- Email: [email protected]
Numerial Taxonomy and DNA-DNA Hybridizations of Aeromonas strains Isolated from Human diarrhoeal Stool, Fish and Environment
System. App!. Microbio!. 20,458-467 (1997) © Gustav Fischer Verlag
Numerial Taxonomy and DNA-DNA Hybridizations of Aeromonas strains Isolated from Human diarrhoeal Stool, Fish and Environment ADAM KAZNOWSKI Department of Microbiology, Institute of Experimental Biology, A. Mickiewicz University, Poznan, Poland Received: March 26, 1997
Summary A numerical taxonomic study and DNA-DNA hybridizations were performed on seventy-one isolates of Aeromonas collected from children diarrhoeal stool, dead and alive fish Rutilus rutilus, and environment. These isolates were compared with the fifteen type and reference strains of the presently recognized Aeromonas genomic species. Results of the phenotypic tests were analysed by numerical taxonomic techniques, using the simple matching coefficient (SSM) and Jaccard coefficient (SJ)' and the unweighted average linkage clustering (UPGMA). At SSM> 85% and SJ > 75% these analyses resolved 6 groups and 9 single strains. DNA-DNA hybridization experiments revealed seven genomic groups among the examined Aeromonas isolates. Clusters were identified as A. hydrophila/A. bestiarum (two groups), A. veronii biotype sobria (two groups), A. caviae/A. media/A. eucrenophila, and A. media. The results obtained with the numerical taxonomy were not in perfect agreement with the DNA-DNA hybridization results and showed genetic heterogeneity of some phena. In some cases strains of one genomic species were found in different clusters or one cluster contained more than one hybridization group (HG). Among isolates of HG 8/10 biovariants were found. Strains of HG 1-3 and of HG 4-6 showed high phenotypic homogeneity. The results of this study emphasise the need to use of molecular methods in addition to phenotypic characteristics for identification of Aeromonas isolates with the genomic species. Key words: Aeromonas - Numerical taxonomy - DNA:DNA hybridization - genomic species
Introduction Bacteria belonging to the genus Aeromonas are ubiquitous aquatic organisms found in freshwater and sewage (SCHUBERT, 1991). They were also cultured from food (HANNINEN and SUTONEN, 1995). Some Aeromonas species are pathogenic for cold and warm-blooded animals and can be responsible for intestinal as well as several extraintestinal human diseases (ALTWEGG and GEISS, 1989; ALTWEGG, 1990; JANDA, 1991). In the Bergey's Manual of Systematic Bacteriology, POPOFF (1984), on the basis of phenotypic analyses, included this genus into Vibrionaceae family and recognized four species: A. hydrophila, A. caviae, A. sobria and A. salmonicida. COLWELL et al. (1986) proposed to separate the genus Aeromonas from Vibrionaceae and created a new family Aeromonadaceae. This proposition was aaccepted by the International Committee on Sys-
tematic Bacteriology, Subcommittee on the Taxonomy of
Vibrionaceae (1992). Furthermore, DNA-DNA hybridization experiments showed significant genetic heterogeneity of this genus. Currently, in the Aeromonas at least 15-16 DNA-DNA hybridization groups (HGs) are recognized (ESTEVE et al. 1995b), equivalent to genomic species comprising strains with approximately 70% or greater DNA-DNA relatedness with 5% or less divergence within the related sequences (WAYNE et al. 1987). In addition to the four species listed by POPOFF (1984), several new species were recognized: A. media (HG 5) (ALLEN et aI., 1983), A. veronii (HG 8110) (HICKMANBRENNER et aI., 1987), A. eucrenophila (HG 6) (SCHUBERT and HEGAZI, 1988), A. schubertii (HG 12) (HICKMAN-BRENNER et al., 1988), A. jandaei (HG 9) (CARNAHAN et aI., 1991a), A. trota (HG 14) (CARNAHAN et al., 1991b), A. allosaccharophila (HG 15) (MARTINEZ-MuRCIA et aI., 1992; ESTEVE et aI., 1995a), A. encheleia
Numerical taxonomy and DNA-DNA hybridizations of Aeromonas
(ESTEVE et ai., 1995b) and recently A. bestiarum (HG 2) (ALI et ai., 1996). Moreover, at least two unnamed genotypes, HG 11 and HG 13, were recognized (CARNAHAN and JOSEPH, 1993). Currently, A. caviae comprises strains of genotype 4, A. sobria includes isolates of HG 7, A. hydrophila encompasses isolates belonging to HG 1 and motile strains of HG 3, whereas A. salmonicida includes un motile isolates of HG 3 (JANDA, 1991; CARNAHAN and JOSEPH, 1993). However, the phenotypic similarity of some of the Aeromonas genomic species causes significant difficulties in their identification on the basis of biochemical tests (KUIJPER et ai., 1989; KAMPFER and ALTWEGG, 1992; ALTWEGG, 1993; CARNAHAN, 1993). Recently, several molecular methods useful in Aeromonas genomic species identification were proposed: mutilocus enzyme electrophoresis (ALTWEGG, 1990; ALTWEGG et ai., 1991), ribotyping (MART/NETTI LUCCHINI and ALTWEGG, 1992; HANNINEN and SIITONEN, 1995), PCR (DORSCH et ai., 1994), colorimetric DNA-DNA hybridization in microplates (KAZNOWSKI, 1995) and AFLP-fingerprinting (Huys et ai., 1996). Most of the numerical taxonomic studies of Aeromonas strains were performed before taxonomic changes were introduced. Only in three recently published papers reference strains of new genomic species were introduced (KAMPFER and ALTWEGG, 1992; CARNAHAN and JOSEPH, 1993; ESTEVE, 1995). Numerical taxonomy of Aeromonas strains, simultaneously defined at the genomic level by DNA-DNA hybridization, was conducted only in one case (KAMPFER and ALTWEGG, 1992), where the authors analysed strains mostly isolated from human feces. The present study was undertaken to cluster by numerical taxonomy strains of Aeromonas isolated from different sources (mostly from environment) and to evaluate results with the genomic species determination by colorimetric DNA-DNA hybridization.
Materials and Methods Bacterial strains: A total of 71 Aeromonas isolates were studied by phenotypic traits and by colorimetric hybridization method for identification with the hybridization groups (HGs). These strains were isolated from children diarrhoeal stool, alive and dead fish Rutilus rutilus, drinking water, recreational lake water, the Warta river water, water of Gdansk Bay in the Baltic Sea, municipal sewage and water-oil aluminium rolling-mill emulsion. The strains used in this study, their designation and sources are shown in Table 1. The isolates were obtained by enrichment in alkaline peptone water (pH 8.6), followed by inoculation onto bile salts brilliant green agar (BBG), xylose - deoxycholate agar (XDCA) (MILLERSHIP and CHATIOPADHYAY, 1984) and blood agar with 15 mg/l of ampicillin. The cultures were maintained on Tryptic Soy Agar (TSA medium) at room temperature. The following type and reference strains were included in this study: A. hydrophila ATCC 7966 T (HG 1), A. bestiarum ATCC 51108 T (HG 2), A. hydrophila CDC 0434-84 (HG 3), A. caviae ATCC 15468 T(HG 4), A. media CDC 0862-83 (HG 5), A. media CDC 9072-83 T (HG 5), A. eucrenophila ATCC 23309 T (HG 6), A. sobria CIP 7433 T(HG 7), A. veronii biotype sobria CDC 0437-84 (HG8/l0), A. veronii biotype veronii
459
ATCC 35624 T (HG 10/8), A. jandaei ATCC 49568 T (HG 9), A. schubertii ATCC 4370QT (HG 12), A. trota ATCC 4965?T (HG 14), A. allosaccharophila CECT 4199 T (HG 15), and A. encheleia CECT 4342T. Phenotypic properties: Each Aeromonas strain was tested for 139 phenotypic properties. Unless otherwise indicated, incubation was done at 30°C. Cell morphology and Gram-straining reaction were tested after 24 hour incubation on TSA medium Production of diffusible brown pigment was examined in 7days cultures on TSA. Motility, salt tolerance, ability to grow in broth at 37°C and 40.5 °C, lecithinase and amylase were determined by means of the techniques described by LEE et al. (1979). Haemolysis was determined on TSA medium supplemented with 5% sheep blood. Anaerobic haemolysis of sheep blood was found out in a jar using gas generating box (bioMerieux). Elastase was determined by the method of SCHARMANN (1972). The test for oxidative and fermentative metabolism of glucose was performed by the method of HUGH and LEIFSON (HOLDING and COLLEE, 1971). Gas production from glucose was tested in the same medium with Durham tube but without agar. H 2 S production from cysteine was determined on cysteine-iron agar (VERON and GASSER, 1963). Self-pelleting and pelleting after boiling were described by JANDA et al. (1987). The following tests were conducted as described by HOLDING and COLLEE (1971): cytochrome oxidase (KOVACS method), catalase, nitrate reductase, arginine dihydrolase (THORNLEY method), lysine and ornithine decarboxylase (MOELLER method), Voges-Proskauer reaction, malonate utilization, urease production (CHRISTENSEN method), phenylalanine deaminase, gluconate oxidation, indole production in peptone water, hydrolysis of gelatine, DNA and Tween 80. Hydrolysis of arbutin was examined by the method of JANDA et al. (1984). Utilization of sole carbon sources was determined on M 70 medium (VERON, 1975) supplemented with 0.05% peptone by means of a multipoint inoculator. The substrates serving as carbon sources were filter-sterilized and bacterial growth was examined for 14 days. The carbon sources used included: L-arabinose, cellobiose, fructose, D-galactose, D-glucose, lactose, saccharose, melibiose, L-rhamnose, salicin, adonitol, D-arabitol, mannitol, sorbitol, acetate, citrate, a-D-galacturonate, methyla-D-glucopyranoside, glucuronate, DL-lactate, DL-3-hydroxybutyrate, a-ketoglutarate, urocanic acid, D-tartrate, glycine, DL-a-alanine, L-arginine, L-asparagine, L-citrulline, L-glutamine, L-histidine, L-isoleucine, L-lysine, L-serine, L-ornithine, L-proline, L-threonine, L-tryptophan, and L-methionine. Fermentation of carbohydrates, glycosides and polyalcohols were assayed in commercial kit API 50CH (bioMerieux, France). Sensitivity to the vibriostatic agent 01129 was detected on TSA medium containing 150 mg/l of this compound. Susceptibility to antibacterial compounds was determined by disk diffusion method on MUELLER-HINTON medium (Difco) by means of BECTON-DICKINSON disks: ampicillin (10 pg), cloxacillin (1 pg), chloramphenicol (30 pg), cephalothin (30 pg), cefazolin (30 pg), clindamycin (2 pg), gentamycin (10 pg), imipenem (10 pg), nalidixic acid (30 pg), neomycin (30 pg), nitrofurantoin (200 pg), rifampicin (10 pg), streptomycin (30 pg), tetracycline (30 pg), vibramycin (30 pg), ticarcillin (75 pg), tobramycin (10 pg), and sulfamethoxazole-trimethoprim. Testing and results interpretation were done according to the manufacturer's instructions. Numerical analysis of phenotypic results: The test error was evaluated by examining 15 strains in duplicate, according to the method described by SNEATH and JOHNSON (1972). The test results were coded 1 for positive results, 0 for negative results and 9 for noncomparable data. Similarities were calculated using the simple matching coefficient (SSM) and Jaccard's coefficient (SJ)' Clustering of the 86 strains was achieved by the unweight-
460
A. KAZNOWSKI
Table 1. Designation and source of strains examined by numerical analysis and DNA-DNA hybridization. Phenon or single strain
HG
Taxon
Strain no.
