Biochemical and molecular characterization of Cronobacter spp . (formerly Enterobacter sakazakii ) isolated from foods

ORIGINAL PAPER

Biochemical and molecular characterization of Cronobacterspp. (formerly Enterobacter sakazakii) isolated from foods

Imrich Turcovsky • Kristına Kunikova •

Hana Drahovska • Eva Kaclıkova

Received: 1 March 2010 / Accepted: 6 July 2010 / Published online: 17 July 2010

� Springer Science+Business Media B.V. 2010

Abstract The aim of this study was to identify and

characterize Cronobacter spp. isolated from a range

of foods. A total of 71 Cronobacter strains were

isolated from 602 foods in our laboratory. The

highest contamination was observed in foods of plant

origin, e.g. spices, teas, chocolate, nuts, pastries and

vegetables. On the basis of genus and species

identification performed using genus-specific PCR,

16S rRNA sequencing and AFLP genotyping, most

of the strains belonged to Cronobacter sakazakii.

Biochemical profiling by the tests included in

API 20E, complemented with relevant additional

tests, classified the strains into 13 biogroups. AFLP

genotyping facilitated discrimination of six main

groups at the 70% similarity level and strain grouping

correlated clearly with species identification. Our

results indicate that molecular typing by AFLP may

be applied as a useful tool not only for direct

comparison of Cronobacter isolates, providing trace-

ability, but also for the reliable species classification.

Moreover, tracing of these bacteria in a wider variety

of foods should be important to enhance the knowl-

edge of their transmission.

Keywords Cronobacter � Characterization �Food isolates � Biotyping � AFLP genotyping

Introduction

Enterobacter sakazakii previously referred to as

‘yellow pigmented E. cloacae’, was defined as a new

species in 1980 (Farmer et al. 1980) and 15 biogroups

were described based on biochemical character-

ization. Members of this species were considered

relatively phenotypically and genotypically hetero-

geneous (Lehner et al. 2004), a 16th biogroup has

been reported and the existence of several genetic

groups has been demonstrated based on 16S rRNA

gene sequence analysis (Iversen et al. 2006). E. sak-

azakii strains were divided into separate groups

based on f-AFLP fingerprints, ribopatterns and full-

length 16S rRNA sequences. DNA–DNA hybridiza-

tion revealed several genomospecies (Iversen et al.

2007) and, subsequently, led to classification of these

bacteria into six species within the new Cronobacter

genus (Iversen et al. 2008a). Subsequently, the six

Cronobacter species were supported by multilocus

sequence analysis (Kuhnert et al. 2009; Baldwin et al.

2009) and a differentiation system based on PCR

targeting the rpoB gene was developed (Stoop et al.

I. Turcovsky � E. Kaclıkova (&)

Department of Microbiology and Molecular Biology,

Food Research Institute, PO Box 25,

82475 Bratislava, Slovakia

e-mail: [email protected]

K. Kunikova � H. Drahovska

Department of Molecular Biology, Faculty of Natural

Sciences, Comenius University, Bratislava, Slovakia

123

Antonie van Leeuwenhoek (2011) 99:257–269

DOI 10.1007/s10482-010-9484-7

2009). The whole genome sequence of the Cronob-

acter sakazakii ATCC BAA-894 has been recently

published and its genome was compared with repre-

sentatives of all Cronobacter species by microarray

analysis. It was shown that about 55% of genes were

common to C. sakazakii strains and 43% were

common to all Cronobacter strains (Kucerova et al.

2010).

Cronobacter spp. are considered to be opportunis-

tic pathogens, implicated in particularly severe food-

borne diseases in neonates and infants (Farber 2004;

Mullane et al. 2007; Townsend et al. 2008). Symp-

toms include bacteraemia, necrotizing enterocolitis

and meningitis, with fatality rates as high as 80%

(Lai 2001). Although these microorganisms are widely

distributed in the environment (Kandhai et al. 2004),

dried infant milk formula has been implicated as the

vehicle of transmission in many clinical manifesta-

tions (Nazarowec-White and Farber 1997; van Acker

et al. 2001; Gurtler et al. 2005). More recently,

infections in adults have been reported, in particular

among the elderly and immunocompromised patients

(Ray et al. 2007; See et al. 2007). More than 500

Cronobacter infections were reported in United

Kingdom during 1999–2007; 90% of them occurred

in persons older than 15 years (FAO/WHO report

2008). For persons with a decreased immunity, in

particular neonates, elderly and persons with severe

underlying diseases, the occurrence of Cronobacter

spp. in food and environment may represent a hazard

to health (Lai 2001; Friedemann 2007).

