MMolecularolecular Identification Methods Identification Methods

for for CronobacterCronobacter spp.spp.

Brendan Healy, Angelika Lehner, Niall Mullane, Carmen Negredo, Shane Cooney,

Stephen O’Brien, Carol Iversen, Roger Stephan & Séamus Fanning,

Centres for Food Safety & Food-borne Zoonomics,

UCD Centre for Veterinary Sciences, University College Dublin &

Institute for Food Safety & Hygiene, VetSuisse Faculty, University of Zurich.

An International Conference on Cronobacter (Enterobacter sakazakii)O’Reilly Hall,

University College Dublin.22nd-23rd January, 2009

Species Type strain Isolated from Clinical source

Synonyms

E. aerogenes ATCC 13048 Yes Aerobacter aerogenesKlebsiella mobilis

E. amnigenus ATCC 33072 Yes

E. asburiae ATCC 35953 Yes CDC enteric group 17

E. cancerogenus ATCC 33241 Yes E. taylorae

CDC enteric group 19

E. cloacae ATCC 13047 Yes Aerobacter cloacae

E. cowanii JCM 10956 Yes Japan NIH group 42

E. gergoviae ATCC 33028 Yes E. cloacae (VP-)

E. hormaechei ATCC 49162 Yes CDC enteric group 75

E. intermedius ATCC 33110) Yes E. intermedium

E. kobei JCM 8580) Yes Japan NIH group 21

E. nimipressuralis ATCC 9912 No Erwinia nimipressuralis

E. pyrinus ATCC 29544 No Erwinia pyrinus

E. radicincitans D5/23T No

E. helveticus DSM 18396(T) No

E. turicensis DSM 18397(T) No

E. pulveris DSM 19144(T) No

- - - -

E. sakazakii ATCC 29544 Yes

- Enterobacter is found in the natural environment(soil, water) and may appear as commensals ofthe human and animal gut

- Enterobacter consists of 16 recognised species

- these organisms have been attracting attention because of links to nosocomial infections

- E. sakazakii is the only food-borne pathogen

- originally defined as a ‘yellow-pigmented’E. cloacae in 1980

- always known to be a heterogenousgroup of organisms at both the pheno- andgenotypic levels

- recent taxonomic revisions led to thecreation of a new genus Cronobacter

-reclassification is supported by AFLP, MLST and OM

E791

C4? E sak C2a?

E797, E680

E sak C3

E sak C2

E sak C4

E sak C1

unidentified C5

z508, z610

E. cloacae

Esch. hermanii

E. hormaechei

E. pyrinus

C. koseri

unidentified C6

Are all E. sakazakii the same?

[Iversen, et al (2004) J. Clin. Microbiol. 42: 5368-5370]

[Iversen, et al (2007) BMC Evol. Biol. 7: 64]

[Iversen et al (2008) Int. J. Syst. Evol. Microbiol. 58: 1442-1447]

Cronobacter species Sources in which detected

C. sakazakii clinical, environmental & food

C. dublinensis clinical, environmental & food

C. malonaticus clinical, environmental & food

C. muytjensii clinical, environmental & food

C. turicensis clinical, environmental & food

C. genomospecies I environmental & food

Other related Enterobacter

E. pulveris dried food, factory

E. helveticus dried food, factory

E. turicensis dried food

Clinical; blood, bone marrow, faeces, spinal fluid, sputum

Environmental; manufacturing facility, water

Food; dried food, baby food, milk powder, whey powder

Food sample

Identification

Phenotype Genotype

Epidemiology(trace-back)

Control

Surveillance/Environmental Monitoring- PFGE

- RAPD- Ribotyping- rep-PCR- MLVA

Detection/Identification- ribosomal DNA- Target gene detection

Chemical analysis

- Antibiogram

- Sero-/Biotyping

Isolate submission

Culture

DNA Isolation

Culture Library

DNA Library

Diagnostic strategies for Diagnostic strategies for CronobacterCronobacterGenotype Phenotype

Plasmid profiles

Identifying the genus Cronobacter & species- molecular approaches

Genus loci Gene targets

ribosomal DNA (rDNA) - 16S rRNA

- 23S rRNA

- tRNAGlu

- FISH

1,6 α-glucosidase gluA

MMS operon dnaG

Zinc-containing metalloprotease zpx

Outer membrane protein ompA

Species loci Gene targets

rfb (O-antigen) wehC [O:1] & wehI [O:2]

β-subunit of RNA polymerase rpoB

Other gene targets

RNaseP

infB (initiation factor)

Detection of Cronobacter by PNA-FISH

� Fluorescence in situ hybridisation (FISH) facilitates the detection of an

organism within a sample using a fluorescent [F]-labelled probe

� Peptide Nucleic Acid (PNA) probes have been developed and applied

to detect several microorganisms• PNA antisense agents act as DNA mimic synthetic polymers

• neutral polyamide [N-(2-aminoethyl)-glycine] backbone with

nucleic acid bases

• normal base-pairing rules apply

• resistant to nuclease/protease degradation

� PNA probe design based on 16S rDNA sequences

• identify conserved sequences among Cronobacter

• evaluate using extensive BLAST searches

• probe synthesis and labelling

� FISH works via specific hybridisation to rRNA targets which then emit

a fluorescent signal

[Almeida, et al (2009) Appl. Environ. Microbiol. In press.]