Source of isolation
1
2
A. bestiarum
3
A. hydrophila
2
1
A. hydrophila
3
2 8110
A. bestiarum A. veronii biotype sobria
AKl,AK41 AK 115 AK 46, AK 50, AK 76, AK 130, AK 131 AK 409, AK 410 AK 400, AK 401, AK 402 AK 106, AK 117, AK 125 CDC 0434-84 AK44 ATCC 7966 T ATCC 51108 T AK 382, AK 389, AK 392 AK 12,AK 59 AK 100, AK 102, AK 120, AK 411, AK412, AK 413 AK279 AK 156, AK 160, AK 164, AK 180 AK 165,AK 167,AK 176 AK 387, AK 391
lake water drinking water lake water drinking water sea water rolling-mill emulsion fresh water lake water canned milk diseased fish human stool lake water drinking water
4
8110
5
4
A. veronii biotype sobria A . caviae
5
A. media
6 5
A. eucrenophila A. media
6
Unclustered strains: 6 7 8110 9 10/8 12 14 15
A. eucrenophila A.sorbria A. veronii biotype sobria A. jandaei A. veronii biotype veronii A. schubertii A. trota A . allosaccharophila A . encheleia
sewage dead fish healthy fish human stool
AK 48, AK 335, AK 338, AK 339 lake water AK 266, AK 276, AK 281, AK 296 sewage AK 404, AK 405 river water AK 104, AK 126 drinking water AK 406, AK 407, AK 408 sea water AK 375, AK 376, AK 377, AK 378, AK 379, human stool AK 380, AK 383, AK 384, AK 385, AK 386, AK 388, AK 390, AK 393 AK 220, AK 232 rolling-mill emulsion ATCC 15468 T guinea pig AK42 lake water AK403 sea water AK65 lake water CDC 0862-83 fish CDC 9072-83 T fresh water ATCC23309 T CIP 7433 T CDC 0437-84 ATCC 49568 T ATCC 35624T ATCC 43700 T ATCC4965?T CECT 4199 T CECT 4342 T
ed pair group method using arithmetic averages (UPGMA), cophenetic correlation (i.e. the correlation between the respective values in the similarity matrix and the corresponding dendogram) was calculated using the correlation coefficient r (SNEATH and SOKAL, 1973). Computing was done using an DTK computer 486 DX2. Genomic species determination: Preparation and purification of DNA was performed as described by ALTWEGG et al. (1990). The colorimetric DNA-DNA hybridizations in CovaLinc microplates (Nunc) were performed as previously described (KAZNOWSKI, 1995). Hybridization method includes immobilization of nonlabelled reference DNA to micro titer wells, hy-
fresh water fish fish human stool sputum abscess human stool diseased elvers fish
bridization of the DNA with DNA probe of examined or reference strain, reacting the probe with streptavidin-peroxidase conjugate, and assaying of enzyme activity with the 3, 3', 5, 5'tetramethylbenzidine at 450 nm with a microplate reader. The nucleic acids probes were constructed by DNA labelling with photo biotin (Vector Lab., USA) according to the manufacturer's instructions. Before hybridization experiments, nucleic acid was fragmented by ten passages through a pipette with a tip possessing capillary (0.3x15 mm) to fragment lengths of 500-800 kDa. The fragment size was checked byelectrophoresis on a 1 % agarose gel in a 0.04 M Tris-acetate buffer (pH 8.0), using as reference restriction fragments Hind III of Lambda DNA (Boeringer, Mannheim). Gels were stained with ethi-
Numerical taxonomy and DNA-DNA hybridizations of Aeromonas dium bromide (0.5 pg/ml) and inspected in a UV transilluminator. For the estimation of DNA relatedness the absorbance of the well of herring sperm DNA was taken as 0% and the absorbance of the well for each reference DNA was taken as 100%. Bacterial strains showing 70% or more relatedness to the reference strain of a given HG were assigned to that HG (WAYNE et al., 1987).
Results Test reproducibility Seventy-one isolates of Aeromonas and fifteen type and reference strains were examined for 139 phenotypic characters. The average probability of an erroneous result, calculated from pooled variance of all the unit characters, scored for the 15 duplicate strains was 3.7%. Values of Sj2 ranged zero for most of the tests through 0.15 for hemolysis reaction on sheep blood agar, growth on glycine, L-arginine, L-histidine, and cellobiose. This is acceptable according to the criteria of SNEATH and JOHNSON (1972).
Data matrix Fifty-nine biochemical tests and 10 antimicrobial susceptibility tests were deleted from the final data matrix because they gave only positive or negative results. All isolates were Gram-negative, positive for catalase, cytochrome oxidase, oxidation and fermentation of glucose, nitrate reductase, hydrolysis of DNA, gelatin, lecithin, starch and Tween 80, growth without NaCI and at 37°C. All strains used D-glucose and D-fructose as sole carbon source and fermented the following substrates in API 50CH: ribose, D-glucose, D-fructose, galactose, trehalose, maltose, N-acetylglucosamine, starch and glycogen. Negative characteristics for all strains were: phenylalanine deaminase, hydrolysis of urea, malonate utilization, growth in 1 % of tryptone water with added 6% NaCI, utilization of adonitol, D-arabitol, glucuronate, DL-3-hydroxybutyrate, D-tartrate, L-Iysine, L-isoleucine, L-tryptophan and L-methionine; fermentation of erythritol, D-arabinose, D- and L-xylose, adonitol, ~ methyl-xyloside, L-sorbose, dulcitol inositol, a-methylD-mannoside, amygdalin, inulin, melezitose, D-raffinose,
No. of strains Phenon HG
% similarity
60 I
70
I
461
80 I
90
I
Identity
100
I
17
213
A. hydrophila / A. besliarum A. hydrophila / A. besliarum
3
2
1/2
19
3
8
A. veronii biotype sobria
2
4
8
A. veronii biotype obria
7 9 14 12 10
A. sobria CIP 7433 T A. jandaei AT C 49568 T A. 17010 AT 49657T A. schuberlii ATCC 43700T A. veronii ATC 35624 T A. encheleia E T 4342 T A. allosaccharophila
15
16
CECT 4199T
Fig. 1. Simplified dendrogram showing relationships among clusters of Aeromonas spp., based on the SSMI UPGMA analysis.
33
5
4/5/6
2
6
5 6 8
A. caviae / A. media / A. eucrenophila
A. media A. eucrenophila AT
43309T
A. veronii biotype sobria CDC 0437-84 T
462
A.
KAZNOWSKI
xylitol, D-Iyxose, D-tagatose, L-fucose, D-fucose, D-arabitol, L-arabitol, 2-ketogluconate and 5-ketogluconate. All strains were sensitive to chloramphenicol, gentamycin, imipenem, neomycin, nalidixic acid, rifampicin, and tobramycin. None of the strains was susceptible to clindamycin, cloxacillin, and vibriostatic agent 0/129. The final data matrix contained information on 86 strains and 70 unit characters.
ysis yielded 6 clusters and 9 unclustered strains at a similarity level of 85%. The simplified dendrogram obtained by the SSM clustering is shown in Fig. 1. Clusters obtained by the SJIUPGMA analysis had similar composition and were recognized at a similarity level of 75% (results not shown). The cophenetic correlation coefficient was 0.89 for SsMIUPGMA and 0.94 for SJIUPGMA. The biochemical characteristics of the clusters have been included in Table 2.
Clustering of strains by numerical taxonomy The 71 Aeromonas isolates and the 15 type and reference strains were numericaly analysed using the simple matching coefficient and the Jaccard coefficient and grouped by the UPGMA method. The SsMIUPGMA anal-
DNA-DNA hybridization The results of DNA-DNA hybridization showed seven genomic species among the examined Aeromonas isolates (Table 1). The majority of the isolates were included in HG
Table 2. Characteristics of the six phenons of Aeromonas sp. defined by SSMIUPGMA analysis. Percentage of strains with positive reaction in the clusters (no. of strains):
1 Test
(17)
2 (3)
3 (19)
4 (2)
5 (33)
6 (2)
Oxidase Catalase Nitrate reductase Glucose fermentation Gas from glucose Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Phenylalanine deaminase Indole Hydrolysis of: arbutin DNA elastin gelatin lecithin starch Tween 80 urea Voges-Proskauer Gluconate oxidation Malonate Motility Growth in 1 % of tryptone water with: 0% of NaCI 6% of NaCI Growth at:
100 100 100 100 94 100 53 0 0 100
100 100 100 100 100 100 67 0 0 100
100 100 100 100 100 100 53 0 0 100
100 100 100 100 50 100 100 0 0 100
100 100 100 100 0 100 0 0 0 97
100 100 100 100 0 100 0 0 0 100
100 100 100 100 100 100 100 0 100 100 0 100
100 100 100 100 100 100 100 0 100 100 0 100
11 100 0 100 100 100 100 0 100 100 0 100
100 100 0 100 100 100 100 0 100 50 0 100
0 100 0 100 100 100 100 0 0 0 0 91
0 100 0 100 100 100 100 0 0 0 0 0
100 0
100 0
100 0
100 0
100 0
100 0
100 0 100 18 18 94 100 0
100 67 100 33 100 100 100 0
100 100 100 26 32 100
100 91 0 0 12 9
0
100 100 100 0 0 0 0 0
3
100 0 0 50 100 0 0 50
24 65 100
67 0 100
5 42 100
50 100 100
30 85 100
0 0 100
3rC 40.5°C H 2S from cysteine self-pelleting pelleting after boiling Haemolysis aerobic Haemolysis anaerobic Brown soluble pigment Utilization as sole carbon source: L-arabinose Cellobiose D-fructose
11
12
Numerical taxonomy and DNA-DNA hybridizations of Aeromonas
463
Table 2. (Continued). Percentage of strains with positive reaction in the clusters (no. of strains):
Test D-galactose D-glucose Lactose Saccharose Melibiose L-rhamnose Adonitol D-arabitol Mannitol Sorbitol Salicin Acetate Citrate a-D-galacturonate Methyl-a-D-glucopyranoside Glucuronate DL-3-hydroxybutyrate a-ketoglutarate DL-Iactate Urocanic acid D-Tartrate Glycine D L-a-alanine L-arginine L-asparagine L-citrulline L-glutamine L-histidine L-iso-Ieucine L-lysine L-ornithine L-serine L-threonine L-tryptophan L-proline L-methionine Acid from: Glycerol Erythritol D-Arabinose L-Arabinose Ribose D-Xylose L-Xylose Adonitol ~-Methyl-xyloside
Galactose D-Glucose D-Fructose D-Mannose L-Sorbose L-Rhamnose Dulcitol Inositol Mannitol Sorbitol a-Methyl-D-mannoside a-Methyl-D-glucoside N-Acetylglucosamine
1 (17)
2
100 100 6 100
100 100
a a a a 100 100 94 29 59
a a a a a a 94
a a
3 (19)
4 (2)
5 (33)
6
100 100 5 100
100 100 50 100
100 100 67 100
100 100
a a
a a a a
a a a a
a a a a
a a a a
100
100
a
a a
100
100 6 100 88 64 24 9
100
(3)
a 100
a 33
67 100
a a 67
a a a
67 33
a
95 89
a 37
a a a a 11
a a
a a 50 50
a a a a a a a a a
a a 21 79 100
a
a 100
a 50
a a a a a a a
50 50
a a
100 53 88
33 100 100 100
a
a
a
a
a
a
100 88
100 100
100 74
100 100
a a
a a
a a
100 73
100 100
a a
a a
a a
100 100 12
67 100 100
100 89
100 100
a 100
5
a
a
a
a a a a
100 100 50
a
a a
94 91 79
100
100
100
a a
a
a a
71
100 100
0 100 100
63 42 95
5 100
50
a 100
a
a 100
a
a
100
100
100
a
a a
a a
100 100
0
a a
a a a a
100 100
100 100 100 36
100 100 100 100
0 100 100
a a a a
a a a a
100 100 100 100
100 100 100 100
100 100 100 100
100 100 100 100
a a a a
a 33
a a
a a a a
a a a a
100 100
100
100
100
a
a a
100 100
67 100
32 100
100 100 100
15
a a a a
a a
27 91 91 91
(2)
a
a a a
100
a a a a
a a a a
a a a a
100 6
100
a a
a a a
100
100
464
A. KAZNOWSKI
Table 2. (Continued). Percentage of strains with positive reaction in the clusters (no. of strains):
Test Amygdalin Arbutin Esculin Salicin Cellobiose Maltose Lactose Melibiose Saccharose Trehalose Inulin Melezitose D-Raffinose Starch Glycogen Xylitol ~-Gentiobiose
D-Turanose D-Lyxose D-Tagatose D-Fucose L-Fucose D-Arabitol L-Arabitol Gluconate 2-Ketogluconate 5-Ketogluconate Susceptibility to: 01129 150 Ilg Ampicillin Chloramphenicol Clindamycin Cephalothin Cefazolin Cloxacillin Gentamicin Imipenem Nalidixic acid Neomycin Nitrofurantoin Rifampicin Streptomycin Tetracycline Vibramycin Ticarcillin Tobramycin Trimethoprim-sulfamethoxazole
1 (17)
2 (3)
3 (19)
4 (2)
5 (33)
6 (2)
0 100 100 94 59 100 53 0 100 100 0 0 0 100 100 0 0 41 0 0 0 0 0 0 18 0 0
0 100 100 100 0 100 0 0 100 100 0 0 0 100 100 0 0 33 0 0 0 0 0 0 33 0 0
0 5 0 0 53 100 11 0 100 100 0 0 0 100 100 0 0 0 0 0 0 0 0 0 5 0 0
0 0 0 0 100 100 0 0 100 100 0 0 0 100 100 0 0 0 0 0 0 0 0 0 100 0 0
0 100 100 100 91 100 70 0 100 100 0 0 0 100 100 0 33 0 0 0 0 0 0 0 100 0 0
0 100 100 100 100 100 100 0 100 100 0 0 0 100 100 0 0 0 0 0 0 0 0 0 100 0 0
0 0 100 0 6 0 0 100 100 100 100 94 100 100 100 100
0 0 100 0 0 0 0 100 100 100 100 100 100 100 100 100 0 100 100
0 0 100 0 100 59 0 100 100 100 100 100 100 100 100 100 0 100 100
0 0 100 0 0 0 0 100 100 100 100 100 100 100 50 100 0 100 100
0 3 100 0 0 9 0 100 100 100 100 85 100 85 94 97 6 100 94
0 0 100 0 0 50 0 100 100 100 100 100 100 100 100 100 0 100 100
12 100 100
4 (42%), HG 8110 (30%) and HG 3 (18%), only one isolate was of HG 1. Strains of diarrhoeal human faecal samples were included in HG 4 and HG 8110. Isolates from the fish Rutilus rutilus were identified as a member of HG 8110. Six isolates from drinking water belonged to HG 8110, two to HG 2 and two to HG 4. The greatest diversity of genomic groups was found in lake water samples.
