APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2000, p. 443–448 0099-2240/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Vol. 66, No. 1
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
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. 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 gramnegative, 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 Dglucose, maltose, or D-arabinose but were produced from Dribose. 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. 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
* 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:
[email protected]. 443
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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 Strain designation
No. 61569 to 103757, a total of 41 isolates 3862, 25191, 50091, 71437 BØ 2, KO 2, KO 3, KO 4, TØ 4, TØ 8, TØ 9, LA 4, LA 7, RA 2 NCIMB 12582 14.80-A 43940 CCUG 526 CCUG 15259 BrY FeRed AIM 14159
Country, yr of isolation
Source or origin
Reference
Denmark, 1994 Denmark, 1995 Denmark, 1994
Human ear infection Human ear infection Seawater
15
Canada, 1987 France, 1980 Denmark, 1994 Sweden, 1969 United States, 1992 United States United States, 1994 Japan
Oil field Flamingo Human bacteremia Human ear secretion, otitis Human ear infection Sediment of Great Bay Surface sediment of marsh Red alga
22 20 8
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%. 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-4chloro-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. RAPD analysis. Ten microliters of a culture grown for 24 h was diluted in 90 l of sterile Millipore water and boiled for 10 min. Five microliters of the lysate was transferred to a PCR tube containing 45 l of PCR mix with final concentrations of 10 mM Tris-HCl, 50 mM KCl, 1% Tween 20, 2.5 mM MgCl2, 4⫻ 200 l of each deoxynucleoside triphosphate (PerkinElmer), 0.01% gelatin, 2.5 U of Taq polymerase (PerkinElmer, Norwalk, Conn.), and 4 M primer. The sequences of primers RAPD1, OPA 10, OPA 18, and OPA 20 were 5⬘-CA
15
4 21 23
Mol% G ⫹ C (this study)
52.7–54.3 52.9–53.5 53.4–54.6 51.9 53.8 53.1 53.2 53.0 52.8 53.7 53.7
ATCGCCGT, 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. 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. 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 repro-
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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.
ducible 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
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TABLE 2. Similarity by different typing methods of 18 strains of S. algae isolated from eight patients with ear infections Patient no.
a
Strain no.
Date of isolation (day.mo.yr)
Whole-cell-protein profiling (% similarity)
Ribotyping results (no. of identical bands/total bands)
RAPD results (no. of identical bands/total bands) with indicated primer RAPD1
OPA 10
OPA 18
OPA 20
1
64500 69559
5.8.94 22.8.94
95
NDa
12/12
10/10
10/10
12/12
2
66038 79895
9.8.94 22.9.94
90
18/18
12/12
10/10
12/12
8/8
3
65733 67597
9.8.94 15.8.94
97
20/20
14/14
8/8
10/10
8/8
4
68116 102445
6.8.94 29.11.94
96
26/26
16/16
8/8
10/10
8/8
7
74757 25191
6.9.94 25.3.95
98
20/20
14/14
8/8
12/12
10/10
8
84145 103757 3862 50091
4.10.94 1.12.94 11.1.95 6.6.95
94
48/48
28/28
12/12
24/24
20/20
5
68702 75438
18.8.94 8.9.94
79
ND
12/20
6/9
6/13
8/17
6
68872 71437
19.8.94 16.8.95
80
16/22
8/14
4/10
6/11
6/7
ND, not determined.
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 sea-
TABLE 3. Typing characteristics of S. algae isolates found identical by either RAPD analysis and/or ribotypinga Strain
66194 65403 61569 65124 65464 25191b 74757b TØ 4
Whole-cell-protein profiling (% similarity)
89
94
Ribotyping results (no. of identical bands/total bands)
RAPD results (no. of identical bands/total bands) with indicated primers RAPD1
OPA 10
OPA 18
OPA 20
RAPD1
OPA 10
OPA 18
OPA 20
84
33/33
14/14
6/6
8/8
8/8
12/20
9/10
8/11
10/14
90
50/50
28/28
16/16
24/24
20/20
32/36
18/22
28/30
26/30
24/24
16/16
10/10
12/12
10/10
16/16
8/8
10/10
8/8
20/24
9/12
15/15
10/14
89
96
69614 KO 2
91
22/22
10/12
2/9
6/10
8/8
64423 68003
84
22/22
8/16
6/7
5/9
4/8
67724 TØ 8 68116b 102445b TØ 9
a b c
92
26/26
c ND
Strains TØ 4, TØ 8, TØ 9, and KO 2 were isolated from seawater, and the remaining strains were isolated from human ear infections. Strains also included in Table 2. ND, not determined.
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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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APPL. ENVIRON. MICROBIOL. This study was supported by the Danish Food Technology Programme. REFERENCES
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.
water 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 Go ¨teborg, Go ¨teborg, Sweden; S. Schæbel, Department of Clinical Microbiology, Hillerød Hospital, Hillerød, Denmark. The excellent assistance of Jette Melchiorsen with the G⫹C determination is acknowledged.
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