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Applied and Environmental Microbiology, January 2000, p. 443-448, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Homogeneity of Danish Environmental and
Clinical Isolates of Shewanella algae
Birte Fonnesbech
Vogel,1,*
Hanne Marie
Holt,2
Peter
Gerner-Smidt,3
Anemone
Bundvad,1
Per
Søgaard,2 and
Lone
Gram1
Danish Institute for Fisheries Research,
Department of Seafood Research, Technical University of Denmark,
Lyngby,1 Department of Clinical
Microbiology, Odense University Hospital,
Odense,2 and Department of
Gastrointestinal Infections, Statens Seruminstitut,
Copenhagen,3 Denmark
Received 4 June 1999/Accepted 1 October 1999
 |
ABSTRACT |
Danish isolates of Shewanella algae constituted by
whole-cell protein profiling a very homogeneous group, and no clear
distinction was seen between strains from the marine environment and
strains of clinical origin. Although variation between all strains was observed by ribotyping and random amplified polymorphic DNA analysis, no clonal relationship between infective strains was found. From several patients, clonally identical strains of S. algae
were reisolated up to 8 months after the primary isolation, indicating that the same strain may be able to maintain the infection.
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TEXT |
Shewanella algae is a
recently defined marine bacterial species (23) which plays a
role in the environment in the turnover Fe(III) and other metal ions
(4, 21). Its ability to reduce Fe(III) and produce
H2S makes it an important cause of corrosion of metal
surfaces in, for example, oil fields (22). It has been suggested that as a dissimilatory metal reducer, it can play a role in
in situ bioremediation (6). S. algae may also
cause a variety of clinical symptoms in humans (3); however,
nothing is known about the relationship between strains isolated from the marine environment and clinical strains. The bacterium was originally isolated from a red alga in Japan (23), and the
original name, Shewanella alga, was recently revalidated as
S. algae (24). The organism is closely related to
the marine bacterium Shewanella putrefaciens but does
constitute a separate species (9, 19). Strains identified as
S. putrefaciens (formerly Pseudomonas
putrefaciens) have been isolated from a number of clinical
specimens, particularly from skin ulcers (1, 7) and ear
infections (11, 14, 15). Most of these infections have
probably been due to S. algae, which by traditional
phenotypic characterization would be misidentified as S. putrefaciens (9, 19). Most cases of S. algae
(putrefaciens) infections have been reported from countries
with a warmer climate than Denmark.
The first descriptions in Denmark of human infections with S. algae were reported in the very warm summer of 1994, when the organism was identified as the cause of two cases of S. algae bacteremia (8) and several cases of ear
infections (15). It was proposed that the infection was
caused by seawater exposure since 49 of 57 patients reported swimming
or bathing in seawater shortly before symptoms developed (8,
15). Seawater was collected from 10 beaches, and S. algae was detected in five locations, including the beaches where
some of the patients had been swimming (15).
Little is known about the epidemiology of S. algae
infections, and the purpose of the present study was to evaluate
whether a link between the marine environment and human infections
could be made and whether isolates of S. algae from ear
infections represent a special group of clones which are infectious and
which differ from environmental isolates of S. algae. Our
strain comparison is based on a polyphasic characterization, including
whole-cell protein, ribotyping, and random amplified polymorphic DNA
(RAPD) profiling.
Physiological characterization of the strains.
A total of 63 strains of S. algae were included in the study (Table
1). The strains were isolated as
described by Holt et al. (15) and characterized
(9). All isolates which were grown in veal infusion broth
(Difco, Detroit, Mich.; catalog no. 0344-17-6) or on iron agar (CM964;
Oxoid, Basingstoke, England) (12) were gram-negative, motile
rods with positive oxidase and catalase reactions. They were unable to
ferment glucose but reduced trimethylamine oxide and produced
hydrogen sulfide. All strains grew in veal infusion broth at 41°C but
not at 4°C. Growth occurred on salmonella-shigella agar and in veal
infusion broth containing 6 and 10% NaCl. The strains exhibited clear
hemolysis on sheep blood agar. Acids were not produced from
D-glucose, maltose, or D-arabinose but were produced from D-ribose. These reactions are consistent with
the characterization of S. algae (9, 19). The
moles % G + C values of the strains were determined by
high-performance liquid chromatography analysis of hydrolyzed DNA as
described previously (9, 18) and ranged from 52 to 55%. The
type strain of S. algae (IAM 14159) reacted similarly to
these strains.
