Jpn. J. Infect. Dis., 65, 203-207, 2012

Original Article

Isolation and Identification of Escherichia albertii froma Patient in an Outbreak of Gastroenteritis
Takayuki Konno*, Jun Yatsuyanagi, Shiho Takahashi, Yuko Kumagai,
Eriko Wada, Machiko Chiba, and Shioko Saito

Health and Hygiene Division, Akita Prefectural Research Center for
Public Health and Environment, Akita 010-0874, Japan(Received December 28, 2011. Accepted March 2, 2012)
SUMMARY: A microbial strain harboring the eae gene, which is known as the virulence gene of enteropathogenic Escherichia coli (EPEC) and most enterohemorrhagic E. coli, was isolated from apatient in a gastroenteritis outbreak that occurred in 22 patients in Akita Prefecture, Japan, in November 2011. The biochemical characteristics of the isolate were more similar to those of a novel Escherichia
sp., E. albertii than E. coli. Partial 16S rRNA gene sequences of the isolate were identical to those of acertain E. albertii strain, but also showed a high degree of similarity to those of E. coli strains. Finally,
we identified this isolate as E. albertii by performing PCR analysis that targeted the uidA, lysP, mdh,
and cdtB genes in addition to stx and eae genes to differentiate between the EPEC and E. albertii
strains.
responsible for the estimated 62,000,000 cases of foodborne illnesses and 3,200 deaths with an unknown etiological origin in the United States (17,18). However, isolation of E. albertii was rarely reported in Japan, andtherefore, its prevalence and possible pathogenic role
are currently unknown.
In November 2011, a gastroenteritis outbreak occurred in 22 patients in Akita Prefecture, Japan. Here,
we report that an EPEC-like strain harboring the eae
gene was isolated from a patient with norovirus infection in this outbreak. We identified the isolate using
biochemical tests and performed genetic identificationon the basis of 16S rRNA gene sequencing and PCR
analysis targeting the stx, eae, uidA, lysP, mdh, andcdtB genes.

INTRODUCTION

Escherichia albertii has been reported to be a novel
Escherichia spp. and a potential diarrheagenic pathogen
in humans (1,2). E. albertii was originally described as
an unusual strain of Hafnia alvei that harbors the eae
gene and encodes intimin, which is associated with theattaching-effacing phenotype of enteropathogenic Escherichia coli (EPEC) and most enterohemorrhagic E. coli
(EHEC), but subsequent phenotypic and genetic studies
suggested that this H. alvei-like strain does not belong
to the genus Hafnia (1,3–5). Further studies by Huys et
al. led to the characterization of these H. alvei-likestrains as a new Escherichia spp., E. albertii, on the basis of DNA-DNA hybridization results and 16S rRNA
gene sequence data (2).
In addition to intimin, cytolethal distending toxin(CDT) has been reported as a putative virulence factor
in E. albertii (6). CDT was first identified in E. coli
O128, which was isolated from the stool of a child who
aged less than 2 years and showing gastroenteritis (7),and the toxin was detected in several pathogenic bacteria, such as Shigella dysenteriae (8), Campylobacter spp.
(9), Haemophilus ducreyi (10), and Actinobacillus actinomycetemcomitans (11). In total, 3 genes, including
cdtA, cdtB, and cdtC, were associated with cytotoxic
activity (12–14); in particular, cdtB was associated withencoding the enzymatically active subunit (12,15).
E. albertii caused diarrheal diseases in children withsymptoms of vomiting, mild dehydration, fever, andabdominal distention (1,16). Sharma et al. describedthat E. albertii could possibly be one of the factors

