VRBGMcConkey McConkey

E. coli control E. coli + budAB genes

Challenge test of E. coli control strain (grey) and strain with budAB genes (black) in tomato juice at 30°C

Appearance of E. coli control strain (left) and strain withbudAB genes (right) on four differential plating media

Do natural butanediol-fermenting stains of E. coli (Salmonella, Shigella…) strains exist?

Appl Environ Microbiol 80:6054 (2014)

Conclusions and outlook

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• Bacteria can develop resistance against processing and preservation hurdles

• This is not a surprise…

• The speed and extent of resistance development varies depending on the stress

• It will be important to continue monitoring resistance development

• Hurdle technology based on synergistic processing/preservation factors cancontrol bacteria that developed resistance to one factor

• Hurdle technology may prevent resistance development because resistancedevelopment against multiple hurdles simultaneously is improbable or impossible

lab food microbiology · lab food microbiology 1.3 control by hurdle technology b. cereus spore...

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