The primary reservoirs for subsequent food con-

tamination remain undefined due to the ubiquitous

nature of these bacteria. Cronobacter strains were

isolated from a wide range of foods including milk,

cheese, dried foods, meats, water, vegetables, rice,

tea, herbs and spices (Iversen and Forsythe 2004;

Edelson-Mammel et al. 2005; Friedemann 2007),

various food production environments and house-

holds (Kandhai et al. 2004; Gurtler et al. 2005). The

spectrum of Cronobacter-contaminated foods covers

both raw and processed foods and the kind of

processing is not restricted to dry products (Friede-

mann 2007).

Isolation and detection of Cronobacter spp. is

based on standardised microbiological procedures of

enrichment followed by the isolation of colonies on

chromogenic media. Confirmation of these colonies

is necessary to verify the identity of the isolates.

Commercial biochemical systems or PCR-based

assays are available for food and clinical microbio-

logical laboratories (Fanjat et al. 2007; Stoop et al.

2009; Druggan and Iversen 2009). Generally, pheno-

type analysis may provide unreliable identification as

long as it is based on unstable expression of the

markers (Drudy et al. 2006; Druggan and Iversen

2009).

DNA-based methods offer an alternative approach,

enabling a direct comparison of isolates in outbreaks.

Molecular methods may facilitate the trace back of

outbreak isolates from clinical samples to the source

of contamination and provide useful tools to target

control strategies to reduce the risk of transmission

(Drudy et al. 2006).

In this study, 98 strains belonging to the genus

Cronobacter, including 71 strains isolated in our

laboratory, were identified and characterized using

biochemical profiling and molecular techniques,

namely genus-specific real-time PCR, AFLP typing

and partial 16S rRNA sequencing. Since Cronobact-

er spp. are ubiquitous in many environments, a wide

range of foods was analysed.

Materials and methods

Isolation of Cronobacter spp. from food

Cronobacter strains were isolated from various food

samples analysed within the frame of a surveillance

programme for coliforms in the State Veterinary and

Food Institute, Bratislava, Slovakia in 2006–2009

(Table 1). Strains were isolated from 5 replicates of a

25 g test portion of each food sample according to the

standard method specified in ISO/TS 22964:2006

using the two-step enrichment consisting of pre-

enrichment (1/10) in Buffered Peptone Water (Merck,

Darmstadt, Germany) and selective enrichment

(1/100) at 45�C in modified Lauryl Sulfate Tryptose

(mLST) broth (Merck) with additional NaCl (Merck)

to the final concentration of 0.5 mol l-1 and vanco-

mycin 10 mg l-1 (Fluka, Buchs, Switzerland). Pre-

sumptive Cronobacter strains were isolated as typical

green–blue colonies grown on Chromogenic Entero-

bacter sakazakii agar, DFI formulation (Oxoid,

Basingstoke, England) or Chromocult Enterobac-

ter sakazakii (Merck) based on the expression of

a-glucosidase activity. Identification of Cronobacter

258 Antonie van Leeuwenhoek (2011) 99:257–269

123

spp. was confirmed by real-time PCR targeting the

dnaG gene according to Seo and Brackett (2005).

Our collection of strains was supplemented

with 22 strains from the American Type Culture

Collection (ATCC, Manassas, VA, USA), Belgian

Co-ordinated Collections of Microorganisms in Gent

(BCCM/LMG Bacteria Collection) and from Czech

Collection of Microorganisms (CCM, Brno, Czech

Republic). Five further strains isolated from fruit

powder, kindly provided by Professor Roger Stephan

from the Institute of Food Safety and Hygiene (ILS),

Vetsuisse Faculty University of Zurich, Switzerland,

were also added to the collection. All strains were

maintained in 20% glycerol solution at -18�C or

freeze-dried for long-term storage. Bacteria were

cultured in Luria–Bertani or Brain Heart broth, and

on Brain Heart agar (Merck) at 37�C. Strains of

Cronobacter spp. used in the study are listed in

Table 2.

