genetic biomarkers for high heat resistance of bacillus spores · genetic biomarkers for high heat...
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Genetic biomarkers for high heat resistance of Bacillus spores:relevance for optimal design of heat treatment
[email protected]’s 12th European Symposium on Food Safety
11-13 May 2016
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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
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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
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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
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• 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
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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!
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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)
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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
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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?
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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!
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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
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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
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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
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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
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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)
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Acknowledgements
• Erwin Berendsen• Jos Boekhorst• Verena Klaus• Rosella Koning
• Thank you for you attention
• Antonina Krawczyk• Robyn Eijlander• Anne de Jong • Oscar Kuipers