Veterinärmedizinische Universität Wien

Monika Ehling-Schulz

Institute of Functional Microbiology, Department of PathobiologyUniversity of Veterinary Medicine Vienna, Austria

Emerging Bacterial Spores: The Good, the Bad, the Ugly

Bacillus sp.

� Gram+ aerobic or facultative anaerobic rods(currently: 144 species described)

� Formation of endospores

(Ball et al., 2008)

Bacillus Spores

(Fricker, Ågren, Segerman, Knutsson, and Ehling-Schulz; IJFM 2011)

Bacilli in food production

X

The Good…

• Biopesticides

• Pre-/ and Probiotics(animals and humans)

• Plant growth promoting Bacilli

X

Bacillus thuringiensis

- Insecticidal toxins (Cry)

(Fricker et al., 2011)

Western corn rootworm

(Fricker)

Isolat 1

Isolat 2

Isolat 3

Typing of B. thuringiensis

by means of FTIR

(according to Ehling-Schulz et al., ASM Press 2011)

The Good…

• Biopesticides

• Pre-/ and Probiotics

• Plant growth promoting Bacilli

(Ågren)

B. cereus – Use as probiotic in piglets

Bacillus – the Bad and the Ugly

Spoilage:economic risk

B. weihenstephanensis /

B. mycoides

B. coagulans

B. licheniformis

B. subtilis

G. sterothermophilus

B. cereus

B. licheniformis

B. mojavensis

B. pumilus

B. subtilis

Poisoning:health risk

Enzymes Toxins

Heat resistance of Bacillus spores inDairy production

Emerging spores

The Bad…

(Unpublished results from an ongoing project funded by the German Ministry of Economics and Technology)

X

The Ugly….

(EFSA Journal 2009 – 271)

The Community Summary Report on Food-BorneOutbreakes in the European Community 2007(EFSA Report April 2009)

2. Emesis (Cereulide, a heat-stable depsipeptide)- Intoxication

Role of Bacillus cereus

in food poisoning

Species Infective Incubation- Durationdose time

Bacillus cereus toxin 0.5-6 h 6-24 h(emetic)

Staphylococcus aureus toxin 0.5-6 h 8-24 h

1. Diarrhoea (HBL, NHE, CytK, heat-labile enterotoxins)- Infection

Species Infective Incubation- Durationdose time

Bacillus cereus 105-107 6-12 h 12-24 h(diarrhoeal type)

Clostridium perfringens 107-108 8-16 h 16-24 h

Bacillus cereus diagnostics

Test of ß-Hemolysis on blood agar

(Ehling-Schulz et al., Microbiology 2005)

F4430/73 INRA SZ

RIVM-BC67F4810/73

emetic

non emetic

International Regulations

XGermany (DGHM)

XAustralia (Food Standards)

XCanada (HPFB)

XUSA (FDA)

MYPbPEMBAa

aPolymyxin- Egg- Yolk- Mannitol- Bromthymol- AgarbMannitol- Egg- Yolk- Polymyxin- Agar

Identification of Bacillus cereus

PEMBA MYPA

1, 5, 8: isolates from emetic outbreak;2: psychrotolerant strain; 3, 4: clinical isolates; 6: isolate from diarrheal outbreak; 7: B. cereus type strain; 9: B. licheniformis

1

2

3

4

5

6

7

8

9

1

2

3

45

6

7

8

9

(Ehling-Schulz et al., Mol Nutr Food Res 2004)

Comparison of two new chromogenic media

CM 1

12

3

45

6

7

8

9

CM 2

12

3

45

6

7

8

9

B. cereus strains 1,5,8: isolates from emetic outbreak; 2: psychrotolerant strain; 3,4: clinical isolates; 6 isolate from diarrheal outbreak; 7: B. cereus type strain; 9: B. licheniformis

(Fricker, Reissbrodt, and Ehling-Schulz, IJFM 2008)

Distance matrix tree - plcR

Atypical strains:

■: PEMBA

▲: MYP

♦: CEI

*: non-hemolytic

(Fricker, Reissbrodt, and Ehling-Schulz, IJFM 2008)

B. cereus knock out mutants

B. cereus WT ∆plcR ∆spo0A

(MYP agar, 24h, 30°C)

(Luecking, Dommel, Scherer, Fouet, and Ehling-Schulz; Microbiology 2009)

Toxin gene profiling Realtime- PCR

Cytotoxicity assayces gene expression

O

NH

O

O

O

NH

O

O

12

34

56

78

9

1011

12

3

O

NH

O

O

O

NH

O

O

12

34

56

78

9

1011

12

3

Toolbox for emetic B. cereus

(© Ehling-Schulz)