Phenotypic characterization and biotyping

Production of the yellow pigment was tested on

Tryptone Soya Agar (Oxoid) supplemented with bile

salts (Fluka) with exposure to light during incubation.

In addition to the results derived from biochemical

reactions included in the biochemical test kit API 20E

(bioMerieux, Marcy l’Etoile, France), the following

tests were performed using conventional methods.

Tests for the acid production from carbohydrates were

examined in broth base (peptone 10 g l-1, yeast

extract 1 g l-1 and NaCl 5 g l-1) with phenol red

indicator (0.018 g l-1). Filter-sterilized dulcitol and

methyl-a-D-glucopyranoside (both from Fluka), at a

Table 1 Occurrence of Cronobacter spp. in analysed food categories

Origin Sample category Number of analysed

samples

Number of

positive samples

Species

Plant Spices 21 13 C. sakazakii, C. malonaticus,

C. muytjensii

Tea 5 3 C. sakazakii

Chocolate products 37 11 C. sakazakii, C. malonaticus

Pastries 9 5 C. sakazakii, C. malonaticus

Nuts 2 2 C. sakazakii

Cereals and derived products 20 0

Instant soups 13 2 C. malonaticus, C. dublinensis

Fruits and derived products 12 0

Vegetables 12 5 C. sakazakii, C. turicensis,

C. muytjensii

Altogether 131 41

Animal Raw meat 64 1 C. sakazakii

Raw meat (spiced or marinated) 48 17 C. sakazakii, C. malonaticus,

C. dublinensis, ni.

Meat semiproducts 29 0

Meat products (ready-to-eat) 81 9 C. sakazakii, C. malonaticus

Eggs and derived products 51 0

Milk powder and derived products 55 1 C. malonaticus

Milk and milk products 98 0

Cheese 32 0

Raw meat swabs 13 1 C. sakazakii

Altogether 471 29

Environmental Dust 2 1 C. sakazakii

Altogether 604 71

Antonie van Leeuwenhoek (2011) 99:257–269 259

123

Table 2 Biochemical and molecular identification of Cronobacter strains used in this study