Discussion Clusters obtained by numerical analysis (Fig. 1; Table 2) were identified by comparing phenotypic properties with the published diagnostic keys and tables (ABBOTT et ai., 1992; ALTWEGG et ai., 1990; ALTWEGG and LOTHYHOTTENSTEIN; 1991; KAMPFER and ALTWEGG, 1992; CARNAHAN and JOSEPH, 1993; HOLT et ai., 1994; HANNINEN
Numerical taxonomy and DNA-DNA hybridizations of Aeromonas
465
and SIITONEN, 1995) as well as by DNA-DNA hybridization. Ph en on 1 (86.4% similarity, SSMIUPGMA), consisted of strains identified as A. hydrophila and A. bestiarum. This cluster included the reference strain of HG 3, three isolates belonging to HG 2 and 13 isolates to HG 3. The results of this study showed a high phenotypic relatedness within strains of HG 2 and 3. Similar results was obtained by CARNAHAN and JOSEPH (1993). They also found strains of HG 2 and 3 in a single phenotypic cluster. The examined strains displayed most of the reactions reported by CARNAHAN and JOSEPH (1993), although phenon 1 showed a higher percentage of isolates fermenting salicin, possessing lysine decarboxylase activity, susceptibility to cefazolin and resistance to ticarcillin than those described by CARNAHAN and JOSEPH (1993). Isolates clustered in phenon 1 displayed the following important phenotypic features differentiating them from the strains of HG 1: lack of growth at 40.5 °C, utilization of urocanic acid but not of DL-lactate as sole carbon source (Table 2). Similar observations were described previously (ALTWEGG and LOTHY-HoTTENSTEIN, 1991; HANNINEN, 1994; JANDA et al., 1996). Phenon 2 contained a reference strain of HG 1 (ATCC 7966 T ), one isolate belonging to HG 1 and a reference strain of HG 2, i.e. A.bestiarum ATCC 51108 T • An identical result, i.e. reference strain of HG 2 clustering with the strains of HG 1, was also obtained by KAMPFER and ALTWEGG (1992), although these authors used a different set of biochemical tests than the set that was used in this study. Since the strain A. bestiarum ATCC 51108 T is genomically closely related to HG 1 (ALTWEGG, 1990; ALI et al., 1996), it was possible to cluster it with the strains of HG 1. The strains of cluster 2 did not ferment and utilize D-sorbitol, D-cellobiose and lactose. The strains of HG 1 also possessed following features characteristic for this DNA group: utilization of DL-lactate and lack of growth on urocanic acid. Also other authors informed about similar findings (ALTWEGG et al., 1990; ALTWEGG and LOTHY-HoTTENSTEIN, 1991; HANNINEN, 1994). Phenon 2 is phenotypically similar to phenon 1 and is linked to it at 78% similarity. Cluster 3 (87.6% similarity, SSMIUPGMA), comprising 19 strains, and cluster 4 (87.1 % similarity, SSMIUPGMA) containing two isolates, were identified as A. veronii biotype sobria (HG 8/10). Althrough, the reference strain for HG 8/10 (CDC 0437-84) was not found in these phenons, all isolates showed at least 70% relatedness with this strain. Clusters 3 and 4 were linked at 81 % similarity. Most isolates of phenon 3 showed a characteristic biochemical profile for A. veronii biotype sobria: lack of hydrolysis of elastin, arbutin, and esculin, positive Voges-Proskauer reaction and gluconate oxidation test, production of gas from glucose, lack of L-arabinose and salicin fermentation, and susceptibility to cephalotin. Phenon 4 comprised two isolates from children's diarrhoeal stool that were received as A. veronii biotype sobria. These strains showed the following atypical features: fermentation of L-arabinose and gluconate and
HG 8110 into two phenons is further evidence that among this single genomic species there may exist more biovars as previously stated KAMPFER and ALTWEGG (1992) and CARNAHAN and JOSEPH (1993). A. veronii biogroup sobria differs from A. veronii biogroup veronii in its positive arginine dihydrolase rection and in its negative recti on for ornithine decarboxylase, hydrolysis of esculin, and acid production from salicin (JANDA, 1991; KAMPFER and ALTWEGG, 1992). Cluster 5 (85% similarity, SSMIUPGMA) contained 30 isolates identified as members of hybridization group 4, two isolates belonging to the HG 5, and one strain of HG 6. However, the reference strains for HG 5 and HG 6 did not cluster with this phenon. Two reference strains of HG 5 were included in phenon 6, these strains displayed reactions reported by other authors (ALLEN et al., 1983; KAMPFER and ALTWEGG, 1992; CARNAHAN and JOSEPH, 1993; ALTWEGG and LOTHY-HoTTENSTEIN, 1991; ESTEVE, 1995). Isolates found in cluster 5 showed phenotypic features characteristic of the group A. caviae/A. media/A. eucrenophila: a negative reaction in lysine decarboxylase, Voges-Proskauer reaction, HzS production from cysteine, hydrolysis of arbutin, positive tests for Larabinose, cellobiose and salicin fermentation and hydrolysis of esculin (ALTWEGG et al., 1990; KAMPFER and ALTWEGG, 1992; CARNAHAN and JOSEPH, 1993). Phenotypic sinilarity of strains of HG 4-6 was also previously stated by ARDUINO et al., (1988) and by KAMPFER and ALTWEGG (1992). The latter authors found phenotypic diversity within HG 5 and showed a necessity to a subdivision of this genomic species. Recently, Huys et al., (1996) stated also significant genetic heterogeneity in A. eucrenophila (HG 6), which may lead to a further subdivision of this genomic species. Numerical taxonomic studies of Aeromonas strains which had been done before taxonomic changes were introduced had usually been able to separate three phenotypically defined species A. hydrophila, A. caviae and A. sobria (POPOFF and VERON, 1976; BRYANT et al., 1986) but had not been satisfactory in separating the various genomic groups. By introducing several new phenotypic tests and reference strains for newly described genomic species it was possible to use numerical taxonomy for grouping and identifying Aeromonas strains at the genomic species level in this and other studies (KAMPFER and ALTWEGG 1992; CARNAHAN and JOSEPH, 1993; EsTEVE, 1995). However, results of this study and other studies (KAMPFER and ALTWEGG, 1992; CARNAHAN and JOSEPH, 1993) still indicate some taxonomic problems, such as phenotypic diversity within one genomic species and phena overlap. The identification of Aeromonas strains to the genomic species level is difficult because strains belonging to different hybridization groups are biochemically very similar. Using DNA hybridization, KUI]PER et al., (1989), found that eight of 26 (31 %) strains isolated from faeces and phenotypically identified as A. hydrophila, in fact belonged to HG 8110 (A. veronii biotype sobria) whereas some strains initially assigned to A. caviae belonged to the genomic species A. media (HG
susceptibility to cephalothin. Clustering the strains of
5) and A. veronii biotype sob ria (HG 811 0). Similar in-
466
A. KAZNOWSKI
consistencies between phenotypic and genotypic data were reported by other authors (ARDUINO et aI., 1988; ALTWEGG et aI., 1990; ALTWEGG, 1993; CARNAHAN, 1993; KAMPFER and ALTWEGG, 1992). These taxonomic problems may be related with the phenotypic homogeneity (KUI]PER et aI., 1989; ALTWEGG et aI., 1990; ALTWEGG, 1993; CARNAHAN, 1993) or genotypic heterogeneity of some genomic species in Aeromonas (Huys et aI., 1996). In conclusion, the numerical analysis of Aeromonas strains of different sources was in most cases helpful in the identification to the genomic species level. However, the results obtained with the numerical taxonomy were not in perfect agreement with the DNA-DNA hybridization results. Computer analysis showed high phenotypic similarity among strains of HG 1-3 and of HG 4-6. Among isolates of HG 8110 biovariants were found. Some differences were noted in the frequency of phenotypic characters in comparison with the literature data and it can be due to different sources of isolates or different geographical areas. The use of molecular methods in addition to phenotypic characteristics for final identification of Aeromonas isolates with the genomic species seems necessary. Acknowledgments I am very grateful to Dr. M. ALTWEGG, Dr. F. W. HICKMANBRENNER, Dr. A. CARNAHAN and Dr. C. ESTEVE for providing the reference bacterial strains. This work was partially supported by grant no. 0158/P2/94/96 from the Polish Committee for Scientific Research and by an Individual grant from Faculty of Biology, Poznan University.