MATERIALS AND METHODS
Isolation of bacteria: Stool samples of 19 patients and
16 restaurant staff members, and 10 environmental
samples from the restaurant were examined to detect
etiological agents, including diarrheagenic E. coli(DEC), Salmonella, Shigella, Staphylococcus aureus,
Bacillus cereus, Clostridium perfringens, Campylobacter, Vibrio, and norovirus, by standard culture procedures and PCR methods.
To detect DEC, we inoculated the fecal samples of thepatient onto DHL agar plate and incubated the plate at
C for 24 h. Isolated colonies were studied to classify379
the isolates as DEC by employing PCR method targeting the major virulence genes of DEC, such as stx for
Shiga toxin-producing (STEC) or EHEC, eae for EPEC
and most of EHEC, aggR and astA for enteroaggregative E. coli (EAggEC), elt and est for enterotoxigenic E.
coli (ETEC), and invE for enteroinvasive E. coli
(EIEC), as described previously with slight modification(19–22). The biochemical characteristics of the isolate
were tested for its ability to produce indole and lysine
decarboxylase, fermentation of lactose and xylose, and*Corresponding author: Mailing address: Akita Prefectural
Research Center for Public Health and Environment, 6-6
Sensyu kubota-machi, Akita 010-0874, Japan. Tel: ＋8118-832-5005, Fax: ＋81-18-832-5938, E-mail: konnotakayuki＠pref.akita.lg.jp
203

Table 1. Primer sequences used in PCR analysis for differentiating E. albertii from EPEC
Targetstx

Shiga toxineaeintimineuidA

beta-Dglucuronidase
lysine-specificpermeasemalatedehydrogenase
cytolethal
distending toxin

lysP
mdh
cdtB

Sequence (5?–3?)

Annealing temp.

ReferenceGAGCGAAATAATTTATATGTGTGATGATGGCAATTCAGTATCAGGATCGCCTTTTTTATACG
CTCTGCAGATTAACCTCTGC
TCAGCGCGAAGTCTTTATACCCGTCGGTAATCACCATTCCC
GGGCGCTGCTTTCATATATTCTTTCCAGATCCAACCGGGAGTATCAGGACTGGAAGGCGCAGATGTGGTACTGATT
CTTGCTGAACCAGATTCTTCACAATACCGGAAAGTAAATGGAATATAAATGTCCG
AAATCACCAAGAATCATCCAGTTA

55

(40)

55
55

This study(41)
This study

65

(25)

65

(25)

55

(35)

Property

motility.
16S rRNA gene sequencing: The forward primer (5?and
GGATCCAGACTTTGATYMTGGCTCAG-3?)
the reverse primer (5?-CCGTCAATTCCTTTRAGTTT3?) were used to amplify bacterial 16S rRNA genes by
PCR under the following conditions: 949C for 5 min; 25
C for 30 s, 559C for 30 s, and 729C for 45
cycles of 949
s; and 729C for 2 min (23,24). Amplified DNA fragments of about 900 bp were separated on a 2z (wt/vol)agarose gel. The gel was stained with ethidium bromide
and visualized on a UV transilluminator, and PCR amplicons were purified using a QIAquick Gel Extraction
Kit (QIAGEN, Tokyo, Japan), according to the
manufacturer's instructions. DNA sequences of the
PCR amplicons were determined using the Big Dye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems,
Tokyo, Japan) on an ABI-3130 apparatus (Applied
Biosystems). A sequence similarity search was performed using an online-system with BLASTN.2.2.24(http://blast.ddbj.nig.ac.jp/top-j.html).
PCR analysis to differentiate E. albertii from EPEC:
To identify the EPEC strain, a set of primers targeting
uidA, which is the specific housekeeping gene in the E.
coli lineage, was used in addition to stx and eae genes.
Two sets of primers targeting lysP and mdh genes,
which were designed to detect E. albertii lineage-specificgenetic polymorphisms (25), were used to identify the E.
albertii strain. The cdtB gene as a putative virulencegene was also amplified. The primer sequences and annealing conditions used for detecting these genes are
summarized in Table 1. Briefly, the PCR stages weredenaturation at 949C for 2 min, followed by 25 amplifiC for 30 s, annealing for 30 s, 729C
cation cycles (949
for 45 s), and a final extension cycle (729C for 2 min).
Analysis of CDT type: CDT typing was performed as
per the method described by Kim et al. (26), with thefollowing modification: the cdt type II R2 primer (5?TTTGTGTTGCCGCCGCTGGTG-3?) and cdt type III
R2 primer (5?-TTTGTGTCGGTGCAGCAGGGA-3?)
were used for PCR analysis instead of reverse primers
for cdt type II and cdt type III.