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TABLE 1.
Origin and moles percent G + C of the type strain of
S. algae (IAM 14159) and 62 strains of S. algae
used in the present study
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Whole-cell protein profiling.
Whole-cell proteins were
extracted from cells grown at 37°C for 24 h. Cells were
harvested, washed, and boiled in sample treatment buffer containing
mercaptoethanol (9). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to the procedure of Laemmli (17). Samples were electrophoresed and gels were stained as described previously (9). Dried gels
were scanned with a DeskTop scanner (Pharmacia Biotech). Detection of
the protein electrophoretic patterns, normalization of the densitometric tracks, and numerical analysis were performed as described by the PC-Windows software package GelCompar (version 4.0;
Applied Maths, Kortrick, Belgium) (25). The levels of
similarity were computed by using the Pearson product moment
correlation coefficient, and data were clustered by using the
unweighted pair group method with arithmetic average algorithm (UPGMA).
The reproducibility of the protein electrophoretic technique was
verified by using the type strain of S. algae (IAM 14159) as
a standard loaded in each fifth lane. Only gels with levels of
similarity of 93% or more (mean, 96%) of the standard profile were
used for the numerical analysis.
Numerical analysis of the whole-cell protein SDS-PAGE revealed that the
S. algae strains formed a very homogeneous group of
bacteria, with a similarity level above 80% for most of the strains
(Fig.
1). In comparison, subgroups of
S. putrefaciens separated
at 60 to 70% similarity
(
9).
S. algae isolates from Danish
seawater did
not separate from strains of human origin. Also,
no systematic
difference was found between strains isolated in
1994 and 1995. Only
one strain, the oil field strain NCIMB 12582,
separated at a similarity
level of 70% ± 8%.
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FIG. 1.
Clustering of SDS-protein electrophoregrams of 57 strains of S. algae listed in Table 1 and the type strain of
S. putrefaciens (ATCC 8071) by using the Pearson
product-moment correlation coefficient and the UPGMA. The horizontal
scale represents the percentage similarities.
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Ribotyping.
Ribotyping of 36 of the human ear isolates and 5 isolates from Danish seawater was performed as described previously
(10). In brief, DNA was extracted by an EDTA-SDS lysis,
phenol-chloroform extraction procedure. Purified DNA was cleaved with
HindIII as described by the manufacturer (GIBCO/BRL,
Life Technologies, Copenhagen, Denmark). Of four restriction enzymes
tested (BamHI, EcoRI, HindIII, PstI), HindIII was the most discriminatory
and was therefore chosen. The restriction fragments were separated by
electrophoresis in an agarose gel. A mixture of phage lambda DNAs
(Boehringer Mannheim, Ercopharm A/S, Kvistgaard, Denmark) cut with
HindIII and StyI was used as a molecular size
marker in every fourth lane. The fragments were vacuum blotted onto a
nylon membrane and hybridized with a digoxigenin-11-dUTP-labelled cDNA
probe derived from a commercially available Escherichia coli
16S and 23S rRNA preparation by random priming with reverse
transcriptase. The hybrids were detected by a color reaction of
nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate
(toluidinium salt) with alkaline phosphatase-labelled antidigoxigenin
antibodies. The banding patterns were compared visually.
Identical ribotyping patterns were found for strains isolated more than
once from the same patient in five of six cases (Table
2). Also, several other strains had
identical ribotyping patterns
(Table
3).
As with SDS-PAGE, marine and clinical isolates were
not systematically
separated. Several marine strains (TØ 4, TØ
8, and KO 2) were
identical by ribotyping to clinical strains.
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TABLE 2.
Similarity by different typing methods of 18 strains of
S. algae isolated from eight patients with ear infections
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RAPD analysis.
