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Identification of "Cronobacter" spp. (Enterobacter sakazaki)

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DOI: 10.1128/JCM.01026-07 · Source: PubMed

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JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2007, p. 3814–3816 Vol. 45, No. 11
0095-1137/07/$08.00⫹0 doi:10.1128/JCM.01026-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of “Cronobacter” spp. (Enterobacter sakazakii)䌤

Carol Iversen,1,2 Angelika Lehner,1 Niall Mullane,3 John Marugg,2 Séamus Fanning,3
Roger Stephan,1* and Han Joosten2
Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zurich, CH-8057, Zurich, Switzerland1; Quality and
Safety Department, Nestlé Research Centre, Vers-chez-les-Blanc, CH-1000 Lausanne, Switzerland2; and Centre for
Food Safety, UCD Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland3
Received 17 May 2007/Returned for modification 29 August 2007/Accepted 6 September 2007

A taxonomic reclassification of the neonatal pathogen Enterobacter sakazakii to consist of five species within
a new genus, “Cronobacter,” has recently been proposed. The correct identification of these organisms is
important to clinicians. Therefore, using 312 Enterobacteriaceae, including 210 “Cronobacter” strains, the
reliabilities of biochemical and genetic confirmation tests were investigated. All “Cronobacter” isolates were
positive using dnaG and gluA PCR protocols, and all expressed ␣-glucosidase activity. ID32E v3.0 identified
99.5% of “Cronobacter” isolates (as the nearest match to E. sakazakii).

Updating the original taxonomy of Enterobacter sakazakii of 5-bromo-4-chloro-3-indolyl-␣-D-glucopyranoside (X-␣-Glc;

has resulted in the clear definition of at least five new species
based on extensive geno- and phenotypic evaluations (7).
These pathogenic species are etiological agents in rare cases of
meningitis, necrotizing enterocolitis, and bacteremia in neo-
nates (11, 13, 15, 21). In order to facilitate their continued
inclusion in schemata for the diagnosis of infection and the
microbiological monitoring of food products, it has been
proposed that these species be moved to a novel genus,
“Cronobacter” (7). Current culture-based isolation methods for
these species include a test for ␣-glucosidase activity (5, 6, 12,
14, 16, 17). However, the reliability of this test has been ques-
tioned (9, 17). The observation of yellow pigment for presump-
tive identification and the efficacy of biochemical test kits have
also been placed in doubt (1, 4, 8). Identification results ob-
tained using the ID32E biochemical gallery (bioMeriéux), with
updated software, v3.0, have recently been reported using a
limited number of “Cronobacter sakazakii” isolates (3).
In this study 102 Enterobacteriaceae from 27 species in nine
genera, as well as 210 “Cronobacter” strains covering the five
species (7), were obtained from food, environmental, and clin-
ical sources. The reliabilities of PCR amplification assays tar-
geting the dnaG (18) and gluA (10) genes, biochemical galler-
ies (API20E and ID32E), ␣-glucosidase assays, and yellow
pigmentation and the diagnostic value of chromogenic isola-
tion media were investigated.
Phenotypic tests. Acid production from methyl-␣-D-gluco-
pyranoside (AMG) was tested in phenol red broth containing
0.5% filter-sterilized carbohydrate solution. Use of 4-methyl-
umbelliferyl-␣-D-glucopyranoside (4-MU-␣-Glc; 44051; Glyco-
synth, United Kingdom) was evaluated by observing fluores-
cence under UV light of cultures previously cultured for 24 h
in buffered peptone-water containing 0.1 g liter⫺1 4-MU-␣-Glc
and of individual colonies grown for 24 h on tryptic soy agar
containing 0.1 g liter⫺1 4-MU-␣-Glc. Constitutive metabolism
* Corresponding author. Mailing address: Institute for Food Safety
and Hygiene, University of Zurich, Winterthurerstrasse 272, CH-8057
Zurich, Switzerland. Phone: 41 44 635 86 51. Fax: 41 44 635 89 80.
E-mail: stephanr@fsafety.uzh.ch.
䌤
Published ahead of print on 19 September 2007.
70051; Glycosynth, United Kingdom) was determined by ob-
serving the formation of an indigo pigment on nonselective
medium containing peptone (7 g), yeast extract (3 g), NaCl (5
g), agar (15 g), X-␣-Glc (0.15 g), and H2O (1,000 ml), incu-
bated at 37°C for 24 h. Growth on selective, differential media
including E. sakazakii isolation agar (ESIA; AEB520010; AES
Laboratoire, France), E. sakazakii chromogenic agar (DFIA;
CM1055; Oxoid Ltd., United Kingdom), E. sakazakii plating
medium (ESPM; R&F Products Inc.), and E. sakazakii screen-
ing plate (ESSP; R&F Products Inc.) was determined using a
10-␮l inoculum from turbid cultures grown in brain heart in-
fusion for 18 to 24 h. Incubation of DFIA, ESPM, and ESSP
was at 37°C, and that of ESIA was at 44°C. All plates were
incubated for 24 h; the ESSP was also read at 6 h, and a
positive result at either time point was recorded. Production of
yellow pigment was determined on tryptic soy agar incubated
at 25°C for 72 h, followed by exposure to natural light for 6 h
at room temperature. Commercial identification biochemical
galleries, API20E and ID32E (bioMérieux, France), were used
according to the manufacturers’ instructions.
dnaG. A real-time PCR assay targeting the dnaG gene, a
component of the macromolecular synthesis operon, was de-
veloped previously by Seo and Brackett (18) and modified by
Drudy et al. (2) using the TaqMan probe (6-carboxyfluores-
cein–ACA GAG TAG TAG TTG TAG AGG CCG TGC TTC
C–) to increase the discriminatory power of the assay.
gluA. The ␣-glucosidase (gluA) gene was amplified using the
following primers: EsAgf, 5⬘-TGA AAG CAA TCG ACA
AGA AG-3⬘, and EsAgr, 5⬘-ACT CAT TAC CCC TCC TGA
TG-3⬘, generating a product of 1,680 bp in size. The amplifi-
cation reaction mixture and the thermal cycling conditions
were described previously (10). The gluA short fragment was
amplified using primers EsAg5f (5⬘-TAT CAG ATC TAC
CCG CGC-3⬘) and EsAg5_5r (5⬘-TTG ATG CCA AGC TGT
TGC-3⬘), resulting in a 105-bp amplicon. The PCR conditions
were as described previously (10) except that annealing was at
62°C for 30 s and extension was at 72°C for 30 s. The ampli-
fication products were analyzed in a 2% agarose gel.
In this study 193/210 “Cronobacter” strains (92%) exhibited