No Designation Source API 20E % id. Cronobacter Biogroup AFLP Species

1 ATCC 29544a Human 3305373 98.40 1 A C. sakazakii

2 ATCC 12868a Human 3305373 98.40 3 A C. sakazakii

3 ATCC 29004 Unknown 3305373 98.40 3 A C. sakazakii

4 ATCC BAA 894a Infant formula 3305173 51.10 2 A C. sakazakii

5 LMG 2758 Human 3305173 51.10 2 A C. sakazakii

6 LMG 2759a Human 3305173 51.10 2 A C. sakazakii

7 LMG 2760 Brush 3305173 51.10 2 A C. sakazakii




11 LMG 2786a Human 3305373 98.40 1 A C. sakazakii

12 CCM 3460 Human 3305373 98.40 1 A C. sakazakii

13 CCM 3479 Human 3304373 98.40 13 A C. sakazakii

14 ILS 744/03a Fruit powder 3305373 98.40 1 A C. sakazakii

15 ILS 759/03 Fruit powder 3305373 98.40 1 A C. sakazakii



18 ILS 1155/04a Fruit powder 3305373 98.40 1 A C. sakazakii

19 121206/23a Ginger 3305373 98.40 1 A C. sakazakii

20 121206/24 Black pepper 3305373 98.40 1 A C. sakazakii


22 201206/17 Chocolate 3305173 51.10 7 A C. sakazakii

23 201206/18a Chocolate 3305173 51.10 3 A C. sakazakii

24 201206/19a Chocolate 3205373 98 4 A C. sakazakii

25 201206/22a Chocolate 3205373 98 4 A C. sakazakii

26 201206/23 Chocolate 3305373 98.40 3 A C. sakazakii

27 201206/25a Chocolate 3305173 51.10 2 A C. sakazakii

28 240107/09 Hazelnut 3305373 98.40 1 A C. sakazakii

29 210307/02 Pastries 3305173 51.10 2 A C. sakazakii



32 210307/19a Chicken liver 3205373 98 4 A C. sakazakii

33 040407/76 Sausage 3305373 98.40 1 A C. sakazakii

34 170407/02 Hot chocolate 3305373 98.40 8 A C. sakazakii

35 170407/05 Fruit tea 3205373 98 4 A C. sakazakii

36 170407/07 Herbal tea 1305373 82.10 1 A C. sakazakii

37 170407/10 Black tea 3305373 98.40 1 A C. sakazakii

38 240407/26a Marinated meat 3305373 98.40 1 A C. sakazakii

39 150507/78 Meat product 3305373 98.40 1 A C. sakazakii

40 120607/02 Nut chocolate 3307173 98.80 4 A C. sakazakii

41 120607/66 Nut chocolate 3307173 98.80 1 A C. sakazakii

42 270607/29 Chicken pate 3305173 51.10 2 A C. sakazakii

43 270607/30 Chicken pate 3305373 98.40 1 A C. sakazakii

44 270607/31 Meat pate 3305373 98.40 1 A C. sakazakii

45 100707/19 Peanuts 3305373 98.40 1 A C. sakazakii


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Table 2 continued


46 140807/15 Marinated meat 3305373 98.40 1 A C. sakazakii

47 210807/15 Meat pate 3305373 98.40 1 A C. sakazakii


49 210807/31 Red pepper 3305373 98.40 1 A C. sakazakii






55 161007/37 Meat product 3305373 98.40 1 A C. sakazakii

56 061107/07a Red pepper 1205363 0.00 4 A C. sakazakii



59 280108/V1 Dust 3305173 51.10 2 A C. sakazakii




63 120308/17 Flavoured meat 3305373 98.40 1 A C. sakazakii



66 290708/08 Dry vegetable 3305373 98.40 1 A C. sakazakii

67 290708/11 Dry vegetable 3305373 98.40 1 A C. sakazakii



70 130808/32 Flavoured meat 3305373 98.40 1 A C. sakazakii



73 100908/15 Pork swab 3305373 98.40 1 A C. sakazakii

74 LMG 23826a Human 3305173 51.10 9 B C. malonaticus

75 121206/29 Black pepper 3305373 98.40 5 B C. malonaticus

76 201206/21a Chocolate 3305373 98.40 5 B C. malonaticus

77 150507/66a Nut chocolate 3325373 98.40 5 B C. malonaticus

78 150507/70 Milk powder 3325373 98.40 5 B C. malonaticus

79 161007/29 Meat product 3305373 98.40 5 B C. malonaticus

80 161007/35a Meat product 3305373 98.40 5 B C. malonaticus

81 290108/11a Marinated meat 3305373 98.40 5 B C. malonaticus

82 120808/24a Pastries 3305373 98.40 5 B C. malonaticus

83 260808/02 Instant soup 3305373 98.40 5 B C. malonaticus

84 LMG 23827a Human 3305373 98.40 16 C C. turicensis

85 290708/07a Dry vegetable 3305373 98.40 16 C C. turicensis

86 260808/05 Dry vegetable 3305373 98.40 16 C C. turicensis

87 LMG 23823a Milk powder 3345373 99.90 12 D C. dublinensis

88 LMG 23824a Water 3345173 99.70 10 D C. dublinensis

89 LMG 23825a Milk powder 3345373 99.90 6 D C. dublinensis

90 CCM 3461a Human 3345173 99.70 10 D C. dublinensis


123

final concentration of 0.5% were used. Gas production

from glucose was determined using Durham tubes.

Malonate utilization was determined using Malonate

broth (ammonium sulphate 1 g l-1, dipotassium phos-

phate 0.6 g l-1, monopotassium phosphate 0.4 g l-1,

NaCl 2 g l-1, sodium malonate 3 g l-1 and bromoth-

ymol blue indicator 0.025 g l-1, pH 6.8). Motility at

36�C was determined in Motility medium (tryptose

10 g l-1, NaCl 5 g l-1, agar 5 g l-1) with 0.05 g l-1

triphenyltetrazoliumchloride allowing the pink col-

our-visualized diffusion of motile cells from stab

inoculation. Nitrate reduction was determined using

Nitrate Reduction Kit (Fluka) and the addition of zinc

dust to confirm the presence of unreduced nitrate in

tubes with negative results. Methyl-red test was

performed by addition of indicator (0.1 g in 300 ml

of 95% ethanol) to 48-h cultures in 5 ml of Glucose

phosphate broth (peptone 10 g l-1, dipotassium phos-

phate 5 g l-1 and glucose 5 g l-1) in tubes.