Fig. 1. PCR-based detection of the major virulence genes ofDEC. The size of PCR amplicons of stx, eae, aggR, astA, elt,
est, and invE are 289 bp, 479 bp, 254 bp, 106 bp, 130 bp, 186
bp, and 382 bp, respectively. Lanes: M, 100-bp size ladder(Bio-Rad, Tokyo, Japan); 1, Positive control (EHEC O157:H7
EDL931 for stx and eae [provided by Tokyo Metropolitan
Research Laboratory of Public Health, Tokyo, Japan],
EAggEC 17-2 for aggR and astA [kindly gifted from James B.
Kaper, University of Maryland School of Medicine, Baltimore,
Md., USA], ETEC JCM-8364 for elt, ETEC JCM-8365 for est,and EIEC JCM-8363 for invE [provided by RIKEN, Kanagawa, Japan]); 2, EC15062.

Furthermore, one strain, designated EC15062, was also
isolated from a patient infected with norovirus by standard culture procedures for DEC. EC15062 was positivefor eae, but negative for stx, aggR, astA, elt, est, andinvE, which are the major virulence genes of DEC (Fig.
1). Therefore, we suspected that EC15062 was an EPEC
strain. However, the eae gene is present not only in
EPEC and most of EHEC, but also in other bacterial
species, such as E. albertii and Citrobacter rodentium(formerly C. freundii biotype 4280) (1,27,28); therefore,
it was neccesary to confirm whether EC15062 is an E.
coli strain or not.
EC15062 produced indole and lysine decarboxylase;
but it did not ferment lactose and xylose and was non-

RESULTS AND DISCUSSION
In the gastroenteritis outbreak that occurred in Akita
Prefecture, Japan, in November 2011, we detected
EPEC and norovirus in 1 and 2 patients, respectively.
204

Table 2. Comparison of biochemical characteristics between
E. coli, E. albertii, and EC15062

Motility
Indole production
Lysin decarboxylase
Lactose fermentation
Xylose fermentation

E. coli 1)

E. coliinactive1)

E. albertii 2)

EC15062

＋
98z
90z
95z
95z

－
80z
40z
25z
70z

－
－
＋
－
－

－
＋
＋
－
－

1): Source: Farmer et al. (42).
2): Source: Abbott et al. (39).