References ABBOTT, S. L., CHEUNG, W. K. W., KROSKE-BRYSTROM, S., MALEKZADEH, T., JANDA, J. M.: Identification of Aeromonas strains to the genospecies level in the clinical laboratory. J. Clin. Microbiol. 30, 1262-1266 (1992). ALI, A., CARNAHAN, A. M., ALTWEGG. M., LUTHy-HOTTENSTEIN, J., JOSEPH, S. W.: Aeromonas bestiarum sp. nov. (formerly genomospecies DNA group 2 A. hydrophila), a new species isolated from non-human sources. Med. Microbiol. Lett 5, 156-165 (1996). ALLEN, D. A., AUSTIN, B., COLWELL, R. R.: Aeromonas media, a new species isolated from river water. Int. J. System. Bacteri01. 33, 599-604 (1983). ALTWEGG, M., GEISS, H. K.: Aeromonas as a human pathogen. CRC Crit. Rev. Microbiol. 16,253-286 (1989). ALTWEGG, M.: Taxonomy and epidemiology of Aeromonas spp.: the value of new typing methods. Habilitationsschrift, Zurich, ADAG Administration & Druck AG 1990. ALTWEGG, M., STEIGERWALT, A. G., ALTWEGG-BISSIG, R., LUTHYHOTTENSTEIN, J., BRENNER, D. J.: Biochemical identification of Aeromonas genospecies isolated from humans. J. Clin. Microbiol. 28, 258-264 (1990). ALTWEGG, M., LUTHy-HOTTENSTEIN, J.: Methods for the identification
of
DNA
hybridization
groups
in
the
genus
Aeromonas. Experientia, 47, 403-406 (1991). ALTWEGG, M., REEVES, M. W., ALTWEGG-BISSIG, R., BRENNER, D. J.: Multilocus enzyme analysis of genus Aeromonas and its use for species identification. Zbl. Bakt. 275, 28-45 (1991).
ALTWEGG, M.: A polyphasic approach to the classification and identification of Aeromonas strains. Med. Microbiol. Lett. 2, 200-205 (1993). ARDUINO, M. J., HICKMAN-BRENNER, F. W., FARMER III, J. J.: Phenotypic analysis of 132 Aeromonas strains representing 12 DNA hybridization groups. J. Diarrh. Dis. Res. 6, 138 (1988). BRYANT, T. N., LEE, J. v., WEST, P. A., COLWELL, R. R.: Numerical classification of species of Vibrio and related genera. J. Appl. Bacteriol. 61,437-467 (1986). CARNAHAN, A. M., FANNING, G. R., JOSEPH, S. W.: Aeromonas jandaei (formerly genospecies DNA group 9 A. sobria), a new sucrose-negative species isolated from clinical specimens. J. Clin. Microbiol. 29, 560-564 (1991a). CARNAHAN, A. M., CHAKRABORTY, T., FANNING, G. R., VERMA, D., ALI, A., JANDA, J. M., JOSEPH, S. W.: Aeromonas trota sp. nov., an ampicillin-susceptible species isolated from clinical specimens. J. Clin. Microbiol. 29, 1206-1210 (1991b). CARNAHAN, A. M.: Aeromonas taxonomy: A sea of change. Med. Microbiol. Lett. 2, 206-211 (1993). CARNAHAN, A. M., JOSEPH, S. W.: Systematic assessment of geographically and clinically diverse aeromonads. System. Appl. Microbiol. 16,72-84 (1993). COLWELL, R. R., MACDONELL, M. T., DE LEY, J.: Proposal to recognize the family Aeromonadaceae fam. nov. Int. J. System. Bacteriol. 36, 473-477 (1986). DORSCH, M., ASHBOLT, N. J., Cox, P. T., GOODMAN, A. E.: Rapid identification of Aeromonas species using 16 rDNA targeted oligonucleotide primers. A molecular approach based on screening of environmental isolates. J. Appl. Bacteriol. 77, 722-726 (1994). ESTEVE, c.: Numerical taxonomy of Aeromonadaceae and Vibrionaceae associated with reared fish and surrounding fresh and brackish water. System. Appl. Microbiol. 18, 391-402 (1995). ESTEVE, c., GUTIERREZ, M. c., VENTOSA, A.: DNA Relatedness among Aeromonas allosaccharophila strains and DNA hybridization groups of the genus Aeromonas. Int. J. System. Bacteriol. 45, 390-391 (1995a). ESTEVE, c., GUTIERREZ, M. c., VENTOSA, A.: Aeromonas encheleia sp. nov., isolated from European eels. Int. J. System. Bacteriol. 45, 462-466 (1995b). HANNINEN, M. L.: Phenotypic characteristics of the three hybridization groups of Aeromonas hydrophila complex isolated from different sources. J. Appl. Bacteriol. 76, 455-462 (1994). HANNINEN, M. L., SIITONEN, A.: Distribution of Aeromonas phenospecies and genospecies among strains isolated from water, foods or from human clinical samples. Epidemiol. Infect. 115,39-50 (1995). HICKMAN-BRENNER, F. W., MACDONALD, K. L., STEIGERWALT, A. G., FANNING, G. R., BRENNER, D. J., FARMER III, J. J.: Aeromonas veronii, a new ornithine decarboxylase-positive species that may cause diarrhea. J. Clin. Microbiol. 25, 900-906 (1987). HICKMAN-BRENNER, F. W., FANNING, G. R., ARDUINO, M. J., BRENNER, D. J., FARMER III, J. J.: Aeromonas schubertii, a new mannitol-negative species found in human clinical specimens. J. Clin. Microbiol. 26, 1561-1564 (1988). HOLDING, A. J., COLLEE, J. G.: Routine biochemical tests, pp. 2-32. In: Methods in microbiology, Vol. 6A, (J. R. NORRIS, D. W. RIBBONS eds.), London-New York, Academic Press 1971.
HOLT, J. G., KRIEG, N. R., SNEATH, P. H. A., STALEY, J. T., WILLIAMS S. T. (eds): Genus Aeromonas, pp. 190-255. In: Bergey's Manual of Determinative Bacteriology, 9th ed., Baltimore, Williams & Wilkins 1994.
Numerical taxonomy and DNA-DNA hybridizations of Aeromonas Huys, G., COOPMAN, R., JANSSEN, P., KERSTERS, K.: High-resolution genotypic analysis of the genus Aeromonas by AFLP fingerprinting. Int. J. System. Bacteriol. 46, 572-580 (1996). International Committee on Systematic Bacteriology, Subcommittee on the Taxonomy of Vibrionaceae. Int. J. System. Bact. 42, 199-201 (1992) . JANDA, J. M., REITANO, M., BOTTONE, E. B. : Biotyping of Aeromonas isolates as a correlation to delineating a species-associated disease spectrum. J. Clin. Microbiol. 19,44-47 (1984). JANDA J. M., OSHIRO L. S., ABBOTT S. L., DUFFEY P.: Virulence markers of mesophilic aeromonads: Association of the autoaglutination phenomenon with mouse pathogenicity and the presence of a peripherial cell-associated layer. Infect. Immun. 55, 3070-3077 (1987). JANDA, J. M. : Recent advances in the study of the taxonomy, pathogenicity, and infectious syndromes associated with the genus Aeromonas. Clin. Microbiol. Rev. 4, 397-410 (1991). JANDA, J. M., ABBOTT, S. L., KHASHE, S., KELLOGG, G. H., SHIMADA, T.: Further studies on biochemical characteristics and serologic properties of the genus Aeromonas. J. Clin. MicrobioI. 34, 1930-1933 (1996). KAZNOWSKI, A.: A method of colorimetric DNA-DNA hybridization in microplates with covalently immobilized DNA for identification of Aeromonas spp. Med. Microbiol. Lett. 4,362-369 (1995). KAMPFER, P., ALTWEGG, M.: Numerical classification and identification of Aeromonas genospecies. J. Appl. Bacteriol. 72, 341-351 (1992). KUI]PER, E. J., STEIGERWALT, A. G., SCHOENMAKERS, B. C. I. M., PEETERS, M. E, ZANEN, H. c., BRENNER, D. J.: Phenotypic characterization and DNA relatedness in human fecal isolates of Aeromonas spp. J. Clin. Microbiol. 27, 132-138 (1989). LEE, J. V., HENDRIE, M. S., SHEWAN, J. M.: Identification of Aeromonas, Vibrio and related organisms, pp. 151-166. In: Identification Methods for Microbiologists (E A. SKINNER, D. W. LOVELOCK, eds.) London, New York, Toronto, Sydney, San Francisco: Acedemic Press 1979. MARTINETTI LUCCHINI, G., ALTWEGG, M.: rRNA gene restriction patterns as taxonomic tools for the genus Aeromonas. Int. J. System. Bacteriol. 42, 384-389 (1992). MARTINEZ-MuRCIA, A. J., ESTEVE, c., GARAY, E., COLLINS, M. D.: Aeromonas allosaccharophila sp. nov., a new mesophilic member of the genus Aeromonas. FEMS Microbiol. Lett. 91, 199-206 (1992).
467
MILLERSHIP, S. E., CHATTOPADHYAY, B.: Methods for the isolation of Aeromonas hydrophila and Plesiomonas shigelloides from faeces. J. Hyg. (Cambridge) 92, 145-152 (1984). POPOFF, M., VERON, M.: A taxonomic study of the Aeromonas hydrophila-Aeromonas punctata group. J. Gen. Microbiol. 94,11-22 (1976). POPOFF, M .: Genus III: Aeromonas, pp. 545-547. In: Bergey's Manual of Systematic Bacteriology Vol 1, (N. R. KRIEG, J. G. HOLT, eds.), Baltimore-London, Wiliams & Wilkins 1984. SCHARMANN, W.: Vorkommen von Elastase bei Pseudomonas und Aeromonas. Zbl. Bakt. Hyg. A 220, 435-442 (1972). SCHUBERT, R. H. W., HEGAZI, M.: Aeromonas eucrenophila species nova and Aeromonas caviae, a later and illegitimate synonym of Aeromonas punctata. Zbl. Bakt. Hyg. A 268, 34-39 (1988). SCHUBERT, R. H. W.: Aeromonads and their significance as potentios pathogens in water. J. Appl. Bacteriol. Symp. Suppl. 70, 131S-135S (1991). SNEATH, P. H. A., JOHNSON, R.: The influence of numerical taxonomic similarities of errors in microbiological tests. J. Gen. Microbiol. 72,377-392 (1972). SNEATH, P. H. A., SOKAL, R. R.: Numerical Taxonomy: The Principles and Practice of Numerical Classification, 2nd ed., San Francisco, W. H. Freeman & Co. 1973. VERON, M., GASSER, E: Sur la detection de l'hydrogene sulfure produit par certaines enterobacteriacees dans les milieux dits de diagnostic rapide. Ann. Inst. Past. 105,524-534 (1963) . VERON, M.: Nutrition et taxonomie des Enterobacteriaceae et bacteries voisines. I. Methods d'etude des auxanogrammes. Ann. Microbiol. Inst. Past. 126 A, 267-274 (1975). WAYNE, L. G., BRENNER, D . J., COLWELL, R. R., GRIMONT, P. A. D., KANDLER, 0., KRICHEVSKY, M. I., MOORE, L. H ., MOORE, W. E. c., MURRAY, R. G. E., STACKER BRANDT, E., STARR, M. P., TROPER, H. P.: Report of the ad hoc Committee on reconciliation of approaches to bacterial systematics. Int. J. System Bacteriol. 37,463-464 (1987).