Fixation step

Paraformaldehyde and Ethanol

Hybridization step

Hybridization solution containing the labelled probe

Washing step

Pre-warmed washing solution

Microscopic visualization

Epifluorescence microscope equipped with suitable filter

Total time required: 2 to 3 h

PNA-FISH protocol

Microorganism

(nº of strains tested)

PNA-FISH

reaction

Cronobacter sakazakii (36)

including ATCC 29544T and 274*

+

Cronobacter dublinensis

subspecies lactaridi (2)

subspecies lausannensis

subspecies dublinensis

+

Cronobacter muytjensii (3)

including ATCC 51329 T

+

Cronobacer malonaticus (6) +

Cronobacter genomospecies 1 +

Cronobacter turicensis +

Enterobacter helveticus (8) -

Enterobacter pulveris (6) -

Enterobacter asburiae (2) -

Enterobacter hormaechei (2) -

Enterobacter turicensis (2) -

Enterobacter cloacae -

Enterobacter aerogenes ATCC 12048 T -

Enterobacter amnigenus ATCC 33072 T -

Microorganism

(nº of strains tested)

PNA-FISH

reaction

Shigella flexineri ATCC 12022 T -

Staphylococcus aureus ATCC 12600 T;

ATCC 6538 T; ATCC 13565 T

-

Staphylococcus epidermidis ATCC

35983 T; ATCC 35984T; ATCC 1798 T

ATCC 14990 T

-

Escherichia coli ATCC 25922; N5*;

N9*

-

Salmonella enterica

serotype Enteritidis ATCC 13076 T;

349*; 355*; 357*; A1*; CC*; Sal4*;

Sal6*

serotype Heidelberg 354* serotype

Typhimurium NCTC 12416 T

-

Pseudomonas fluorescens ATCC 13525T; N3*

-

Pseudomonas aeruginosa ATCC 10145 T -

Serratia plymuthica F4* -

Listeria monocytogenes 747*; 925*;

930*; 994*; 1562*

-

Bacillus cereus -

PNA-probe specificity

Detection in Pure Culture

Detection in Powdered Infant Formula after 8 h enrichment – Hybridization on glass slide

Detection in Mixed culture in Powdered Infant Formula Cronobacter population in lower numbers

C - C. sakazakii, S. Enteritidis (100-fold concentrated) and

P. aeruginosa (100-fold concentrated)

D - C. sakazakii and S. Enteritidis (100-fold concentrated)

• PNA-FISH is an alternative to existing gel-based/real-time molecular methods because:

- Presents high specificity and sensitivity-

- Detects less than 1 CFU per 10 g of Cronobacter in infant formula

in less than 12 h:

8 h for the enrichment step

2 to 3 h for the PNA-FISH procedure

- Allows direct cell visualization

- Less technically demanding

• This method can be successfully applied to different samples (such as blood and water), and support material (slides and filtration membranes)

• Samples can be analysed by flow cytometry avoiding the need of an epifluorescence microscope.

Sensitivity (%) Specificity (%)

Experimental 100 100

Theoretical 100 90

Conclusions –PNA FISH

Denaturation

Annealing

Extension

Cycle-1

Po

lym

era

se

Ch

ain

Re

ac

tion

(PC

R)

Cronobacter genus -specific

α-glucosidase-based PCR

Cronobacter species –specific

rpoB-based PCR

M 1 2 3 N M

M 1 2 3 4 5 6 7 N M

C.

mu

ytj

en

sii

C.

sakazakii

E. clo

acae

C. tu

ricen

sis

E.

pu

lveri

s

E.

helv

eti

cu

s

C.

du

blin

en

sis

C.

mu

ytj

en

sii

C.

sakazakii

C. tu

ricen

sis

[Lehner, et al (2006) BMC Microbiol. 6: 15]

[Stoop (2009) Unpublished]

- α-Glucosidase (α-Glu) an important biochemical feature used to differentiate between Cronobacter and other Enterobacteriaceae

- some Enterobacteriaceae can give +-ve reactions on culture media

- molecular detection provides a more specific diagnostic protocol

- following the characterisation of a BAC clone demonstrating α−Glu activity in aheterologous background a Cronobacter-genus specific PCR was developed

‘real-time’ Polymerase Chain Reaction (PCR)

rpsU dnaG rpoD

ES 205ES NCTC 11467ES 305ES 76ES 08155

E. cloacae

E. aerogenes

K. pneumoniaeC. freundii

E. asburiae 135

No template control

Cycle number

Flu

ore

sce

nce

(a

rbit

rary

)