Ten microliters of a culture grown for 24 h was diluted in 90 µl of sterile Millipore water and boiled for 10 min. Five microliters of the lysate was transferred to a PCR tube
containing 45 µl of PCR mix with final concentrations of 10 mM
Tris-HCl, 50 mM KCl, 1% Tween 20, 2.5 mM MgCl2, 4× 200 µl of each deoxynucleoside triphosphate (Perkin-Elmer), 0.01%
gelatin, 2.5 U of Taq polymerase (Perkin-Elmer, Norwalk,
Conn.), and 4 µM primer. The sequences of primers RAPD1, OPA 10, OPA
18, and OPA 20 were 5'-CAATCGCCGT, 5'-GTGATCGCAG, 5'-AGGTGACCGT, and 5'-GTTGCGATCC,
respectively (DNA-Technology, Aarhus, Denmark). The PCR was run
in a thermocycler (model 2400; Perkin-Elmer) for 45 cycles of 1 min at
95°C, 2 min at 35°C, and 1 min at 72°C followed by 10 min at
72°C. Twenty microliters of product was subjected to electrophoresis
in a 2% agarose gel at 90 V for 4 h and visualized by staining
with ethidium bromide. RAPD reaction mixtures without bacterial DNA
acted as negative controls and a 100-bp ladder (Pharmacia Biotech) was
included three times in each agarose gel as a standard. Photos of RAPD patterns were scanned with a Pharmacia DeskTop scanner. Data were treated as described by the PC-Windows software package GelCompar (version 4.0; Applied Maths), and grouping was performed by using the
Dice coefficient and UPGMA cluster analysis. Band tolerance (maximum
tolerance in percent of the curve to match bands) was 1%.
RAPD patterns were generated by primers RAPD1, OPA 10, OPA 18, and OPA
20 for 63, 61, 57, and 62 of the strains listed in
Table
1,
respectively. The number of DNA bands varied from 3
to 11. A total of
51 different RAPD profiles were reproducibly
obtained for primer RAPD1
(Fig.
2). The same degree of similarity
or differentiation was obtained with the three other primers (Tables
2
and
3), except for two cases in which the profiles were identical
with
one primer only. The RAPD profile of seawater isolate TØ
9 by primer
OPA 18 was equal to strains from patient 4 (strain
no. 68116 and
102445) (Table
3). When primer OPA 20 was used,
isolate 69614 matched
KO 2. No systematic grouping depending on
year of isolation or origin
(environmental versus clinical) was
found.
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FIG. 2.
Dendrogram of RAPD patterns generated by primer RAPD1 of
the 63 S. algae strains listed in Table 1 obtained after
UPGMA analysis of the Dice coefficient (SD). The
horizontal numbers represents the percentage similarities.
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From six of the eight patients, identical RAPD profiles of the isolates
were generated by all four primers; conversely, four
isolates from two
patients obtained different amplification patterns
with the four
primers (Table
2). An example of this contrast
of DNA profiles for four
S. algae isolates from patients 6 and
7 is illustrated in
Fig.
3. Identical RAPD profiles were also
generated by the four primers from strains isolated from different
patients (Table
3). The seawater isolate TØ 8 profile was similar
to
that of the human isolate 67724.
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FIG. 3.
RAPD patterns of four isolates of S. algae
from two patients generated with four different primers. Strain 68872 and 71437 from patient 6, strain 74757 and 25191 from patient 7. Lanes
1, 5, 9, and 13, strain 68872; lanes 2, 6, 10, and 14, strain 71437;
lanes 3, 7, 11, and 15, strain 74757; lanes 4, 8, 12, and 16, strain
25191; lane S, 100-bp ladder (Pharmacia). Lanes 1 to 4, primer RAPD1;
lanes 4 to 8, primer OPA 10; lanes 9 to 12, primer OPA 18; lanes 13 to
16, primer OPA 20.