Sequencing of 16S rRNA gene

The chromosomal DNA was isolated using the

DNeasy Blood and Tissue kit (Qiagen, Hilden,

Germany) according to manufacturers instructions.

A fragment of the 16S rRNA gene was ampli-

fied with bacterial universal primers 27f/685r (Lane

1991). DNA sequencing was performed using the

automatic DNA analyzer ABI Prism 3130 Avant

(Applied Biosystems, Foster City, CA, USA) using

Big Dye Terminator 3.1 kit (Applied Biosystems).

Final sequences (one representative for each unique

sequence) were deposited into the EMBL database

under accession numbers FN539026–FN539035.

Amplified fragment length polymorphism

AFLP was performed according to the procedure

described previously (Pangallo et al. 2008). 200 ng of

the genomic DNA was digested with 5 U of EcoRI

and 2 U of MseI in a 10 ll reaction mixture

containing 19 T4 DNA ligase buffer (New England

Biolabs, Ipswich, USA), 0.05 mol l-1 NaCl and

0.1 mg ml-1 bovine serum albumin at 37�C for 1 h.

Then, 5 ll of the fresh ligation mixture containing

50 pmol each of the EcoRI adapter (ctcgtagactgcg-

tacc/aattggtacgcagtctac) and MseI adapter (gacgat-

gagtcctgag/tactcaggactcat), and 1 U of T4 DNA

ligase were added and the incubation continued for

3 h. Subsequently, the mixture was diluted 20 times

with TE buffer (10 mmol l-1 Tris HCl, 0.1 mmol l-1

EDTA, pH 8) and stored at -20�C. PCR was per-

formed in a 20 ll volume, which contained 10 pmol

of EcoA primer (gactgcgtaccaattca, FAM-labelled),

10 pmol of MseC primer (gatgagtcctgagtaac),

200 lmol l-1 of each dNTP (Promega, Madison,

USA), 1 U Taq DNA polymerase (New England

Biolabs), 19 PCR buffer and 2 ll template DNA.

The thermocycler program consisted of 2 min at

72�C, 10 cycles (30 s at 94�C, 30 s at 65�C, 90 s at

72�C, the annealing temperature decreased by 1�C

with each cycle), 25 cycles (30 s at 94�C, 30 s at

56�C, 90 s at 72�C), and a final polymerization for

30 min at 60�C. PCR products were separated in the

automatic genetic analyzer ABI Prism 3130 with

LIZ-500 size standard (Applied Biosystems). The

AFLP profiles were analysed by the BioNumerics

Software (Applied Maths, Belgium). The levels of

similarity between AFLP fingerprints were calculated

Table 2 continued


91 140807/04 Marinated meat 3345373 99.90 6 D C. dublinensis

92 260808/01a Instant soup 3345173 99.70 10 D C. dublinensis

93 040407/32a Spiced meat 3305373 98.40 1 E Ni

94 ATCC 51329a Unknown 3345373 99.90 15 F C. muytjensii

95 LMG 2787 Human 3345373 99.90 15 F C. muytjensii

96 LMG 2788a Human 3345373 99.90 15 F C. muytjensii

97 051206/07 Caraway 3345373 99.90 15 F C. muytjensii

98 260808/07a Dry vegetable 3247373 99.40 15 nt C. muytjensii

a Strains were analyzed by 16S rRNA gene sequencing; ni not identified; nt untypable


123

on the basis of Dice’s coefficient (at a position

tolerance of 0.2% and optimization at 0) and cluster

analysis was done by UPGMA.

Results

Isolation, identification and phenotypic

characterization of Cronobacter strains

Seventy-one Cronobacter strains were isolated and

identified from 602 food and 2 environmental sam-

ples sorted for this study to 19 sample categories

(Table 1) by processing of five test portions from

each sample. Among the analysed samples, Cronob-

acter strains were isolated from 41 of 131 (31.3%)

samples of plant origin and from 29 of 471 (6.2%)

samples of animal origin; one strain was isolated

from dust.

