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

IAFP’s 12th European Symposium on Food Safety [email protected] 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

• Impact on spore germination

• Conclusions

• Impact for food industry Spore forming Growth, Sporulation, Germination and Outgrowth

Growth Dormant/ Growth resistant spore Germination (i.e. by nutrients)

Growing cells Sporulation Dormant Germinated Outgrowth inert spore spore Exponential (phase bright) (phase dark) cell division

Resistance against: Heat, desiccation, chemicals, radiation, acids Spores of concern in foods

• Pathogenic -> foodborne illness e.g. , Clostridium botulinum, C. perfringens

• Spoilage bacteria -> reduced shelf life, spoilage other 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 High-level heat resistant spores

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

Hot springs Soil Decaying plants/compost Bean / fiber fermentations Processing equipment

• 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 € Highly heat resistant spores

Non-sterility issues in heat treated foods

Present in low numbers, but little inactivation 1 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 °C Geobacillus 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. amyloliquefaciens not all strains produce heat resistant spores! Spore heat resistance B. subtilis – large variation

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

Spores of Average time for 1 log reduction at 18 isolates (duplicate) 100 °C 112.5 °C

9 low heat 2.9 min 3.6 s resistant

9 high heat 630 min 600 s resistant (10.5 h) (10 min)

Average >100-fold more time to inactivate spores of group 2 than group 1 Is there a genetic basis for spore HR?

18 strains: Genomes sequenced and analysed 9 strains - Heat resistant ( HR ) spores 9 strains - Heat sensitive ( HS ) spores One specific genetic element ( Tn 1546 transposon) – only in HR strains

Tn 1546 backbone, related to class II cointegrative Tn 3 E. faecium (AB vanr) - fragmented tnpA, 93% ID na E. feacium tnpA K G G K G - 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 sporulation sigmaK - mother cell, sigmaG - forespore Does Tn 1546-like element directly confer spore HR? Introduction element in lab strain 168 168

Heat sensitive spores

168 + Tn element

Heat resistant spores

Tn 1546-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 - Operon 3 crucial Operon 1 N-acetylmuramoyl-L-alanine amidase Ger(x)A Ger(x)C Operon 2 Unknown Putative Manganese catalase Operon 3 Unknown Unknown YchN/YlaJ domain spoVAC spoVAD spoVAEb unknown unknown Gene 4 yetF N terminal yetF C terminal Gene 5 Light bars N0 10 min 80 °C Putative cardiolipin synthetase 12 Dark bars Nt 1h 100 °C

10

8 Insertion Tn 1546: HR spores

6 Deletion operon 3 HR strain:

4 loss HR

2 Insertion operon 3 in HS strain: * HR spores 0 Viable spore count spore CFUViable (log10 mL-1) -> gene products responsible!

* Calculated inactivation 17.4 log spoVA2mob operon -> high level spore HR

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

• Genome analysis: Some B. subtilis strains multiple copies Tn 1546 and/or spoVA 2mob

• 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

• What about other Bacillus spp? spoVA operons widely distributed in Bacillaceae

B. sporothermodurans, B. thermoamylovorans, Caldibacillus debilis

Geobacillus spp Anoxybacillus spp

SpoVA1 B. subtilis group incl . Nearly always present B. licheniformis SpoVA2 non-mobile B. amyloliquefaciens B. cereus Geobacillus Anoxybacillus B. subtilis B. thermoamylovorans B. sporothermodurans

SpoVA2 mob B. subtilis group B. cereus B. thermoamylovorans B. cereus B. sporothermodurans No major differences ability to grow at different temperatures

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

HR spores: delayed germination

Other important finding: spores of strains with 168HR Tn 1546 delayed germination! 168 Demonstrated: due to spoVA2 mob 168HR ∆Tn

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

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

• Kinetics directly linked to presence/absence Tn1546 element / spoVA 2 operon When 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

• spoVA 2mob operon found in species producing HR spores B. subtilis, B. amyloliquefaciens, B. licheniformis B. 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 bacteria Food borne pathogens ( B. cereus , Clostridia) Acknowledgements

• Erwin Berendsen • Antonina Krawczyk • Jos Boekhorst • Robyn Eijlander • Verena Klaus • Anne de Jong • Rosella Koning • Oscar Kuipers • Thank you for you attention