Corresponding author: Dr. ADAM KAZNOWSKI, Department of Microbiology, Institute of Experimental Biology, A. Mickiewicz University, ul. Fredry 10, 61-701 Poznan, Poland. Tel. (048 ) 61 524953; Fax (048) 61 8523615; e-mail: [email protected]
Numerial Taxonomy and DNA-DNA Hybridizations of Aeromonas strains Isolated from Human diarrhoeal Stool, Fish and Environment ADAM KAZNOWSKI Department of Microbiology, Institute of Experimental Biology, A. Mickiewicz University, Poznan, Poland Received: March 26, 1997
Summary A numerical taxonomic study and DNA-DNA hybridizations were performed on seventy-one isolates of Aeromonas collected from children diarrhoeal stool, dead and alive fish Rutilus rutilus, and environment. These isolates were compared with the fifteen type and reference strains of the presently recognized Aeromonas genomic species. Results of the phenotypic tests were analysed by numerical taxonomic techniques, using the simple matching coefficient (SSM) and Jaccard coefficient (SJ)' and the unweighted average linkage clustering (UPGMA). At SSM> 85% and SJ > 75% these analyses resolved 6 groups and 9 single strains. DNA-DNA hybridization experiments revealed seven genomic groups among the examined Aeromonas isolates. Clusters were identified as A. hydrophila/A. bestiarum (two groups), A. veronii biotype sobria (two groups), A. caviae/A. media/A. eucrenophila, and A. media. The results obtained with the numerical taxonomy were not in perfect agreement with the DNA-DNA hybridization results and showed genetic heterogeneity of some phena. In some cases strains of one genomic species were found in different clusters or one cluster contained more than one hybridization group (HG). Among isolates of HG 8/10 biovariants were found. Strains of HG 1-3 and of HG 4-6 showed high phenotypic homogeneity. The results of this study emphasise the need to use of molecular methods in addition to phenotypic characteristics for identification of Aeromonas isolates with the genomic species. Key words: Aeromonas - Numerical taxonomy - DNA:DNA hybridization - genomic species
Introduction Bacteria belonging to the genus Aeromonas are ubiquitous aquatic organisms found in freshwater and sewage (SCHUBERT, 1991). They were also cultured from food (HANNINEN and SUTONEN, 1995). Some Aeromonas species are pathogenic for cold and warm-blooded animals and can be responsible for intestinal as well as several extraintestinal human diseases (ALTWEGG and GEISS, 1989; ALTWEGG, 1990; JANDA, 1991). In the Bergey's Manual of Systematic Bacteriology, POPOFF (1984), on the basis of phenotypic analyses, included this genus into Vibrionaceae family and recognized four species: A. hydrophila, A. caviae, A. sobria and A. salmonicida. COLWELL et al. (1986) proposed to separate the genus Aeromonas from Vibrionaceae and created a new family Aeromonadaceae. This proposition was aaccepted by the International Committee on Sys-
tematic Bacteriology, Subcommittee on the Taxonomy of
Vibrionaceae (1992). Furthermore, DNA-DNA hybridization experiments showed significant genetic heterogeneity of this genus. Currently, in the Aeromonas at least 15-16 DNA-DNA hybridization groups (HGs) are recognized (ESTEVE et al. 1995b), equivalent to genomic species comprising strains with approximately 70% or greater DNA-DNA relatedness with 5% or less divergence within the related sequences (WAYNE et al. 1987). In addition to the four species listed by POPOFF (1984), several new species were recognized: A. media (HG 5) (ALLEN et aI., 1983), A. veronii (HG 8110) (HICKMANBRENNER et aI., 1987), A. eucrenophila (HG 6) (SCHUBERT and HEGAZI, 1988), A. schubertii (HG 12) (HICKMAN-BRENNER et al., 1988), A. jandaei (HG 9) (CARNAHAN et aI., 1991a), A. trota (HG 14) (CARNAHAN et al., 1991b), A. allosaccharophila (HG 15) (MARTINEZ-MuRCIA et aI., 1992; ESTEVE et aI., 1995a), A. encheleia
Numerical taxonomy and DNA-DNA hybridizations of Aeromonas
(ESTEVE et ai., 1995b) and recently A. bestiarum (HG 2) (ALI et ai., 1996). Moreover, at least two unnamed genotypes, HG 11 and HG 13, were recognized (CARNAHAN and JOSEPH, 1993). Currently, A. caviae comprises strains of genotype 4, A. sobria includes isolates of HG 7, A. hydrophila encompasses isolates belonging to HG 1 and motile strains of HG 3, whereas A. salmonicida includes un motile isolates of HG 3 (JANDA, 1991; CARNAHAN and JOSEPH, 1993). However, the phenotypic similarity of some of the Aeromonas genomic species causes significant difficulties in their identification on the basis of biochemical tests (KUIJPER et ai., 1989; KAMPFER and ALTWEGG, 1992; ALTWEGG, 1993; CARNAHAN, 1993). Recently, several molecular methods useful in Aeromonas genomic species identification were proposed: mutilocus enzyme electrophoresis (ALTWEGG, 1990; ALTWEGG et ai., 1991), ribotyping (MART/NETTI LUCCHINI and ALTWEGG, 1992; HANNINEN and SIITONEN, 1995), PCR (DORSCH et ai., 1994), colorimetric DNA-DNA hybridization in microplates (KAZNOWSKI, 1995) and AFLP-fingerprinting (Huys et ai., 1996). Most of the numerical taxonomic studies of Aeromonas strains were performed before taxonomic changes were introduced. Only in three recently published papers reference strains of new genomic species were introduced (KAMPFER and ALTWEGG, 1992; CARNAHAN and JOSEPH, 1993; ESTEVE, 1995). Numerical taxonomy of Aeromonas strains, simultaneously defined at the genomic level by DNA-DNA hybridization, was conducted only in one case (KAMPFER and ALTWEGG, 1992), where the authors analysed strains mostly isolated from human feces. The present study was undertaken to cluster by numerical taxonomy strains of Aeromonas isolated from different sources (mostly from environment) and to evaluate results with the genomic species determination by colorimetric DNA-DNA hybridization.
Materials and Methods Bacterial strains: A total of 71 Aeromonas isolates were studied by phenotypic traits and by colorimetric hybridization method for identification with the hybridization groups (HGs). These strains were isolated from children diarrhoeal stool, alive and dead fish Rutilus rutilus, drinking water, recreational lake water, the Warta river water, water of Gdansk Bay in the Baltic Sea, municipal sewage and water-oil aluminium rolling-mill emulsion. The strains used in this study, their designation and sources are shown in Table 1. The isolates were obtained by enrichment in alkaline peptone water (pH 8.6), followed by inoculation onto bile salts brilliant green agar (BBG), xylose - deoxycholate agar (XDCA) (MILLERSHIP and CHATIOPADHYAY, 1984) and blood agar with 15 mg/l of ampicillin. The cultures were maintained on Tryptic Soy Agar (TSA medium) at room temperature. The following type and reference strains were included in this study: A. hydrophila ATCC 7966 T (HG 1), A. bestiarum ATCC 51108 T (HG 2), A. hydrophila CDC 0434-84 (HG 3), A. caviae ATCC 15468 T(HG 4), A. media CDC 0862-83 (HG 5), A. media CDC 9072-83 T (HG 5), A. eucrenophila ATCC 23309 T (HG 6), A. sobria CIP 7433 T(HG 7), A. veronii biotype sobria CDC 0437-84 (HG8/l0), A. veronii biotype veronii
459
ATCC 35624 T (HG 10/8), A. jandaei ATCC 49568 T (HG 9), A. schubertii ATCC 4370QT (HG 12), A. trota ATCC 4965?T (HG 14), A. allosaccharophila CECT 4199 T (HG 15), and A. encheleia CECT 4342T. Phenotypic properties: Each Aeromonas strain was tested for 139 phenotypic properties. Unless otherwise indicated, incubation was done at 30°C. Cell morphology and Gram-straining reaction were tested after 24 hour incubation on TSA medium Production of diffusible brown pigment was examined in 7days cultures on TSA. Motility, salt tolerance, ability to grow in broth at 37°C and 40.5 °C, lecithinase and amylase were determined by means of the techniques described by LEE et al. (1979). Haemolysis was determined on TSA medium supplemented with 5% sheep blood. Anaerobic haemolysis of sheep blood was found out in a jar using gas generating box (bioMerieux). Elastase was determined by the method of SCHARMANN (1972). The test for oxidative and fermentative metabolism of glucose was performed by the method of HUGH and LEIFSON (HOLDING and COLLEE, 1971). Gas production from glucose was tested in the same medium with Durham tube but without agar. H 2 S production from cysteine was determined on cysteine-iron agar (VERON and GASSER, 1963). Self-pelleting and pelleting after boiling were described by JANDA et al. (1987). The following tests were conducted as described by HOLDING and COLLEE (1971): cytochrome oxidase (KOVACS method), catalase, nitrate reductase, arginine dihydrolase (THORNLEY method), lysine and ornithine decarboxylase (MOELLER method), Voges-Proskauer reaction, malonate utilization, urease production (CHRISTENSEN method), phenylalanine deaminase, gluconate oxidation, indole production in peptone water, hydrolysis of gelatine, DNA and Tween 80. Hydrolysis of arbutin was examined by the method of JANDA et al. (1984). Utilization of sole carbon sources was determined on M 70 medium (VERON, 1975) supplemented with 0.05% peptone by means of a multipoint inoculator. The substrates serving as carbon sources were filter-sterilized and bacterial growth was examined for 14 days. The carbon sources used included: L-arabinose, cellobiose, fructose, D-galactose, D-glucose, lactose, saccharose, melibiose, L-rhamnose, salicin, adonitol, D-arabitol, mannitol, sorbitol, acetate, citrate, a-D-galacturonate, methyla-D-glucopyranoside, glucuronate, DL-lactate, DL-3-hydroxybutyrate, a-ketoglutarate, urocanic acid, D-tartrate, glycine, DL-a-alanine, L-arginine, L-asparagine, L-citrulline, L-glutamine, L-histidine, L-isoleucine, L-lysine, L-serine, L-ornithine, L-proline, L-threonine, L-tryptophan, and L-methionine. Fermentation of carbohydrates, glycosides and polyalcohols were assayed in commercial kit API 50CH (bioMerieux, France). Sensitivity to the vibriostatic agent 01129 was detected on TSA medium containing 150 mg/l of this compound. Susceptibility to antibacterial compounds was determined by disk diffusion method on MUELLER-HINTON medium (Difco) by means of BECTON-DICKINSON disks: ampicillin (10 pg), cloxacillin (1 pg), chloramphenicol (30 pg), cephalothin (30 pg), cefazolin (30 pg), clindamycin (2 pg), gentamycin (10 pg), imipenem (10 pg), nalidixic acid (30 pg), neomycin (30 pg), nitrofurantoin (200 pg), rifampicin (10 pg), streptomycin (30 pg), tetracycline (30 pg), vibramycin (30 pg), ticarcillin (75 pg), tobramycin (10 pg), and sulfamethoxazole-trimethoprim. Testing and results interpretation were done according to the manufacturer's instructions. Numerical analysis of phenotypic results: The test error was evaluated by examining 15 strains in duplicate, according to the method described by SNEATH and JOHNSON (1972). The test results were coded 1 for positive results, 0 for negative results and 9 for noncomparable data. Similarities were calculated using the simple matching coefficient (SSM) and Jaccard's coefficient (SJ)' Clustering of the 86 strains was achieved by the unweight-
460
A. KAZNOWSKI
Table 1. Designation and source of strains examined by numerical analysis and DNA-DNA hybridization. Phenon or single strain
HG
Taxon
Strain no.