3814
VOL. 45, 2007 NOTES 3815

TABLE 1. Differentiation of Cronobacter spp. using phenotypic and genotypic tests

% Positive strains by assay % Strains identified
as a match by
No. of ␣-Glucosidase substrate a
Chromogenic agar b
gluA PCR dnaG biochemical galleryc
Species reverse
isolates
4-NP-␣- 4-MU-␣- X-␣- transcriptase
AMG ESIA DFIA ESPM Full Short API20E ID32E v3.0
Glc Glc Glc PCR

“C. sakazakii” 185 100 100 100 99 95 96 99.5 100 100 100 66 89
“C. turicensis” 8 100 100 100 100 100 100 100 100 100 100 100 100
“C. muytjensii” 7 100 100 100 0 100 100 100 100 100 100 100 100
“Cronobacter dublinensis” 8 100 100 100 100 100 87.5 100 100 100 100 100 100
“Cronobacter” genomospecies 1 2 100 100 100 100 100 100 100 100 100 100 100 100
All “Cronobacter” spp. 210 100 100 100 96 95.7 96.2 99.5 100 100 100 70 90
Other Enterobacteriaceae (after 102 35 46 32 37 24.5 (7)d 32.4 (8)d 38.2 (11)d 0 0 0 7 0
ESSP)
a
Substrate assays were for yellow pigment (4-NP-␣-Glc), fluorescence (4-MU-␣-Glc), blue-green pigment (X-␣-Glc), and acid production (AMG).
b
Chromogenic agar assays were for blue-green colonies (ESIA and DFIA), blue-black or blue-gray colonies (ESPM), and fermentation of sucrose and melibiose
(ESSP).
c
Percent strains identified as a good to excellent match with E. sakazakii.
d
Value in parentheses is the new percent positive after screening for sucrose and melibiose fermentation; values for Cronobacter isolates remain unchanged.