All 71 putative Cronobacter strains which grew as

green–blue colonies on the chromogenic medium

were positively identified as Cronobacter spp. by the

genus-specific real-time PCR. All but one were

determined to be ‘‘E. sakazakii’’ using API 20E. All

strains produced a yellow pigment.

The panel of biochemical tests contained in the

API 20E gave 10 different profiles resulting in per-

centage identification (% id.) values from 51.1% for

19 inositol-negative C. sakazakii and Cronobact-

er malonaticus to 99.9% for most Cronobacter

muytjensii and some Cronobacter dublinensis isolates.

One strain (061107/07) was misidentified as Esche-

richia vulneris at 61.5% id and a 0.0% id. value for

‘‘E. sakazakii’’. However, this isolate was positive in

the genus-specific real time PCR and clustered with

C. sakazakii by AFLP (Fig. 1). All 22 Cronobacter

spp. culture collection strains and five strains from

ILS grew as green–blue colonies on the chromogenic

medium, produced the yellow pigment on Tryptone

soya agar with bile salts and were positive in the real-

time PCR confirmation. By API 20E, these strains

were identified as ‘‘E. sakazakii’’ with 51.1–99.9% id.

Additional biochemical tests such as acid production

from dulcitol and methyl-a-D-glucopyranoside, mal-

onate utilization, gas production from glucose,

methyl red and motility tests facilitated classification

of the strains into 13 different biogroups according to

Farmer et al. (1980) and Iversen et al. (2006)

(Table 2).

AFLP subtyping

The clonality of 71 Cronobacter spp. food isolates, 22

collection strains and 5 identified isolates was deter-

mined by AFLP. By repeated analysis of the strain

C. sakazakii ATCC 12868 (CCM 1902), 95–100%

similarity of Dice’s coefficient was obtained for

all runs. AFLP-profile similarity of different strains

reached values from 60 to 100%. Strains were

grouped into 46 clusters at a similarity level of 90%.

Six main groups (designated A–F) were clearly

distinguished at the 70% similarity level; these strain

grouping being in concordance with their species

identification and biochemical properties (Fig. 1).

From the total number of 98 Cronobacter strains,

the largest AFLP cluster A contained 73 C. sakazakii

strains including the type strain ATCC 29544 (LMG

5740). Other Cronobacter species were separated into

the remaining AFLP clusters: ten strains of C. malo-

naticus in cluster B, three Cronobacter turicensis

strains in cluster C, six C. dublinensis strains in

cluster D and four C. muytjensii strains in cluster F.

One strain (040407/32) had a low similarity to all

other Cronobacter strains and was separated to its

own cluster E, while API 20E identified this strain as

‘E. sakazakii’ with 98.4% id. value corresponding to

C. sakazakii type strains profile (3305373) and bio-

type 1 using additional biochemical tests. The strain

C. muytjensii 260808/07 was untypable by AFLP.

Partial sequencing of 16S rRNA gene

For confirmation of species identification, a fragment

of 657 bp from the beginning of the 16S rRNA gene

was sequenced from 27 Cronobacter strains. Eight

additional sequences of collection strains were

downloaded from the NCBI database (Fig. 2). The

similarity of all Cronobacter spp. strains reached

95.5% and the sequences were grouped according to

the species: C. dublinensis (5 strains, 100% similar-

ity), C. turicensis (2 strains, 99.9% similarity),

C. muytjensii (3 strains, 99.7% similarity). C. sak-

azakii (18 strains) and C. malonaticus (6 strains)

could not be distinguished by partial 16S rRNA

sequencing and formed one common cluster with

98.3% similarity. Cronobacter sp. 040407/32, which

had a unique profile in AFLP, formed an independent

branch also in the 16S rRNA sequence analysis being

adjacent to C. muytjensii sequences.


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Discussion

Cronobacter spp. (formerly E. sakazakii) have

emerged as a rare cause of life-threatening neonatal

infections. The microorganism has been detected in

powdered infant milk formula products and improper

handling during its rehydration has been implicated

in several clinical cases. Considering the indication

that Cronobacter spp. may cause infection in both

infants and adults, evaluation of a wide variety of

foods may be important to reveal the possible routes

for its transmission.