Source of isolation
1
2
A. bestiarum
3
A. hydrophila
2
1
A. hydrophila
3
2 8110
A. bestiarum A. veronii biotype sobria
AKl,AK41 AK 115 AK 46, AK 50, AK 76, AK 130, AK 131 AK 409, AK 410 AK 400, AK 401, AK 402 AK 106, AK 117, AK 125 CDC 0434-84 AK44 ATCC 7966 T ATCC 51108 T AK 382, AK 389, AK 392 AK 12,AK 59 AK 100, AK 102, AK 120, AK 411, AK412, AK 413 AK279 AK 156, AK 160, AK 164, AK 180 AK 165,AK 167,AK 176 AK 387, AK 391
lake water drinking water lake water drinking water sea water rolling-mill emulsion fresh water lake water canned milk diseased fish human stool lake water drinking water
4
8110
5
4
A. veronii biotype sobria A . caviae
5
A. media
6 5
A. eucrenophila A. media
6
Unclustered strains: 6 7 8110 9 10/8 12 14 15
A. eucrenophila A.sorbria A. veronii biotype sobria A. jandaei A. veronii biotype veronii A. schubertii A. trota A . allosaccharophila A . encheleia
sewage dead fish healthy fish human stool
AK 48, AK 335, AK 338, AK 339 lake water AK 266, AK 276, AK 281, AK 296 sewage AK 404, AK 405 river water AK 104, AK 126 drinking water AK 406, AK 407, AK 408 sea water AK 375, AK 376, AK 377, AK 378, AK 379, human stool AK 380, AK 383, AK 384, AK 385, AK 386, AK 388, AK 390, AK 393 AK 220, AK 232 rolling-mill emulsion ATCC 15468 T guinea pig AK42 lake water AK403 sea water AK65 lake water CDC 0862-83 fish CDC 9072-83 T fresh water ATCC23309 T CIP 7433 T CDC 0437-84 ATCC 49568 T ATCC 35624T ATCC 43700 T ATCC4965?T CECT 4199 T CECT 4342 T
ed pair group method using arithmetic averages (UPGMA), cophenetic correlation (i.e. the correlation between the respective values in the similarity matrix and the corresponding dendogram) was calculated using the correlation coefficient r (SNEATH and SOKAL, 1973). Computing was done using an DTK computer 486 DX2. Genomic species determination: Preparation and purification of DNA was performed as described by ALTWEGG et al. (1990). The colorimetric DNA-DNA hybridizations in CovaLinc microplates (Nunc) were performed as previously described (KAZNOWSKI, 1995). Hybridization method includes immobilization of nonlabelled reference DNA to micro titer wells, hy-
fresh water fish fish human stool sputum abscess human stool diseased elvers fish
bridization of the DNA with DNA probe of examined or reference strain, reacting the probe with streptavidin-peroxidase conjugate, and assaying of enzyme activity with the 3, 3', 5, 5'tetramethylbenzidine at 450 nm with a microplate reader. The nucleic acids probes were constructed by DNA labelling with photo biotin (Vector Lab., USA) according to the manufacturer's instructions. Before hybridization experiments, nucleic acid was fragmented by ten passages through a pipette with a tip possessing capillary (0.3x15 mm) to fragment lengths of 500-800 kDa. The fragment size was checked byelectrophoresis on a 1 % agarose gel in a 0.04 M Tris-acetate buffer (pH 8.0), using as reference restriction fragments Hind III of Lambda DNA (Boeringer, Mannheim). Gels were stained with ethi-
Numerical taxonomy and DNA-DNA hybridizations of Aeromonas dium bromide (0.5 pg/ml) and inspected in a UV transilluminator. For the estimation of DNA relatedness the absorbance of the well of herring sperm DNA was taken as 0% and the absorbance of the well for each reference DNA was taken as 100%. Bacterial strains showing 70% or more relatedness to the reference strain of a given HG were assigned to that HG (WAYNE et al., 1987).
Results Test reproducibility Seventy-one isolates of Aeromonas and fifteen type and reference strains were examined for 139 phenotypic characters. The average probability of an erroneous result, calculated from pooled variance of all the unit characters, scored for the 15 duplicate strains was 3.7%. Values of Sj2 ranged zero for most of the tests through 0.15 for hemolysis reaction on sheep blood agar, growth on glycine, L-arginine, L-histidine, and cellobiose. This is acceptable according to the criteria of SNEATH and JOHNSON (1972).
Data matrix Fifty-nine biochemical tests and 10 antimicrobial susceptibility tests were deleted from the final data matrix because they gave only positive or negative results. All isolates were Gram-negative, positive for catalase, cytochrome oxidase, oxidation and fermentation of glucose, nitrate reductase, hydrolysis of DNA, gelatin, lecithin, starch and Tween 80, growth without NaCI and at 37°C. All strains used D-glucose and D-fructose as sole carbon source and fermented the following substrates in API 50CH: ribose, D-glucose, D-fructose, galactose, trehalose, maltose, N-acetylglucosamine, starch and glycogen. Negative characteristics for all strains were: phenylalanine deaminase, hydrolysis of urea, malonate utilization, growth in 1 % of tryptone water with added 6% NaCI, utilization of adonitol, D-arabitol, glucuronate, DL-3-hydroxybutyrate, D-tartrate, L-Iysine, L-isoleucine, L-tryptophan and L-methionine; fermentation of erythritol, D-arabinose, D- and L-xylose, adonitol, ~ methyl-xyloside, L-sorbose, dulcitol inositol, a-methylD-mannoside, amygdalin, inulin, melezitose, D-raffinose,
No. of strains Phenon HG
% similarity
60 I
70
I
461
80 I
90
I
Identity
100
I
17
213
A. hydrophila / A. besliarum A. hydrophila / A. besliarum
3
2
1/2
19
3
8
A. veronii biotype sobria
2
4
8
A. veronii biotype obria
7 9 14 12 10
A. sobria CIP 7433 T A. jandaei AT C 49568 T A. 17010 AT 49657T A. schuberlii ATCC 43700T A. veronii ATC 35624 T A. encheleia E T 4342 T A. allosaccharophila
15
16
CECT 4199T
Fig. 1. Simplified dendrogram showing relationships among clusters of Aeromonas spp., based on the SSMI UPGMA analysis.
33
5
4/5/6
2
6
5 6 8
A. caviae / A. media / A. eucrenophila
A. media A. eucrenophila AT
43309T
A. veronii biotype sobria CDC 0437-84 T
462
A.
KAZNOWSKI
xylitol, D-Iyxose, D-tagatose, L-fucose, D-fucose, D-arabitol, L-arabitol, 2-ketogluconate and 5-ketogluconate. All strains were sensitive to chloramphenicol, gentamycin, imipenem, neomycin, nalidixic acid, rifampicin, and tobramycin. None of the strains was susceptible to clindamycin, cloxacillin, and vibriostatic agent 0/129. The final data matrix contained information on 86 strains and 70 unit characters.
ysis yielded 6 clusters and 9 unclustered strains at a similarity level of 85%. The simplified dendrogram obtained by the SSM clustering is shown in Fig. 1. Clusters obtained by the SJIUPGMA analysis had similar composition and were recognized at a similarity level of 75% (results not shown). The cophenetic correlation coefficient was 0.89 for SsMIUPGMA and 0.94 for SJIUPGMA. The biochemical characteristics of the clusters have been included in Table 2.
Clustering of strains by numerical taxonomy The 71 Aeromonas isolates and the 15 type and reference strains were numericaly analysed using the simple matching coefficient and the Jaccard coefficient and grouped by the UPGMA method. The SsMIUPGMA anal-
DNA-DNA hybridization The results of DNA-DNA hybridization showed seven genomic species among the examined Aeromonas isolates (Table 1). The majority of the isolates were included in HG
Table 2. Characteristics of the six phenons of Aeromonas sp. defined by SSMIUPGMA analysis. Percentage of strains with positive reaction in the clusters (no. of strains):
1 Test
(17)
2 (3)
3 (19)
4 (2)
5 (33)
6 (2)
Oxidase Catalase Nitrate reductase Glucose fermentation Gas from glucose Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Phenylalanine deaminase Indole Hydrolysis of: arbutin DNA elastin gelatin lecithin starch Tween 80 urea Voges-Proskauer Gluconate oxidation Malonate Motility Growth in 1 % of tryptone water with: 0% of NaCI 6% of NaCI Growth at:
100 100 100 100 94 100 53 0 0 100
100 100 100 100 100 100 67 0 0 100
100 100 100 100 100 100 53 0 0 100
100 100 100 100 50 100 100 0 0 100
100 100 100 100 0 100 0 0 0 97
100 100 100 100 0 100 0 0 0 100
100 100 100 100 100 100 100 0 100 100 0 100
100 100 100 100 100 100 100 0 100 100 0 100
11 100 0 100 100 100 100 0 100 100 0 100
100 100 0 100 100 100 100 0 100 50 0 100
0 100 0 100 100 100 100 0 0 0 0 91
0 100 0 100 100 100 100 0 0 0 0 0
100 0
100 0
100 0
100 0
100 0
100 0
100 0 100 18 18 94 100 0
100 67 100 33 100 100 100 0
100 100 100 26 32 100
100 91 0 0 12 9
0
100 100 100 0 0 0 0 0
3
100 0 0 50 100 0 0 50
24 65 100
67 0 100
5 42 100
50 100 100
30 85 100
0 0 100
3rC 40.5°C H 2S from cysteine self-pelleting pelleting after boiling Haemolysis aerobic Haemolysis anaerobic Brown soluble pigment Utilization as sole carbon source: L-arabinose Cellobiose D-fructose
11
12
Numerical taxonomy and DNA-DNA hybridizations of Aeromonas
463
Table 2. (Continued). Percentage of strains with positive reaction in the clusters (no. of strains):
Test D-galactose D-glucose Lactose Saccharose Melibiose L-rhamnose Adonitol D-arabitol Mannitol Sorbitol Salicin Acetate Citrate a-D-galacturonate Methyl-a-D-glucopyranoside Glucuronate DL-3-hydroxybutyrate a-ketoglutarate DL-Iactate Urocanic acid D-Tartrate Glycine D L-a-alanine L-arginine L-asparagine L-citrulline L-glutamine L-histidine L-iso-Ieucine L-lysine L-ornithine L-serine L-threonine L-tryptophan L-proline L-methionine Acid from: Glycerol Erythritol D-Arabinose L-Arabinose Ribose D-Xylose L-Xylose Adonitol ~-Methyl-xyloside
Galactose D-Glucose D-Fructose D-Mannose L-Sorbose L-Rhamnose Dulcitol Inositol Mannitol Sorbitol a-Methyl-D-mannoside a-Methyl-D-glucoside N-Acetylglucosamine
1 (17)
2
100 100 6 100
100 100
a a a a 100 100 94 29 59
a a a a a a 94
a a
3 (19)
4 (2)
5 (33)
6
100 100 5 100
100 100 50 100
100 100 67 100
100 100
a a
a a a a
a a a a
a a a a
a a a a
100
100
a
a a
100
100 6 100 88 64 24 9
100
(3)
a 100
a 33
67 100
a a 67
a a a
67 33
a
95 89
a 37
a a a a 11
a a
a a 50 50
a a a a a a a a a
a a 21 79 100
a
a 100
a 50
a a a a a a a
50 50
a a
100 53 88
33 100 100 100
a
a
a
a
a
a
100 88
100 100
100 74
100 100
a a
a a
a a
100 73
100 100
a a
a a
a a
100 100 12
67 100 100
100 89
100 100
a 100
5
a
a
a
a a a a
100 100 50
a
a a
94 91 79
100
100
100
a a
a
a a
71
100 100
0 100 100
63 42 95
5 100
50
a 100
a
a 100
a
a
100
100
100
a
a a
a a
100 100
0
a a
a a a a
100 100
100 100 100 36
100 100 100 100
0 100 100
a a a a
a a a a
100 100 100 100
100 100 100 100
100 100 100 100
100 100 100 100
a a a a
a 33
a a
a a a a
a a a a
100 100
100
100
100
a
a a
100 100
67 100
32 100
100 100 100
15
a a a a
a a
27 91 91 91
(2)
a
a a a
100
a a a a
a a a a
a a a a
100 6
100
a a
a a a
100
100
464
A. KAZNOWSKI
Table 2. (Continued). Percentage of strains with positive reaction in the clusters (no. of strains):
Test Amygdalin Arbutin Esculin Salicin Cellobiose Maltose Lactose Melibiose Saccharose Trehalose Inulin Melezitose D-Raffinose Starch Glycogen Xylitol ~-Gentiobiose
D-Turanose D-Lyxose D-Tagatose D-Fucose L-Fucose D-Arabitol L-Arabitol Gluconate 2-Ketogluconate 5-Ketogluconate Susceptibility to: 01129 150 Ilg Ampicillin Chloramphenicol Clindamycin Cephalothin Cefazolin Cloxacillin Gentamicin Imipenem Nalidixic acid Neomycin Nitrofurantoin Rifampicin Streptomycin Tetracycline Vibramycin Ticarcillin Tobramycin Trimethoprim-sulfamethoxazole
1 (17)
2 (3)
3 (19)
4 (2)
5 (33)
6 (2)
0 100 100 94 59 100 53 0 100 100 0 0 0 100 100 0 0 41 0 0 0 0 0 0 18 0 0
0 100 100 100 0 100 0 0 100 100 0 0 0 100 100 0 0 33 0 0 0 0 0 0 33 0 0
0 5 0 0 53 100 11 0 100 100 0 0 0 100 100 0 0 0 0 0 0 0 0 0 5 0 0
0 0 0 0 100 100 0 0 100 100 0 0 0 100 100 0 0 0 0 0 0 0 0 0 100 0 0
0 100 100 100 91 100 70 0 100 100 0 0 0 100 100 0 33 0 0 0 0 0 0 0 100 0 0
0 100 100 100 100 100 100 0 100 100 0 0 0 100 100 0 0 0 0 0 0 0 0 0 100 0 0
0 0 100 0 6 0 0 100 100 100 100 94 100 100 100 100
0 0 100 0 0 0 0 100 100 100 100 100 100 100 100 100 0 100 100
0 0 100 0 100 59 0 100 100 100 100 100 100 100 100 100 0 100 100
0 0 100 0 0 0 0 100 100 100 100 100 100 100 50 100 0 100 100
0 3 100 0 0 9 0 100 100 100 100 85 100 85 94 97 6 100 94
0 0 100 0 0 50 0 100 100 100 100 100 100 100 100 100 0 100 100
12 100 100
4 (42%), HG 8110 (30%) and HG 3 (18%), only one isolate was of HG 1. Strains of diarrhoeal human faecal samples were included in HG 4 and HG 8110. Isolates from the fish Rutilus rutilus were identified as a member of HG 8110. Six isolates from drinking water belonged to HG 8110, two to HG 2 and two to HG 4. The greatest diversity of genomic groups was found in lake water samples.