Identification of emetic B. cereus

cesH cesP cesT cesA cesB cesC cesD

Genetic locus of cereulide synthetase(Ehling-Schulz et al., 2005, 2006)

Multiplex PCR for B. cereus group food poisoning toxin genes

M 1 2 3 4 5 6 7 8 9

ceshbl

nhe

cytK

M Marker1 F4810/72 (emetic reference

strain; isolated from vomit)2 NVH 0391-98 (cytK reference

strain)3 NVH 0075-95 (NHE-reference

strain)4 F837/76 (HBL-reference strain,

isolated from wound infection)5 WSBC10914 (emetic isolate

from baby food)6 WSBC10897 (isolate from butter

crème)7 WSBC10898 (isolate from milk

rice)8 WSBC10900 (isolate from fruits)9 M14 (isolate from blood culture)

(Ehling-Schulz et al., 2006)

Toxin gene profiling

33 %100 %100 %85 %67 %0 %0 %

non-emetic food strains

(n = 27)

70 %97 %97 %77 %77 %0 %0 %

diarrhoeal strains

(n = 30)

22 %100 %100 %0 %0 %0 %0 %

emetic like strains

(n = 9)

8 %100 %71 %0 %0 %100 %100 %emetic strains

(n = 24)

cytKnhetoxin pro-duction

hbltoxin pro-duction

cestoxin pro-duction

cytKNHEHBLCereulide

(Ehling-Schulz et al., ASM Press 2011)

Identification of emetic B. cereus II

cesH cesP cesT cesA cesB cesC cesD

Identification of the etiological agent in food borne outbreaks by real-time PCR

0

20

40

60

80

100

120

70 75 80 85 90degrees celsius

ricedishcauli-flower

B

100

120

0

20

40

60

80

100

120

70 75 80 85 90degrees celsius

flu

ore

scen

ce ´

F4810/72

WS2608duplex

A

(Fricker et al., AEM 2007)

pXen1[cesP-P1/luxABCDE]

11701 bp

amp

cat

cesP-P1

luxA

luxB

luxC

luxD

luxE

Lux Reportersystem -Monitoring of promoter activities

Ultra sensitive IVIS camera system

Vanilla -sauceRice

hig

h r

isk

Reconstituted infant formula

Curd cheese with herbs Whey drink with fruits Fruit yoghurt

low

ris

k

Soft cheese Latte Macchiato Chocolate milk

ris

k

Ces expression – extrinsic factors

(Dommel et al., AEM 2010)

Cereulide Toxin: Detection and quantification

Cytotoxicity test

Cell culture SIDA

m/z%

isotopologueanalyte

inte

nsity

LC-MS

Hep2 cells LC/ESI-TOF-MS

Development of a Stable Isotope Dilution Analysis (SIDA)

72.0 172.2

200.2

286.2

314.2

357.2

385.2

556.4

698.4

940.8

1082.8

1025.8

173.2

202.2

288.2

316.4

359.2

387.2

559.4

702.6

945.8

1089.0

1032.0

Comparison of product ion scan (MS2) of L-O-Val chain of unlabeled and labeled chain

Analysis of cereulide

(Bauer et al., 2010)

Comparison of valinomycin and 13C cereulide as internal standard

CEN (Comité Européen de Normalisation) Mandat

Consortium

� Coordination: NVWA , Netherlands

� Partners: Universities, European governmental and private laboratories, (FDA)

� Countries: Austria/Germany, Belgium, Finland, France, Netherlands, Spain, Switzerland, UK

Bacilli in food production…

� Bacilli show rather strains dependent characteristics than species specific ones

� Generally, high amount of thermophilic Bacillus sp.present in foods but spoilage potential of mesophilicspecies tends to be higher

� Toxinogenic potential of heat resistant Bacillus sp. outside the B. cereus group probably rather low

� Diagnostic Toolbox – paves the way for new diagnostic strategy, taking into consideration the pathogenic potential rather than taxonomy.

Tobias Bauer

Viktoria Doll

(Monika Dommel)

Elrike Frenzel

Martina Fricker

Tom Grunert

Rugaia Idris

Genia Lücking

Mathias Pauthner

Romy Renner

The serious B. cereus group:

Agnes FouetBirgitta Svensson

Marie Helene GuinebretièreFrederic Carlin

LGL

Ute Messelhäuser

This work was supported by funding from the European Commission (FP6/ FP7), theGerman Research Council (DFG), the Ministry of Economics and Technology, and by

funding from the Bavarian Ministry for Agriculture and Forestry.

Thomas Hoffmann Timo StarkSiegfried SchererRoger Vogelmann

Zeynap AtamarJörg Hinrichs

Rickard KnutssonJoakim Agren

Thanks’ to….