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(Unpublished results from an ongoing project funded by the German Ministry of Economics and Technology)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