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|
We found RAPD analysis to be a powerful method for typing
S. algae at the clonal level since the four primers distinguished
almost the same isolates and indicated a large degree of clonal
variability. RAPD typing of
S. putrefaciens isolated from
the
water column of the central Baltic Sea yielded fewer different
genotypes (
26) than those observed in our study. However,
most
strains were obtained by selective or indicative isolation and
a
strong correlation between method of isolation and genotype
was seen
(
26). It was possible to obtain reproducible results
only
with strict standardization. Kerr et al. (
16) found that
using only one primer in RAPD analysis may fail to distinguish
between
strains and that at least three primers are required to
differentiate
between clones. We similarly found (Table
3) that
more than one primer
was required to
discriminate.
Concluding remarks.
The present study shows that, in
accordance with other studies (9, 19), strains of S. algae constitute a very homogeneous group of bacteria when
examined by whole-cell protein profiling but that great clonal
variability exists in both environmental and clinical isolates. The two
molecular subtyping methods, ribotyping and RAPD, gave very similar
results, although RAPD had the greater discriminatory power, which has
also been found for other bacterial species (2).
We found no systematic differences distinguishing between environmental
and clinical strains by these typing methods, and
similarly, Høi et
al. (
13), who investigated Danish
Vibrio
vulnificus strains from seawater and patients, could not by
ribotyping or
RAPD differentiate between the two groups. However, a
yet-undiscovered
virulence factor may be present exclusively in the
clinical
strains.
Our results indicate that a large degree of clonal variability exists
in Danish strains of
S. algae even though they were
isolated
from a narrow time span (1994 to 1995) and from a small
geographical
area. Also, humans are infected by strains of widely
different clonal
origin. In previous studies (
8,
14), it
has been proposed
that
S. algae infections originate from seawater
exposure.
Seawater isolates were found to be, in general, very
similar to the
clinical isolates by all of the analyses. Since
one strain isolated
from seawater (TØ 8) was indistinguishable
from one human ear
infection isolate (67724) by all of the typing
methods used, it is not
unlikely that seawater was the source
of the
infection.
The same clone was isolated more than once from six patients, and from
two of these patients, the same clone was isolated
6 and 8 months after
diagnosis. This is an indication that the
infection with
S. algae may be maintained by the same isolate.
Such persistence may
be facilitated by adhesion (e.g., to ear
drains) and biofilm formation,
and although this behavior has
not been investigated specifically for
S. algae,
S. algae has
been shown to adhere
readily to surfaces of flocs from activated
sludge (
5). In
two patients (patients 5 and 6), two different
clones were found when
isolates were recovered 3 weeks and 1 year
later, respectively. The
clones were in both cases isolated from
different ears, but whether the
patients were infected on two
different occasions or whether the clones
had persisted in parallel
during the infection remains
unknown.
The epidemiology of
S. algae infections has not been studied
before, and the purpose of the present study was to evaluate
whether
isolates of
S. algae from ear infections represent a special
group of clones that differ from environmental (marine) isolates
of
S. algae. We have found that strains of
S. algae
isolated from
Danish seawater and Danish human ear infections
constitute a very
homogeneous group of bacteria at the species level;
however, humans
are infected by strains of different clonal origins.
RAPD typing
revealed a high degree of clonal variability, but despite
the
high discriminatory power of this method, no particular lineage
could be associated with clinical
origin.
| |
ACKNOWLEDGMENTS |
We are grateful to the following individuals who kindly provided
bacterial strains: P. J. M. Bouvet, Centre National des
Salmonella et Shigella, Unit des Enterobacteries, Institut Pasteur,
Paris, France; E. Falsen, Culture Collection, University of
Göteborg, Göteborg, Sweden; S. Schæbel, Department of
Clinical Microbiology, Hillerød Hospital, Hillerød, Denmark. The
excellent assistance of Jette Melchiorsen with the G+C determination is acknowledged.
This study was supported by the Danish Food Technology Programme.
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FOOTNOTES |
*
Corresponding author. Mailing address: Danish Institute
for Fisheries Research, Department of Seafood Research, Technical University of Denmark, Bldg. 221, DK-2800 Lyngby, Denmark. Phone: 45 45 88 33 22. Fax: 45 45 88 47 74. E-mail: bfv{at}dfu.min.dk.
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Applied and Environmental Microbiology, January 2000, p. 443-448, Vol. 66, No. 1
0099-2240/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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