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

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1 Genetic biomarkers for high heat resistance of Bacillus spores: relevance for optimal design of heat treatment IAFP s 12th European Symposium on Food Safety marjon.wells-bennik@nizo.com May 2016

2 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

3 Spore forming bacteria Growth, Sporulation, Germination and Outgrowth Growth Dormant/ resistant spore Germination (i.e. by nutrients) Growth Growing cells Sporulation Dormant inert spore Germinated spore (phase bright) (phase dark) Outgrowth Exponential cell division Resistance against: Heat, desiccation, chemicals, radiation, acids

4 Spores of concern in foods Pathogenic species -> foodborne illness e.g. Bacillus cereus, 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

biofilms/fouling/growth; heating sections /

5 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

per packaging unit -> spoilage / recalls Commonly encountered

spore inactivation 30 min 100 C Geobacillus spp, Anoxybacillus

subtilis, B.sporothermodurans, B. thermoamylovorans, B.

6 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 > 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!

7 Spore heat resistance B. subtilis large variation Two distinct groups within B. subtilis with respect to spore heat resistance Spores of 18 isolates (duplicate) 9 low heat resistant Average time for 1 log reduction at 100 C C 2.9 min 3.6 s 9 high heat resistant 630 min (10.5 h) 600 s (10 min) Average >100-fold more time to inactivate spores of group 2 than group 1

8 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 (Tn1546 transposon) only in HR strains K K G G G 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 sporulation sigmak - mother cell, sigmag - forespore Does Tn1546-like element directly confer spore HR?

9 Introduction element in lab strain Tn element Heat sensitive spores Tn1546-like element confers spore heat resistance Heating 1h 100 C: HS spores ~10 log HR spores 0.1 log Heat resistant spores Which genes in this element critical?

Tn1546 essential for HR - Operon 3 crucial Viable spore count (log10 CFU ml-1) 12 10 8 6 4 2 0 Light bars N0 10 min 80 C Dark bars Nt 1h 100 C * Operon 1 N-acetylmuramoyl-L-alanine amidase Ger(x)A

10 Tn1546 essential for HR - Operon 3 crucial Viable spore count (log10 CFU ml-1) Light bars N0 10 min 80 C Dark bars Nt 1h 100 C * 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 Putative cardiolipin synthetase Insertion Tn1546: HR spores Deletion operon 3 HR strain: loss HR Insertion operon 3 in HS strain: HR spores -> gene products responsible! * Calculated inactivation 17.4 log

11 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 Tn1546 and/or spova2mob More copies -> higher HR 0 Tn: D ,2 min 1 Tn: D ,2 min 2 Tn: D ,8 min 2 Tn +spova2: D ,6 min What about other Bacillus spp?

12 spova operons widely distributed in Bacillaceae B. sporothermodurans, B. thermoamylovorans, Caldibacillus debilis Geobacillus spp Anoxybacillus spp B. subtilis group incl. B. licheniformis B. amyloliquefaciens B. subtilis B. cereus SpoVA1 Nearly always present SpoVA2 non-mobile B. cereus Geobacillus Anoxybacillus B. thermoamylovorans B. sporothermodurans SpoVA2mob B. subtilis group B. cereus B. thermoamylovorans B. sporothermodurans

13 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 Tn1546 delayed germination! HR Demonstrated: due to spova2mob 168HR Tn Delayed germination HR spores, but vegetative cells grow similar: -> delayed/unpredictable spoilage upon high heat treatment

14 Conclusions Heat inactivation kinetics spores B. subtilis, licheniformis, amyloliquefaciens Distinctly different for different groups Kinetics directly linked to presence/absence Tn1546 element /spova2 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 spova2mob 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

15 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)

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

University of Groningen. Heat resistance of Bacillus spores Berendsen, Erwin Mathijs

University of Groningen. Heat resistance of Bacillus spores Berendsen, Erwin Mathijs University of Groningen Heat resistance of Bacillus spores Berendsen, Erwin Mathijs IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please