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Journal of Clinical Microbiology, August 1999, p. 2399-2401, Vol. 37, No. 8
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Prototypal Diarrheagenic Strains of Hafnia
alvei Are Actually Members of the Genus
Escherichia
J. Michael
Janda,1,*
Sharon L.
Abbott,1 and
M. John
Albert2
Microbial Diseases Laboratory, Division of
Communicable Disease Control, California Department of Health Services,
Berkeley, California 94704-1011,1 and
International Centre for Diarrhoeal Disease Research,
Bangladesh, Dhaka-1000, Bangladesh2
Received 8 February 1999/Returned for modification 9 April
1999/Accepted 19 April 1999
 |
ABSTRACT |
We analyzed five bacterial strains, designated 19982, 9194, 10457, 10790, and 12502, that were isolated from stool specimens of
individuals with diarrheal illness by the International Centre for
Diarrhoeal Disease Research in Dhaka, Bangladesh (M. J. Albert, S. M. Faruque, M. Ansaruzzaman, M. M. Islam, K. Haider, K. Alam, I. Kabir, and R. Robins-Browne, J. Med. Microbiol.
37:310-314, 1992). The strains were initially identified as
Hafnia alvei with a commercial identification system and
were reported to contain the eae gene of enteropathogenic
Escherichia coli. Results of conventional biochemical
analyses, testing of susceptibility to cephalothin, lysis by a
Hafnia-specific phage, and amplification of the outer
membrane protein gene phoE with species-specific primers
support the identification of these strains as members of the genus
Escherichia rather than Hafnia alvei. These
strains varied from typical E. coli strains by their
inability to produce acid from lactose or D-sorbitol and
failure to elaborate the enzyme 
-D-glucuronidase. PCR
analysis confirmed previous findings that the strains were positive for
the eae gene and negative for other virulence markers
present among recognized categories of diarrheagenic E. coli. Our findings support the hypothesis that these strains are
a new category of diarrheagenic isolates belonging to the genus
Escherichia and illustrate the importance of using multiple methodologies when identifying new bacterial agents of diarrheal disease.
| |
INTRODUCTION |
Over the past three decades the
number of bacterial taxa proven to cause gastrointestinal disease has
risen dramatically. In addition to bona fide enteric pathogens, a
number of reputed agents of gastroenteritis which had previously been
thought to be simple commensals of the gastrointestinal tract have been
described (1). One such agent is Hafnia alvei. In
1991, John Albert and colleagues at the International Centre for
Diarrhoeal Disease Research, Bangladesh (ICDDRB), reported on
laboratory investigations concerning a strain of H. alvei
(designated 19982) recovered from a 9-month-old girl seen at ICDDRB
with vomiting, mild dehydration, fever, abdominal distention, and
diarrhea of 72 h duration (2). This fecal isolate was
found to induce diarrhea in 8 of 12 adult rabbits by using the
removable intestinal tie-adult rabbit diarrhea model. Electron
microscopic studies of the apical surface of the rabbit's infected
gastrointestinal epithelium revealed classic attachment-effacement
lesions traditionally associated with enteropathogenic Escherichia coli (EPEC). Subsequent investigations by the
same investigators at ICDDRB identified an additional six strains of H. alvei with similar properties (3). Like EPEC,
all seven H. alvei strains were fluorescent actin staining
positive and hybridized with the EPEC attaching-and-effacing
(eae) probe (3). These findings led to the
hypothesis that the eae locus is highly conserved among
diverse species within the family Enterobacteriaceae (16).
On the basis of these investigations, several case reports and
case-control studies followed, and they documented H. alvei as an enteric pathogen (18-20, 25). However, a 1996 Canadian study failed to detect virulence characteristics present in
H. alvei 19982 in any of nine H. alvei isolates
recovered from children with diarrhea at The Hospital for Sick Children
in Toronto or in two strains previously implicated in outbreaks of
gastroenteritis (12). That study also noted that the outer
membrane, plasmid, and pulsed-field gel electrophoresis (PFGE) profiles
of Canadian isolates differed significantly from those of H. alvei 19982. Finally, recent collaborative studies between ICDDRB
and the University of Helsinki found a low level of 16S rRNA sequence
homology (92%) between eae-positive and
eae-negative H. alvei strains, raising concerns
about the correct taxonomic position of these strains (20).
Here we report on the results for five of these strains and provide
data indicating that these isolates are, in fact, unusual biotypes of
E. coli or represent a new species in the genus
Escherichia.
| |
MATERIALS AND METHODS |
H. alvei 19982, 9194, 10457, 10790, and 12502 were
provided by M. John Albert (Dhaka, Bangladesh). The enterovirulence
characteristics of these strains have been described previously
(2, 3). Isolates were kept as working cultures on motility
deeps, with permanent stock cultures maintained at 
70°C.
Conventional biochemical tests were performed as described previously
(13), and susceptibility to cephalothin was determined by
the AB Biodisk E test (Remel, Lexena, Kans.) according to the
manufacturer's instructions.
Testing of individual bacterial strains for Hafnia-specific
phage susceptibility (9) and for E. coli-specific
genes and virulence characteristics were determined by multiplex PCR
through the courtesy of the Centers for Communicable Disease Control by previously described methods (5, 7, 8, 11, 14, 17, 22-24).
