Genetic biomarkers for high heat resistance of Bacillus spores:relevance for optimal design of heat treatment

marjon.wells-bennik@nizo.comIAFP’s 12th European Symposium on Food Safety

11-13 May 2016

Outline

• Short intro on spores

• Highly heat resistant spores in food – issues

• Breakthrough insight on high-level spore heat resistance Matching genomic info with phenotypic traits!

• Occurence of HR elements in Bacillaceae

• Impact on spore germination

• Conclusions

• Impact for food industry

Spore forming bacteriaGrowth, Sporulation, Germination and Outgrowth

OutgrowthGerminatedspore

(phase dark)

Growing cells Sporulation Dormantinert spore

(phase bright)

Dormant/ resistant

spore

Growth

Germination (i.e. by nutrients)

Growth

Resistance against: Heat, desiccation, chemicals, radiation, acids

Exponential

cell division

Spores of concern in foods

• Pathogenic species -> foodborne illnesse.g. Bacillus cereus, Clostridium botulinum, C. perfringens

• Spoilage bacteria -> reduced shelf life, spoilageother Bacillus, Paenibacillus, Geobacillus, Clostridium species

• Things to consider in foods:- Survival of spores during inactivation treatment- Germination spores and outgrowth potential vegetative cells

• Focus talk: highly heat resistant spores (survival > 30 min 100°C)able to grow at temperatures up to 60 °C

• No inactivation >30 min 100°C - Ubiquitous in nature!

• Introduction in food chain: Soil / dust / spontaneous (heap) fermentation processes During manufacturing - biofilms/fouling/growth; heating sections / evaporators

• SPOILAGE – product loss in final products, recalls €• Meeting SPECIFICATIONS (e.g. powders) – downtime manufacturing €

Bean / fiber fermentations Processing equipmentSoil Decaying plants/compost

High -level heat resistant spores

Hot springs

Highly heat resistant spores

Non-sterility issues in heat treated foods

Present in low numbers, but little inactivation1 per packaging unit -> spoilage / recalls

Commonly encountered species, surviving > 3 min 121°C

Growth at T > 45-65 °C, no spore inactivation 30 min 100 °CGeobacillus spp, Anoxybacillus spp.

Growth at T 10~60 °C, no spore inactivation 30 min 100 °C:B.subtilis, B.sporothermodurans, B. thermoamylovorans, B. licheniformis, B. amyloliquefaciensnot all strains produce heat resistant spores!

Two distinct groups within B. subtilis with respect to spore heat resistance

Spore heat resistance B. subtilis– large variation

Average >100-fold more time to inactivate spores of group 2 than group 1

Spores of 18 isolates (duplicate)

Average time for 1 log reduction at

100 °C 112.5 °C

9 low heat resistant

2.9 min 3.6 s

9 high heat resistant

630 min (10.5 h)

600 s (10 min)

18 strains: Genomes sequenced and analysed9 strains - Heat resistant (HR) spores 9 strains - Heat sensitive (HS) spores

One specific genetic element (Tn1546 transposon) – only in HR strains

Is there a g enetic basis for spore HR?

GGKK KKGG

GG

Does Tn1546-like element directly confer spore HR?

Tn1546 backbone, related to class II cointegrative Tn3 E. faecium (AB vanr)- fragmented tnpA, 93% ID na

E. feacium tnpA- tnpR resolvase only in 3 strains- two 38 bp imperfect inv repeats- 5 bp direct repeat at integration site

~12 kb, five transcriptional units, uniquely expression during sporulationsigmaK - mother cell, sigmaG - forespore

Introduction element in lab strain 168168

Heat sensitive spores

168 + Tn element

Heat resistant spores

Tn1546-like element confers spore heat resistance

Heating 1h 100°C: HS spores ~10 log ↓HR spores 0.1 log ↓

Which genes in this element critical?

Tn1546 essential for HR

* Calculated inactivation 17.4 log

*

Light bars N0 10 min 80°CDark bars Nt 1h 100°C

Via

ble

spor

e co

unt (

log1

0 C

FU

mL-

1)

0

2

4

6

8

10

12

Gene 5Putative cardiolipin synthetase

Operon 1N-acetylmuramoyl-L-alanine amidaseGer(x)AGer(x)C

Operon 3UnknownUnknown YchN/YlaJ domainspoVACspoVADspoVAEbunknownunknownGene 4yetF N terminalyetF C terminal

Operon 2UnknownPutative Manganese catalase

Deletion operon 3 HR strain: loss HR

- Operon 3 crucial

Insertion Tn1546: HR spores

Insertion operon 3 in HS strain: HR spores-> gene products responsible!

spoVA2mob operon -> high level spore HR

• What about other Bacillus spp?

• More copies -> higher HR• 0 Tn: D112.5 0,2 min• 1 Tn: D112.5 1,2 min• 2 Tn: D112.5 8,8 min• 2 Tn +spoVA2: D112.5 25,6 min

• Three homologs spoVAA-AF operon B. subtilis in operon 3: spoVA2mob

• Genome analysis:Some B. subtilis strains multiple copies Tn1546 and/or spoVA2mob

spoVA operons widely distributed in Bacillaceae

B. sporothermodurans, B. thermoamylovorans, Caldibacillus debilis

Geobacillus sppAnoxybacillus spp

B. subtilis group incl . B. licheniformis

B. amyloliquefaciens

B. subtilis

B. cereus

SpoVA1Nearly always present

SpoVA2 non-mobileB. cereusGeobacillusAnoxybacillus B. thermoamylovoransB. sporothermodurans

SpoVA2mobB. subtilis groupB. cereusB. thermoamylovoransB. sporothermodurans

Two distinct groups within B. subtilis with respect to spore heat resistanceVegetative cells span similar range growth temperatures

No major differences ability to grow at different temperatures

168HR

168

168HR∆Tn

Other important finding: spores of strains with Tn1546 delayed germination!

Delayed germination HR spores, but vegetative cells grow similar : -> delayed/unpredictable spoilage upon high heat treatment

Demonstrated: due to spoVA2mob

HR spores: delayed germination

Conclusions

• Heat inactivation kinetics spores B. subtilis, licheniformis, amyloliquefaciensDistinctly different for different groups

• Kinetics directly linked to presence/absence Tn1546 element /spoVA2 operonWhen present: highly heat resistance spores

• Discovery based on genomes natural isolates and phenotypes

• Presence spoVA2: also delayed germination When HR spores survive – delayed spoilage in products

• Bacillus strains with/without element span similar growth temp range

• Transfer element can occur during vegetative growth / stress

• spoVA2mob operon found in species producing HR sporesB. subtilis, B. amyloliquefaciens, B. licheniformisB. sporothermodurans, B. thermoamylovorans, Geobacillus species

• Now possible to detect ‘trouble’ spores within species! e.g. in ingredients, track and trace

Challenges and consequences for the food industry

Control of heat resistant spores

• Direct detection strains producing HR spores possible

• Modelling spore inactivation / Calculating spore heat inactivation - Take differences between strains into account- When SpoVA2mob present: high heat resistance kinetics

• Prevent spread of mobile genetic element possible (in vegetative state)e.g. avoid rework heat treated streams

• Extending knowledge to other spore forming bacteriaFood borne pathogens (B. cereus, Clostridia)

Acknowledgements

• Erwin Berendsen• Jos Boekhorst• Verena Klaus• Rosella Koning

• Thank you for you attention

• Antonina Krawczyk• Robyn Eijlander• Anne de Jong • Oscar Kuipers