Discussion Clusters obtained by numerical analysis (Fig. 1; Table 2) were identified by comparing phenotypic properties with the published diagnostic keys and tables (ABBOTT et ai., 1992; ALTWEGG et ai., 1990; ALTWEGG and LOTHYHOTTENSTEIN; 1991; KAMPFER and ALTWEGG, 1992; CARNAHAN and JOSEPH, 1993; HOLT et ai., 1994; HANNINEN
Numerical taxonomy and DNA-DNA hybridizations of Aeromonas
465
and SIITONEN, 1995) as well as by DNA-DNA hybridization. Ph en on 1 (86.4% similarity, SSMIUPGMA), consisted of strains identified as A. hydrophila and A. bestiarum. This cluster included the reference strain of HG 3, three isolates belonging to HG 2 and 13 isolates to HG 3. The results of this study showed a high phenotypic relatedness within strains of HG 2 and 3. Similar results was obtained by CARNAHAN and JOSEPH (1993). They also found strains of HG 2 and 3 in a single phenotypic cluster. The examined strains displayed most of the reactions reported by CARNAHAN and JOSEPH (1993), although phenon 1 showed a higher percentage of isolates fermenting salicin, possessing lysine decarboxylase activity, susceptibility to cefazolin and resistance to ticarcillin than those described by CARNAHAN and JOSEPH (1993). Isolates clustered in phenon 1 displayed the following important phenotypic features differentiating them from the strains of HG 1: lack of growth at 40.5 °C, utilization of urocanic acid but not of DL-lactate as sole carbon source (Table 2). Similar observations were described previously (ALTWEGG and LOTHY-HoTTENSTEIN, 1991; HANNINEN, 1994; JANDA et al., 1996). Phenon 2 contained a reference strain of HG 1 (ATCC 7966 T ), one isolate belonging to HG 1 and a reference strain of HG 2, i.e. A.bestiarum ATCC 51108 T • An identical result, i.e. reference strain of HG 2 clustering with the strains of HG 1, was also obtained by KAMPFER and ALTWEGG (1992), although these authors used a different set of biochemical tests than the set that was used in this study. Since the strain A. bestiarum ATCC 51108 T is genomically closely related to HG 1 (ALTWEGG, 1990; ALI et al., 1996), it was possible to cluster it with the strains of HG 1. The strains of cluster 2 did not ferment and utilize D-sorbitol, D-cellobiose and lactose. The strains of HG 1 also possessed following features characteristic for this DNA group: utilization of DL-lactate and lack of growth on urocanic acid. Also other authors informed about similar findings (ALTWEGG et al., 1990; ALTWEGG and LOTHY-HoTTENSTEIN, 1991; HANNINEN, 1994). Phenon 2 is phenotypically similar to phenon 1 and is linked to it at 78% similarity. Cluster 3 (87.6% similarity, SSMIUPGMA), comprising 19 strains, and cluster 4 (87.1 % similarity, SSMIUPGMA) containing two isolates, were identified as A. veronii biotype sobria (HG 8/10). Althrough, the reference strain for HG 8/10 (CDC 0437-84) was not found in these phenons, all isolates showed at least 70% relatedness with this strain. Clusters 3 and 4 were linked at 81 % similarity. Most isolates of phenon 3 showed a characteristic biochemical profile for A. veronii biotype sobria: lack of hydrolysis of elastin, arbutin, and esculin, positive Voges-Proskauer reaction and gluconate oxidation test, production of gas from glucose, lack of L-arabinose and salicin fermentation, and susceptibility to cephalotin. Phenon 4 comprised two isolates from children's diarrhoeal stool that were received as A. veronii biotype sobria. These strains showed the following atypical features: fermentation of L-arabinose and gluconate and
HG 8110 into two phenons is further evidence that among this single genomic species there may exist more biovars as previously stated KAMPFER and ALTWEGG (1992) and CARNAHAN and JOSEPH (1993). A. veronii biogroup sobria differs from A. veronii biogroup veronii in its positive arginine dihydrolase rection and in its negative recti on for ornithine decarboxylase, hydrolysis of esculin, and acid production from salicin (JANDA, 1991; KAMPFER and ALTWEGG, 1992). Cluster 5 (85% similarity, SSMIUPGMA) contained 30 isolates identified as members of hybridization group 4, two isolates belonging to the HG 5, and one strain of HG 6. However, the reference strains for HG 5 and HG 6 did not cluster with this phenon. Two reference strains of HG 5 were included in phenon 6, these strains displayed reactions reported by other authors (ALLEN et al., 1983; KAMPFER and ALTWEGG, 1992; CARNAHAN and JOSEPH, 1993; ALTWEGG and LOTHY-HoTTENSTEIN, 1991; ESTEVE, 1995). Isolates found in cluster 5 showed phenotypic features characteristic of the group A. caviae/A. media/A. eucrenophila: a negative reaction in lysine decarboxylase, Voges-Proskauer reaction, HzS production from cysteine, hydrolysis of arbutin, positive tests for Larabinose, cellobiose and salicin fermentation and hydrolysis of esculin (ALTWEGG et al., 1990; KAMPFER and ALTWEGG, 1992; CARNAHAN and JOSEPH, 1993). Phenotypic sinilarity of strains of HG 4-6 was also previously stated by ARDUINO et al., (1988) and by KAMPFER and ALTWEGG (1992). The latter authors found phenotypic diversity within HG 5 and showed a necessity to a subdivision of this genomic species. Recently, Huys et al., (1996) stated also significant genetic heterogeneity in A. eucrenophila (HG 6), which may lead to a further subdivision of this genomic species. Numerical taxonomic studies of Aeromonas strains which had been done before taxonomic changes were introduced had usually been able to separate three phenotypically defined species A. hydrophila, A. caviae and A. sobria (POPOFF and VERON, 1976; BRYANT et al., 1986) but had not been satisfactory in separating the various genomic groups. By introducing several new phenotypic tests and reference strains for newly described genomic species it was possible to use numerical taxonomy for grouping and identifying Aeromonas strains at the genomic species level in this and other studies (KAMPFER and ALTWEGG 1992; CARNAHAN and JOSEPH, 1993; EsTEVE, 1995). However, results of this study and other studies (KAMPFER and ALTWEGG, 1992; CARNAHAN and JOSEPH, 1993) still indicate some taxonomic problems, such as phenotypic diversity within one genomic species and phena overlap. The identification of Aeromonas strains to the genomic species level is difficult because strains belonging to different hybridization groups are biochemically very similar. Using DNA hybridization, KUI]PER et al., (1989), found that eight of 26 (31 %) strains isolated from faeces and phenotypically identified as A. hydrophila, in fact belonged to HG 8110 (A. veronii biotype sobria) whereas some strains initially assigned to A. caviae belonged to the genomic species A. media (HG
susceptibility to cephalothin. Clustering the strains of
5) and A. veronii biotype sob ria (HG 811 0). Similar in-
466
A. KAZNOWSKI
consistencies between phenotypic and genotypic data were reported by other authors (ARDUINO et aI., 1988; ALTWEGG et aI., 1990; ALTWEGG, 1993; CARNAHAN, 1993; KAMPFER and ALTWEGG, 1992). These taxonomic problems may be related with the phenotypic homogeneity (KUI]PER et aI., 1989; ALTWEGG et aI., 1990; ALTWEGG, 1993; CARNAHAN, 1993) or genotypic heterogeneity of some genomic species in Aeromonas (Huys et aI., 1996). In conclusion, the numerical analysis of Aeromonas strains of different sources was in most cases helpful in the identification to the genomic species level. However, the results obtained with the numerical taxonomy were not in perfect agreement with the DNA-DNA hybridization results. Computer analysis showed high phenotypic similarity among strains of HG 1-3 and of HG 4-6. Among isolates of HG 8110 biovariants were found. Some differences were noted in the frequency of phenotypic characters in comparison with the literature data and it can be due to different sources of isolates or different geographical areas. The use of molecular methods in addition to phenotypic characteristics for final identification of Aeromonas isolates with the genomic species seems necessary. Acknowledgments I am very grateful to Dr. M. ALTWEGG, Dr. F. W. HICKMANBRENNER, Dr. A. CARNAHAN and Dr. C. ESTEVE for providing the reference bacterial strains. This work was partially supported by grant no. 0158/P2/94/96 from the Polish Committee for Scientific Research and by an Individual grant from Faculty of Biology, Poznan University.