motile at 359C, indicating that the biochemical characteristics of EC15062 were similar to those of E. albertii
rather than E. coli (Table 2). Although the indoleproduction characteristics of EC15062 were different
from those of E. albertii isolated from Bangladeshi children, this difference did not eliminate the possibility
that EC15062 was an E. albertii strain, since in a study
by Oaks et al., indole production was seen in the E. albertii strains isolated from birds (29).
The partial 16S rRNA sequences of EC15062 (823 nt)
were identical to those of an E. albertii strain (accession
no. HM194877), which was isolated from a teal, andshowed 99.9z (822 of 823 nt) similarity to the sequences in E. coli strains. These findings suggested thatthe similarity analysis of 16S rRNA gene sequences between the strains was insufficient to differentiate E. albertii from E. coli.
The uidA gene has been often used for the identification of E. coli strains in previous studies since uidA is a
specific housekeeping gene in E. coli and Shigella spp.
(30–33). On the other hand, Hyma et al. reported a PCR
method targeting conserved housekeeping genes, including clpX, lysP, and mdh, to identify E. albertii strains(25). Primers of lysP and mdh have been designed on
the basis of E. albertii-specific nucleotide polymorphisms (25). We examined the presence of uidA, lysP,and mdh, in addition to stx and eae, to determine
whether EC15062 is EPEC or E. albertii strain by comparative PCR analysis with a reference strain of EPEC,
termed E2348/69 (Fig. 2). We confirmed that both the
EPEC strain and EC15062 were negative for stx, butwere positive for eae (same results as in Fig. 1). In contrast to the EPEC strain, EC15062 was negative for
uidA, and positive for both lysP and mdh. On the basisof these results, EC15062 was identified as E. albertii.
We detected cdtB, a putative virulence gene of E. albertii in EC15062 (Fig. 2). Furthermore, we examined
the CDT type of EC15062 by PCR analysis designed toclassify E. coli CDTs. In E. coli, CDTs are classified
into 5 types, on the basis of differences in the sequencesof cdt (34–36). As shown in Fig. 3, EC15062 was positive for cdt type III and cdt type V. These results are
consistent with those obtained by Hyma et al., whoreported that the cdt sequences of E. albertii lineage
were most similar to those of cdt type V and cdt type III(25). Hyma et al. suggested that there was no significant
association between the presence of CDT and diarrhea(25). Okuda et al., however, showed direct experimental
evidence for the role of CDT in diarrhea by using an
animal model with a recombinant E. coli cloned with S.

Fig. 2. Comparison of the presence of the virulence genes andhousekeeping genes between EPEC and EC15062. The size of
PCR amplicons of stx, eae, uidA, lysP, mdh, and cdtB are 518
bp, 479 bp, 248 bp, 252 bp, 115 bp, and 466 bp, respectively.
Lanes: M, 100-bp size ladder (Bio-Rad); 1, EPEC (E2348/69,
kindly gifted from James B. Kaper); 2, EC15062.

Fig. 3. CDT typing of EC15062. The size of PCR amplicons of
cdt type I, II, III, IV, and V are 411 bp, 556 bp, 555 bp, 350
bp, and 1363 bp, respectively. Lanes: M, 100-bp size ladder(Takara, Shiga, Japan).

dysenteriae cdt operon (37). In Campylobacter spp.,
CDT was associated with the property of invasiveness(38). Although further studies are required to warrant
the role of CDT in the diarrheagenicity of E. albertii,
the cdtB gene may be one of the important virulencemarkers in E. albertii.
Huys et al. reported that E. albertii was associatedwith diarrhea in Bangladeshi children (2), but there islimited epidemiological data suggesting that E. albertii
was the etiological agent of diarrheal outbreaks. In thisstudy, we isolated E. albertii strain from a patient of the205gastroenteritis outbreak, which occurred in Akita
Prefecture, Japan. To the best of our knowledge, this is
the first report that describes the isolation of E. albertii
from a diarrheal patient in Japan. We, however, could
not determine whether E. albertii was the etiologicalagent in our case, because the patient was co-infectedwith norovirus, and only 1 patient was positive for E. albertii in this case.
The prevalence, epidemiology, and clinical relevanceof E. albertii still remain unclear, in part, because E. albertii is either likely to remain often unidentified or be
misidentified by routine biochemical identification
methods employed in clinical laboratories (39). In thepresent study, we could successfully identify the strain
as E. albertii by using PCR analysis to differentiate between E. albertii and EPEC, while the similarity analysis of 16S rRNA gene sequences was insufficient to identify and conclude the isolate as E. albertii. Our results
suggested that the optimal method to isolate E. albertii
strains was to detect the presence of eae on selective agar
plates for E. coli, select lactose and xylose nonfermenting and nonmotile colonies, and differentiate E. albertii
from EPEC by the PCR analysis. Isolation of a large
number of E. albertii strains and proper identificationof these isolates as E. albertii will be highly important tofurther elucidate the significance of E. albertii as a diarrheagenic pathogen.

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Conflict of interest None to declare.
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