[Seo and Brackett (2005) J. Food Protect. 68: 59-63]

[Drudy, et al (2006) Int. J. Food Microbiol. 110: 127-134]

MMS operon

Summary of the Summary of the gluAgluA & & dnaGdnaG molecular detection molecular detection

methods for methods for CronobacterCronobacter

[Iversen, et al (2007) J. Clin. Microbiol. 45: 3814-3816]

- 312 Enterobacteriaceae tested

- 210 Cronobacter

- all were positive for gluA

- all positive for dnaG

- in a strain collection of approx. 800 Cronobacter, all are positive

for dnaG

- gluA & dnaG are the only two molecular targets that have been fully validated

Molecular Molecular serotypingserotypingLip

id A

Inner

Core

O-s

pecific

chain

rfb

-gene locus

Oute

r C

ore

� Bacterial typing can be performed by serotyping

� Serotyping relies on a series of agglutination tests with antibodies

directed against the O-antigens, the surface polysaccharide

� Genes encoding the O-specific antigen are clustered between the galF

and gnd genes, flanking the rfb-locus

� Identifying and characterisation of the rfb-locus permits the

development of serotype-specific detection assays based on PCR

� Molecular serotyping can be used both for detection and typing

purposes

� rfb region encodes genes required for O-antigen synthesis

� Highly variable region in other Enterobacteriaceae

� Comparative genome analysis located this region in E.

sakazakii BAA-894

Genetic characterisation of the Genetic characterisation of the

OO--antigen locusantigen locus

gnd, 6-phosphgluconate dhse

galF, UDP transferase

NC

TC

8

155

NC

TC

11467

DE

S-9

0 [

en

v]

[3305173, in

dole

/

dulc

itol/ m

alo

nate

-

neg]

DE

S-3

7 [

en

v]

[3305173, in

dole

/

dulc

itol/ m

alo

nate

-

neg]

M M15-kbp

1517

1200

1000900800700600

517 / 500

400

3000

2000

1500

1250

1000

750

500

250

M1 M1 M21 2 3 4 5 6 7 128 9 10 11

300

MboIIMboII RFLP analysis of complete RFLP analysis of complete rfbrfb locus locus

[Mullane, et al (2008) Appl. Environ. Microbiol. 74: 3783-3794]

rfb

1

00

9

0

8

0

7

0

6

0

rfb

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

.

turicensis

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

malonaticus

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

genomospecies 1

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

muytjensii

sakazakii

sakazakii

sakazakii

sakazakii

sakazakii

malonaticus

sakazakii

malonaticus

malonaticus

dublinensis

E866

102

E899

343

80

E901

E775

228N

109

E787

E892

NCTC 8155

CFS10

79

CFS176

71

CFS136

E844

E837

ATCC 12868

E604

E900

CFS149

E846

NCTC 9238

CFS175

E830

NCTC 29004

NCTC 9529

E824

CFS06

NCTC 11467

44

90

93

CFS104

CFS131

CFS122

E891

E827

E770

E784

CFS1001

CFS153

CFS164

305N

ATCC BAA894

ATCC BAA893

88

E632

E828

ATCC 51329

CFS173

82

E826

366

344

CFS129

E839

E829

E825

CFS237

Species Strain

Serotype O:2

Serotype O:1

*

*

*

*

*

*

**

..sakazakii

a) O:1 antigen locus

1000 30002000 50004000 6000 7000 8000 9000 10000 11000 12000

galF rmlB

rmlA fdtB

wehBfdtC

fdtA

wehA wzx gndwehD

wehC

wzy

1000 30002000 50004000 6000 7000 8000 9000 10000 11000 12000

galF rmlB

rmlD

wehF

rmlCrmlA wzx

wehE gnd

wehHwehGwzy

wehI

b) O:2 antigen locus

M M M MM 161011127654321 1514138 9 23222120191817

O:1 serotype-specific PCR

O:2 serotype-specific PCR

341 bp

329 bp

Serotype specific PCR to detect Serotype specific PCR to detect CronobacterCronobacter sakazakiisakazakii O:1 & O:2O:1 & O:2

wehC

wehI

Future molecular diagnostic Future molecular diagnostic

strategies for strategies for CronobacterCronobacter

Cronobacter

turicensissakazakii dublinensis

- dublinensis

- lactaridi- lausannensis

malonaticus muytijensii

Genomospecies 1

rRNA

dnaG

gluA

rpoB

recN

Others

Enrichment

Nucleic acid

purification

Detection- FISH- PCR- OligoArray

Brendan Healy

Shane Cooney

Carol Iversen

Teresa Quinn

Niall Mullane

Patrick Wall

Carmen Negredo

Stephen O’Brien

Acknowledgements

Angelika Lehner

Roger Stephan

Carina Almeida

Maria Vivera

Catherine Molloy

Geraldine Duffy

Benedict Arku

Kieran Jordan

Ed Fox

Ben Tall

Mahendra Kothary

Barbara McCardell

Han Joosten