yellow or pale yellow colony pigmentation with the remaining
17 strains (8%) appearing cream-white. Also, 34% of the other
Enterobacteriaceae in this data set produced yellow-pigmented
colonies, clearly illustrating the lack of confidence in this fea-
ture as a reliable identification criterion.
All “Cronobacter” strains demonstrated biochemical activity
consistent with the constitutive expression of functional ␣-glu-
cosidase and metabolized the substrates X-␣-Glc, 4-MU-␣-
Glc, and 4-nitrophenyl-␣-D-glucopyranoside (4-NP-␣-Glc)
(Table 1). All were positive for the presence of the ␣-glucosi-
dase gene (gluA) using both primer sets. Unlike other
“Cronobacter” species, “Cronobacter muytjensii” was negative
for acid production from AMG. This may indicate a difference
between “Cronobacter” spp. in either specificity or inducibility
of the ␣-glucosidase enzyme and/or transport mechanisms for
the different glucopyranosides. The fluorogenic compound
4-MU-␣-Glc was not as specific as the other substrates for the
presumptive identification of “Cronobacter” spp., with 46% of
other Enterobacteriaceae in this data set displaying a positive
reaction. One strain (E770) did not grow on any of the commer-
cial chromogenic media. Additionally 4.3% of “Cronobacter”
strains were sensitive to crystal violet and/or the elevated
incubation temperature (44°C) and failed to grow on ESIA.
Interestingly 3.8% of “Cronobacter” strains grew but did not
produce blue-green-pigmented colonies on DFIA containing
X-␣-Glc at a final concentration of 0.1 g liter⫺1. However,
when this substrate was used at a concentration of 0.15 g
liter⫺1 in nonselective medium, these strains produced blue-
green colonies. For the non-Cronobacter strains in this data set,
the proportion producing typical colonies on the chromogenic
media ranged from 24.5% (ESIA) to 38.2% (ESPM). Screen-
ing the presumptive positive colonies for fermentation of su-
crose on ESSP did not reduce the number of positive
“Cronobacter” strains but did successfully reduce the number
of false-presumptive-positive Enterobacteriaceae. The melibi-
ose fermentation test reduced false positives on ESPM by one
additional strain; melibiose fermentation also appeared tran-
sient for some strains with positive results at 6 h but not at 24 h.
The false-positive strains on ESIA and DFIA were identified
as Enterobacter helveticus and Enterobacter turicensis (19) and a
proposed novel species, Enterobacter pulveris (20). In addition,
the false-positive strains from ESPM included Enterobacter as-
buriae, Enterobacter amnigenus, Enterobacter cloacae, and Enter-
obacter hormaechei.
The API20E gallery positively identified only 70% of the
“Cronobacter” strains to species level (as E. sakazakii) with
28% of strains identified to the genus Enterobacter. No false
identifications were obtained for “Cronobacter” strains; how-
ever, seven strains previously identified as E. cloacae, E. hor-
maechei, or E. asburiae using 16S rRNA gene sequencing (7)
and/or the ID32E gallery were identified as E. sakazakii by
using API20E. These strains were among those positive on
ESPM and ESSM but negative on ESIA and DFIA. They were
also negative for ␣-glucosidase activity using the different sub-
strates and negative in the gluA and dnaG PCR assays. Mis-
identification of these types of Enterobacter strains using lim-
ited biochemical galleries may have contributed to the reports
of ␣-glucosidase-negative E. sakazakii isolates (9, 17). Using
the ID32E gallery with version 3.0 of the apiweb database,
189/210 “Cronobacter” strains (90%) were identified to the
species level (as E. sakazakii) with “good-excellent” identifica-
tion. The nearest significant taxon for all but one of the re-
maining strains (20/21) was E. sakazakii, and none of the other
Enterobacteriaceae were misidentified as E. sakazakii with this
gallery.
In conclusion, using this extensive data set of “Cronobacter”
strains, all target isolates demonstrated ␣-glucosidase activity,
the chromogenic media were 95.7 to 99.5% sensitive, and the
ID32E v3.0 gallery was 99.5% successful for identifying target
strains to a correct nearest match. Notably for the purposes of
confident identification, the dnaG and gluA gene PCR assays
were 100% sensitive and specific.
We acknowledge Nicole Garofalo (NRC) for assistance with the
biochemical tests and Maya Grimm (UZH) for designing the primers
for the short ␣-glucosidase PCR product and thank Patrick Druggan
(Oxoid Ltd., United Kingdom) for advice on microbiological medium
composition.
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