References ABBOTT, S. L., CHEUNG, W. K. W., KROSKE-BRYSTROM, S., MALEKZADEH, T., JANDA, J. M.: Identification of Aeromonas strains to the genospecies level in the clinical laboratory. J. Clin. Microbiol. 30, 1262-1266 (1992). ALI, A., CARNAHAN, A. M., ALTWEGG. M., LUTHy-HOTTENSTEIN, J., JOSEPH, S. W.: Aeromonas bestiarum sp. nov. (formerly genomospecies DNA group 2 A. hydrophila), a new species isolated from non-human sources. Med. Microbiol. Lett 5, 156-165 (1996). ALLEN, D. A., AUSTIN, B., COLWELL, R. R.: Aeromonas media, a new species isolated from river water. Int. J. System. Bacteri01. 33, 599-604 (1983). ALTWEGG, M., GEISS, H. K.: Aeromonas as a human pathogen. CRC Crit. Rev. Microbiol. 16,253-286 (1989). ALTWEGG, M.: Taxonomy and epidemiology of Aeromonas spp.: the value of new typing methods. Habilitationsschrift, Zurich, ADAG Administration & Druck AG 1990. ALTWEGG, M., STEIGERWALT, A. G., ALTWEGG-BISSIG, R., LUTHYHOTTENSTEIN, J., BRENNER, D. J.: Biochemical identification of Aeromonas genospecies isolated from humans. J. Clin. Microbiol. 28, 258-264 (1990). ALTWEGG, M., LUTHy-HOTTENSTEIN, J.: Methods for the identification
of
DNA
hybridization
groups
in
the
genus
Aeromonas. Experientia, 47, 403-406 (1991). ALTWEGG, M., REEVES, M. W., ALTWEGG-BISSIG, R., BRENNER, D. J.: Multilocus enzyme analysis of genus Aeromonas and its use for species identification. Zbl. Bakt. 275, 28-45 (1991).
ALTWEGG, M.: A polyphasic approach to the classification and identification of Aeromonas strains. Med. Microbiol. Lett. 2, 200-205 (1993). ARDUINO, M. J., HICKMAN-BRENNER, F. W., FARMER III, J. J.: Phenotypic analysis of 132 Aeromonas strains representing 12 DNA hybridization groups. J. Diarrh. Dis. Res. 6, 138 (1988). BRYANT, T. N., LEE, J. v., WEST, P. A., COLWELL, R. R.: Numerical classification of species of Vibrio and related genera. J. Appl. Bacteriol. 61,437-467 (1986). CARNAHAN, A. M., FANNING, G. R., JOSEPH, S. W.: Aeromonas jandaei (formerly genospecies DNA group 9 A. sobria), a new sucrose-negative species isolated from clinical specimens. J. Clin. Microbiol. 29, 560-564 (1991a). CARNAHAN, A. M., CHAKRABORTY, T., FANNING, G. R., VERMA, D., ALI, A., JANDA, J. M., JOSEPH, S. W.: Aeromonas trota sp. nov., an ampicillin-susceptible species isolated from clinical specimens. J. Clin. Microbiol. 29, 1206-1210 (1991b). CARNAHAN, A. M.: Aeromonas taxonomy: A sea of change. Med. Microbiol. Lett. 2, 206-211 (1993). CARNAHAN, A. M., JOSEPH, S. W.: Systematic assessment of geographically and clinically diverse aeromonads. System. Appl. Microbiol. 16,72-84 (1993). COLWELL, R. R., MACDONELL, M. T., DE LEY, J.: Proposal to recognize the family Aeromonadaceae fam. nov. Int. J. System. Bacteriol. 36, 473-477 (1986). DORSCH, M., ASHBOLT, N. J., Cox, P. T., GOODMAN, A. E.: Rapid identification of Aeromonas species using 16 rDNA targeted oligonucleotide primers. A molecular approach based on screening of environmental isolates. J. Appl. Bacteriol. 77, 722-726 (1994). ESTEVE, c.: Numerical taxonomy of Aeromonadaceae and Vibrionaceae associated with reared fish and surrounding fresh and brackish water. System. Appl. Microbiol. 18, 391-402 (1995). ESTEVE, c., GUTIERREZ, M. c., VENTOSA, A.: DNA Relatedness among Aeromonas allosaccharophila strains and DNA hybridization groups of the genus Aeromonas. Int. J. System. Bacteriol. 45, 390-391 (1995a). ESTEVE, c., GUTIERREZ, M. c., VENTOSA, A.: Aeromonas encheleia sp. nov., isolated from European eels. Int. J. System. Bacteriol. 45, 462-466 (1995b). HANNINEN, M. L.: Phenotypic characteristics of the three hybridization groups of Aeromonas hydrophila complex isolated from different sources. J. Appl. Bacteriol. 76, 455-462 (1994). HANNINEN, M. L., SIITONEN, A.: Distribution of Aeromonas phenospecies and genospecies among strains isolated from water, foods or from human clinical samples. Epidemiol. Infect. 115,39-50 (1995). HICKMAN-BRENNER, F. W., MACDONALD, K. L., STEIGERWALT, A. G., FANNING, G. R., BRENNER, D. J., FARMER III, J. J.: Aeromonas veronii, a new ornithine decarboxylase-positive species that may cause diarrhea. J. Clin. Microbiol. 25, 900-906 (1987). HICKMAN-BRENNER, F. W., FANNING, G. R., ARDUINO, M. J., BRENNER, D. J., FARMER III, J. J.: Aeromonas schubertii, a new mannitol-negative species found in human clinical specimens. J. Clin. Microbiol. 26, 1561-1564 (1988). HOLDING, A. J., COLLEE, J. G.: Routine biochemical tests, pp. 2-32. In: Methods in microbiology, Vol. 6A, (J. R. NORRIS, D. W. RIBBONS eds.), London-New York, Academic Press 1971.
HOLT, J. G., KRIEG, N. R., SNEATH, P. H. A., STALEY, J. T., WILLIAMS S. T. (eds): Genus Aeromonas, pp. 190-255. In: Bergey's Manual of Determinative Bacteriology, 9th ed., Baltimore, Williams & Wilkins 1994.
Numerical taxonomy and DNA-DNA hybridizations of Aeromonas Huys, G., COOPMAN, R., JANSSEN, P., KERSTERS, K.: High-resolution genotypic analysis of the genus Aeromonas by AFLP fingerprinting. Int. J. System. Bacteriol. 46, 572-580 (1996). International Committee on Systematic Bacteriology, Subcommittee on the Taxonomy of Vibrionaceae. Int. J. System. Bact. 42, 199-201 (1992) . JANDA, J. M., REITANO, M., BOTTONE, E. B. : Biotyping of Aeromonas isolates as a correlation to delineating a species-associated disease spectrum. J. Clin. Microbiol. 19,44-47 (1984). JANDA J. M., OSHIRO L. S., ABBOTT S. L., DUFFEY P.: Virulence markers of mesophilic aeromonads: Association of the autoaglutination phenomenon with mouse pathogenicity and the presence of a peripherial cell-associated layer. Infect. Immun. 55, 3070-3077 (1987). JANDA, J. M. : Recent advances in the study of the taxonomy, pathogenicity, and infectious syndromes associated with the genus Aeromonas. Clin. Microbiol. Rev. 4, 397-410 (1991). JANDA, J. M., ABBOTT, S. L., KHASHE, S., KELLOGG, G. H., SHIMADA, T.: Further studies on biochemical characteristics and serologic properties of the genus Aeromonas. J. Clin. MicrobioI. 34, 1930-1933 (1996). KAZNOWSKI, A.: A method of colorimetric DNA-DNA hybridization in microplates with covalently immobilized DNA for identification of Aeromonas spp. Med. Microbiol. Lett. 4,362-369 (1995). KAMPFER, P., ALTWEGG, M.: Numerical classification and identification of Aeromonas genospecies. J. Appl. Bacteriol. 72, 341-351 (1992). KUI]PER, E. J., STEIGERWALT, A. G., SCHOENMAKERS, B. C. I. M., PEETERS, M. E, ZANEN, H. c., BRENNER, D. J.: Phenotypic characterization and DNA relatedness in human fecal isolates of Aeromonas spp. J. Clin. Microbiol. 27, 132-138 (1989). LEE, J. V., HENDRIE, M. S., SHEWAN, J. M.: Identification of Aeromonas, Vibrio and related organisms, pp. 151-166. In: Identification Methods for Microbiologists (E A. SKINNER, D. W. LOVELOCK, eds.) London, New York, Toronto, Sydney, San Francisco: Acedemic Press 1979. MARTINETTI LUCCHINI, G., ALTWEGG, M.: rRNA gene restriction patterns as taxonomic tools for the genus Aeromonas. Int. J. System. Bacteriol. 42, 384-389 (1992). MARTINEZ-MuRCIA, A. J., ESTEVE, c., GARAY, E., COLLINS, M. D.: Aeromonas allosaccharophila sp. nov., a new mesophilic member of the genus Aeromonas. FEMS Microbiol. Lett. 91, 199-206 (1992).
467
MILLERSHIP, S. E., CHATTOPADHYAY, B.: Methods for the isolation of Aeromonas hydrophila and Plesiomonas shigelloides from faeces. J. Hyg. (Cambridge) 92, 145-152 (1984). POPOFF, M., VERON, M.: A taxonomic study of the Aeromonas hydrophila-Aeromonas punctata group. J. Gen. Microbiol. 94,11-22 (1976). POPOFF, M .: Genus III: Aeromonas, pp. 545-547. In: Bergey's Manual of Systematic Bacteriology Vol 1, (N. R. KRIEG, J. G. HOLT, eds.), Baltimore-London, Wiliams & Wilkins 1984. SCHARMANN, W.: Vorkommen von Elastase bei Pseudomonas und Aeromonas. Zbl. Bakt. Hyg. A 220, 435-442 (1972). SCHUBERT, R. H. W., HEGAZI, M.: Aeromonas eucrenophila species nova and Aeromonas caviae, a later and illegitimate synonym of Aeromonas punctata. Zbl. Bakt. Hyg. A 268, 34-39 (1988). SCHUBERT, R. H. W.: Aeromonads and their significance as potentios pathogens in water. J. Appl. Bacteriol. Symp. Suppl. 70, 131S-135S (1991). SNEATH, P. H. A., JOHNSON, R.: The influence of numerical taxonomic similarities of errors in microbiological tests. J. Gen. Microbiol. 72,377-392 (1972). SNEATH, P. H. A., SOKAL, R. R.: Numerical Taxonomy: The Principles and Practice of Numerical Classification, 2nd ed., San Francisco, W. H. Freeman & Co. 1973. VERON, M., GASSER, E: Sur la detection de l'hydrogene sulfure produit par certaines enterobacteriacees dans les milieux dits de diagnostic rapide. Ann. Inst. Past. 105,524-534 (1963) . VERON, M.: Nutrition et taxonomie des Enterobacteriaceae et bacteries voisines. I. Methods d'etude des auxanogrammes. Ann. Microbiol. Inst. Past. 126 A, 267-274 (1975). WAYNE, L. G., BRENNER, D . J., COLWELL, R. R., GRIMONT, P. A. D., KANDLER, 0., KRICHEVSKY, M. I., MOORE, L. H ., MOORE, W. E. c., MURRAY, R. G. E., STACKER BRANDT, E., STARR, M. P., TROPER, H. P.: Report of the ad hoc Committee on reconciliation of approaches to bacterial systematics. Int. J. System Bacteriol. 37,463-464 (1987).
Corresponding author: Dr. ADAM KAZNOWSKI, Department of Microbiology, Institute of Experimental Biology, A. Mickiewicz University, ul. Fredry 10, 61-701 Poznan, Poland. Tel. (048 ) 61 524953; Fax (048) 61 8523615; e-mail: [email protected]