In our study, 71 strains of Cronobacter genus

isolated from a variety of foods in our laboratory and

5 other isolates were identified and compared with 22

strains from collections. All strains were analysed

using biochemical profiling and molecular tech-

niques, namely genus-specific real-time PCR, AFLP

typing and, for selected strains, sequencing of

16S rRNA.

During food screening, more than 600 food

products other than infant formula showed on average

12% positivity. However, this value may be an

underestimate, as the method specified in ISO/TS

22964 was used for isolation as the best alternative

available in 2006, when the study has started. This

method is now known to miss some strains unable to

grow in mLST media and/or at 45�C (Iversen and

Forsythe 2007; Druggan and Iversen 2009). Advanced

procedures have been recently recommended for

effective isolation of Cronobacter spp., e.g. combi-

nation of Cronobacter screening broth (CSB) and

chromogenic agar (Iversen et al. 2008b), and alter-

natively Cronobacter enrichment broth (CEB) may

be effectively used in conjunction with selective-

differential agar (O’Brien et al. 2009a).

Cronobacter spp. occurrence in samples was

unevenly distributed in different food types, the

highest contamination being observed in foods of

plant origin, e.g. spices, teas, chocolate, nuts, pastries

and vegetables. It is also interesting that the majority

of positive samples of animal origin contained plant

ingredients, namely herbs and spices. This corre-

sponds to the observations of other authors:

Cronobacter contamination was particularly detected

in fresh and dried vegetables and spices (Iversen and

Forsythe 2004; Baumgartner et al. 2009; Jaradat et al.

2009) and in cereal-based infant drinks and foods

(O’Brien et al. 2009b; Chap et al. 2009). The ability

of Cronobacter strains to associate with plants was

recently experimentally confirmed for environmental

as well as clinical strains (Schmid et al. 2009). Their

presence in dried foods (spices, chocolate, pastries)

may be due to the increased desiccation resistance of

Cronobacter strains (Breeuwer et al. 2003; Edelson-

Mammel et al. 2005; Osaili and Forsythe 2009).

Real-time PCR targeting the dnaG gene of the

MMS operon according to Seo and Brackett (2005) is

an extensively utilized Cronobacter spp. identifica-

tion method (Druggan and Iversen 2009). In our study

it was used as a principal identification test for

Cronobacter genus confirmation and it 100% agreed

with AFLP and 16S rRNA sequencing classification.

API 20E biochemical identification system (bio-

Merieux) was employed as a simple commercially

available tool. It proved to be highly accurate for

Cronobacter identification at the genus level as all

but one isolate were correctly identified (99% relative

sensitivity based upon the analysis of 98 Cronobacter

strains). Most of the analysed Cronobacter strains

(80%) fell within the range from 98.0 to 99.9% id.

values. At the same time, the id. value of 98.4% for

C. sakazakii type strain ATCC 29544 was lower than

for C. muytjensii strains with id. values of 99.9%.

Surprisingly, the lowest % id. value of 51.1%, which

is considered a doubtful result by the API 20E

software, was associated with the group of 19

inositol-negative C. sakazakii and C. malonaticus

strains, as negative inositol fermentation is a typical

attribute of about 25% of Cronobacter strains

(Farmer et al. 1980). Inability to correctly identify

some Cronobacter strains by the API20E was previ-

ously reported (Iversen et al. 2004). For this reason,

additional tests such as production of acid from

dulcitol and methyl-a-D-glucopyranoside, as well as

utilization of malonate according to Iversen et al.

(2007) were necessary for reliable genus level

classification. Further additional tests such as motility

test, gas production and methyl red tests allowed the

identification of Cronobacter spp. (E. sakazakii)

biogroups (Farmer et al. 1980; Iversen et al. 2006).

It was interesting that apart from all the C. malo-

naticus isolates, a relatively high proportion of

Fig. 1 Dendrogram of AFLP profiles of Cronobacter strains.

AFLP profiles of 97 Cronobacter strains with species

separation at the 70% similarity level are shown in the

dendrogram. E. cloacae CCM 1903 is used as an outgroup

b


123

malonate-positive C. sakazakii strains (10%) was

also present in our collection, five of seven such

strains being very similar in the AFLP analysis.