The PFGE profiles of selected strains were determined with a CHEF II
system (Bio-Rad, Hercules, Calif.) according to the manufacturer's
instruction with the restriction endonuclease XbaI.
| |
RESULTS AND DISCUSSION |
All five ICDDRB strains were oxidase and indole negative,
nonmotile, lysine and ornithine decarboxylase positive,
o-nitrophenyl--D-galactopyranoside positive,
and methyl red positive. Key differential tests useful in the
separation of hafniae from E. coli are listed in Table 1. Several reactions, including failure
to produce aceytlmethycarbinol (Voges-Proskauer), lack of growth in KCN
broth, and failure to utilize acetate, suggested that the ICDDRB
strains were not H. alvei. Among the common sugars, only
D-glucose, D-mannitol, L-arabinose, trehalose, and maltose were fermented. The inability to produce acid
from lactose and D-sorbitol and the failure to elaborate the enzyme -D-glucuronidase (MUG) did not, however, fit
the typical pattern exhibited by most E. coli strains.
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TABLE 1.
Comparison of properties of Albert's diarrheal isolates
to characteristic properties of H. alvei and E. coli strains
|
|
By PCR, all five of the ICDDRB strains were positive for the
eaeA gene, as reported previously (14), but were
negative by multiplex PCR (5, 7, 8, 11, 17, 22, 23) for the following virulence characteristics: enterohemolysin (18),
bundle-forming pilus A (bfpA) (20, 25), EAF
plasmid (21), Shiga toxins stx1 and
stx2 (22, 24), E. coli
invasion plasmid antigen H (ipaH) (23), and
enterotoxigenic E. coli heat-labile and heat-stable enterotoxins (22).
Evidence indicating that these strains were in fact members of the
genus Escherichia comes from several lines of evidence. Almost all H. alvei strains are resistant to cephalothin
(6), but all of these strains were partially or completely
susceptible to cephalosporin by the E test. A genus-specific
Hafnia phage, originally described by Guinée and
Valkenburg in 1968 (9), failed to lyse any of these strains.
Furthermore, a PCR probe encoding the phoE outer membrane
protein gene, which is found only in E. coli and
Shigella species, hybridized to all five strains but not to
true H. alvei isolates (24). Finally, when all
five of the ICDDRB strains were typed by PFGE with XbaI they
generated a typical series of 10 to 15 large DNA fragments since only a few restriction sites are available (Fig.
1). However, when H. alvei
ATCC 13337 and ATCC 29927 were typed by PFGE they produced a large
series of small DNA bands, indicating that many XbaI
restriction sites were present in true hafniae.
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|
FIG. 1.
PFGE profile (XbaI) of Albert's strains and
reference strains of H. alvei. Lanes 1 and 9, bacteriophage
lambda markers; lanes 2, Albert's strain 12502; lane 3, Albert's
strain 10790; lane 4, Albert's strain 10457; lane 5, Albert's strain
9194; lane 6, Albert's strain 19982; lane 7, H. alvei ATCC
13337; and lane 8, H. alvei ATCC 29927.
|
|
Cumulative results from the present investigation indicate that the
diarrheagenic H. alvei isolates originally described by Albert and associates (2, 3) either are unusual biotypes of
E. coli or represent a new species in the genus
Escherichia. This conclusion is based upon biochemical and
antimicrobial susceptibility differences between true hafniae and the
ICDDRB "H. alvei-like" strains and the failure of the
latter group to be lysed by Hafnia-specific phage and
homology between the phoE genes of the latter group and that
of E. coli. Recent sequence data on a 353-bp fragment from
the 5' end of the 16S rRNA sequences of 10 eae-positive
H. alvei strains indicates homology closest to an EPEC
strain designated E2348/69 (20), further supporting the
identification of these isolates as E. coli.
When these strains were initially characterized as H. alvei
they were identified on the basis of API 20E (bioMérieux Vitek, Hazelwood, Mo.) reactions. Although the likelihood of a correct identification was not stated (e.g., excellent identification, acceptable identification), several reactions, notably a negative Voges-Proskauer reaction and the inability to ferment
L-rhamnose, could have suggested the possibility that these
strains were not true hafniae. Because of these initial
misidentifications, the association of "H. alvei" with
bacterial gastroenteritis was subsequently reinforced and was
disseminated in a series of case reports, clinical studies, and a
review (10, 18, 19, 21, 25).
Although many new potential enteric pathogens have been described over
the past decade, current data do not strongly support a role for
hafniae in infectious gastroenteritis. Furthermore, the present report
points out the critical importance of bacterial taxonomy and the
complete biochemical characterization of isolates in this process
(4, 15). Without such analysis the impact of molecular
characterization may be lost or provide incorrect information if the
isolate is not initially placed in the correct taxa. Therefore,
infrequently isolated groups, particularly those with aberrant
properties, should receive close systematic scrutiny prior to the
reporting of new disease associations or virulence properties linked to
such groups.
| |
ACKNOWLEDGMENTS |
We thank Nancy Strockbine, Evan Sowers, and Caroline O'Hara for
fruitful discussions and for assistance in the genotyping of our strains.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Microbial
Diseases Laboratory, 2151 Berkeley Way, Berkeley, CA 94704-1011. Phone:
(510) 540-2242. Fax: (510) 540-2374. E-mail:
JJANDA{at}DHS.CA.GOV.
| |
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Journal of Clinical Microbiology, August 1999, p. 2399-2401, Vol. 37, No. 8
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