Clonal relationships between strains were analysed

by AFLP. This method is well established for

population studies of bacteria including Cronobacter

C. sakazakii 091007/14







C. sakazakii ATCC 12868

C. malonaticus 161007/35





C. sakazakii ATCC BAA 894

C. sakazakii LMG 2759



C. malonaticus LMG 23826

C. sakazakii ILS 1155/04



C. sakazakii ILS 744/03

C. sakazakii LMG 2786

C. sakazakii ATCC 29544

C. turicensis LMG 23827

C. turicensis 290708/07

C. dublinensis LMG 23823

C. dublinensis 260808/01

C. dublinensis CCM 3461



E. cloacae CCM 1903

E. cloacae ATCC 13047

C. sp. 040407/32

C. muytjensii LMG 2788

C. muytjensii ATCC 51329

C. muytjensii 260808/07

E. coli ATCC 11775

99

75

100

99

100

83

75

0.0000.0050.0100.0150.020


123

spp. (Iversen et al. 2007). In our study, a high

discriminatory power of AFLP was confirmed, as the

98 strains were separated into 46 different clusters at

a similarity level of 90%. With a few exceptions,

different strains simultaneously isolated from differ-

ent batches of the same food did not share any genetic

similarity, which may reflect the fact that Cronob-

acter spp. contamination occurs at various occasions

from different sources. At a similarity level of 70%,

six main groups were clearly distinguished in the

AFLP dendrogram, and strain grouping was in

concordance with their species identification as

confirmed by biogroups and 16S rRNA sequencing.

With a few exceptions, strains that were highly

similar according to the AFLP analysis had the same

biogroup assignation. Despite using different restric-

tion endonucleases in their AFLP protocol, Iversen

et al. (2007) observed similar species specific clus-

tering of the AFLP profiles of Cronobacter strains.

The AFLP-based similarity between strains belong-

ing to the same species reached 73–83%, the most

heterogeneous being three strains of C. turicensis. In

C. dublinensis, no clear delineation between subspe-

cies dublinensis, lactaridi and lausannensis was

observed, which might have been due to the low

number of analysed strains. Our results indicate that

AFLP typing may be applied as a useful tool not only

for direct comparison of Cronobacter isolates pro-

viding the traceability, but also for the reliable

species identification and classification. Partial

sequencing of 16S rRNA gene was further used for

confirmation of species identification and in all cases

we observed strain clustering identical with main

species delineating AFLP groups. The only excep-

tions were C. sakazakii and C. malonaticus, which

belonged to one common 16S rRNA cluster.

However, due to low variability in the sequenced

region and due to some polymorphism between 16S

rRNA copies in the same strain (e.g. 1–6 nucleotide

variants are present between six 16S rRNA genes in

the genome of C. sakazakii ATTC BAA 894,

Kucerova et al. 2010), the relationship between these

two species could not be properly distinguished from

the dendrogram (many branches are supported with

low bootstrap values).

Among Cronobacter spp. food isolates, strains of

C. sakazakii dominated with 79% occurrence. This

species was followed by C. malonaticus (12% iso-

lates), while representatives of other species were

isolated only rarely. Similar results were published

previously, e.g., preferential isolation of C. sakazakii

from dairy products was observed by El-Sharoud

et al. (2009). This confirms the predominance of this

species among food isolates (Iversen et al. 2007;

Chap et al. 2009).

One strain (040407/32) isolated from spiced meat

possessed a unique AFLP profile and differed from

all other Cronobacter spp. also in 16S rRNA gene

sequence. Detailed taxonomic classification of this

strain will be the subject of our further study.

In conclusion, we can conclude that Cronobacter

spp. are frequently present in various foods, in

particular fresh and dry foods of plant origin.

Although it is harmless for most of the population,

it can pose risks for immunocompromised consumers.

Tracing of these bacteria in a wide variety of foods is

important to reveal the possible routes for its

transmission.

Acknowledgments This work was supported by Slovak

Research and Development Agency under the contract no.

APVV-27-009705 and by Slovak Ministry of Education under

the contract no. VEGA 1/0344/10.

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