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1 Institut für Angewandte Mikrobiologie, Justus-Liebig-Universität Giessen, Germany
2 Microbiology Unit, Faculty of Medicine, University of Louvain, Brussels, Belgium
3 Department of Clinical Chemistry, Microbiology and Immunology, University of Ghent, Ghent, Belgium
Correspondence
Mario Vaneechoutte
Mario.Vaneechoutte{at}UGent.be
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of Wautersiella falsenii genomovar 1 strains NF 993T, NF 1182, NF 880, NF 777, NF 869, NF 289, NF 1240, NF 1241, NF 835, NF 1242, NF 1243 and NF 1244 are AM084341 and AM238680–AM238690, respectively, those of Wautersiella falsenii genomovar 2 strains NF 770 (reference strain), NF 622, NF 1159, NF 1249, NF 1140, NF 1084, NF 1080, NF 1136, NF 203, NF 316 and NF 58 are AM084342 and AM238670–AM238679, respectively, and those of Empedobacter brevis strain LMG 4011T and group CDC II-h strain NF 802 are AM177497 and AM261868, respectively.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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, comprises several genera of which Bergeyella, Chryseobacterium and Riemerella form a separate branch on the basis of rRNA cistron similarity (Vandamme et al., 1994; Bernardet et al., 2002) and whereby the genus Empedobacter (Vandamme et al., 1994) represents a separate line of descent comprising only one species, Empedobacter brevis. We propose a new genus within the Flavobacteriaceae, designated Wautersiella gen. nov., comprising the species Wautersiella falsenii sp. nov., with genomovars 1 and 2 to accommodate two groups of closely related isolates from clinical origins and with phenotypic resemblance to isolates of the genera Chryseobacterium, Weeksella and Empedobacter and of CDC groups II-e and II-h (Schreckenberger et al., 2003).
The 26 isolates, which were obtained between 1980 and 2004 by at least ten clinical laboratories in Belgium, originated from various human clinical samples, including blood, wounds, pus, respiratory tract, ear discharge, vaginal swab and pleural fluid (Table 1) and were cultivated on tryptic soy agar containing 5 % sheep blood. Strains of Empedobacter brevis, Weeksella virosa and CDC group II-h used in this study were isolated from different patients in various Belgian hospitals.
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) and tree construction was done as described by Nemec et al. (2001).
The results of sequence determination and cluster analysis of the 16S rRNA genes are presented in Fig. 1. The Wautersiella falsenii isolates clearly affiliated separately and the 16S rRNA gene sequences showed 94–95 % similarity with the type strain of Empedobacter brevis as the closest relative, indicating that the isolates might belong to a separate genus. Furthermore, the 16S rRNA gene sequences of the isolates were similar, but two clusters (genomovars) could be distinguished. The sequence similarities were 99.5–100 % for the 14 isolates of genomovar 1 and 99.4–100 % for the 12 isolates of genomovar 2. The similarity between the two clusters was 98.3–99.5 %.
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tRNA intergenic length polymorphism analysis (tDNA-PCR) was carried out as described previously (Baele et al., 2000). Fig. 2 presents the tDNA-PCR fingerprints as obtained for representative strains of Wautersiella falsenii genomovars 1 and 2, Empedobacter brevis, Weeksella virosa and CDC group II-h. The tDNA-PCR fingerprints of isolates from the two genomovars had the following fragments in common: 57.6 bp (SD for 11 isolates, 0.3 bp), 90.7 (0.1), 101.3 (0.1) and 221.4 bp (0.2 bp), but all genomovar 2 isolates had an additional tRNA spacer of 132.9 bp (0.1 bp for 5 isolates).
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The three Weeksella virosa strains tested (NF 135, NF 309 and NF 323) had tRNA spacers with lengths of 57.6 (0.5), 88.0 (0.1), 98.4 (0.5), 99.3 (0.5) and 104.0 bp (0.1 bp) in common, with strains NF 135 and NF 309 having a spacer of 91.3 bp (0.1 bp) in common and NF 135 and NF 323 having spacers of 230.4 (0.1) and 291.1 bp (0.0 bp) in common.
All eight CDC II-h strains tested (NF 675, NF 690, NF 696, NF 802, NF 974, NF 1141, NF 1146 and NF 1335) had tRNA spacers with lengths of 57.5 (0.2), 81.1 (0.1), 90.2 (0.1), 92.3 (0.1), 110.6 (0.1) and 218.5 bp (0.1 bp) in common. As such, tDNA-PCR enabled differentiation between Wautersiella falsenii and the most closely related species.
Biochemical and morphological tests were performed as described previously (Laffineur et al., 2002) and by Schreckenberger et al. (2003). Assimilation–alkalinization of organic compounds was detected on Simmons' citrate agar base by replacing citrate with 0.2 % (w/v) of various organic substrates, according to Martin et al. (1981). For casein hydrolysis, 1 % (v/v) of skimmed milk was added to nutrient agar plates. Enzymic reactions were carried out using diagnostic tablets from Rosco. Utilization of carbohydrates was performed using API 50 CH strips (bioMérieux), which were read after 1, 2 and 3 days. The KOH test was used to detect flexirubin pigment (Bernardet et al., 2002).
All isolates were Gram-negative, non-motile, non-fermenting rods and were oxidase- and catalase-positive. Some strains displayed yellow-pigmented colonies after prolonged incubation, but the pigment was not of the flexirubin type. Acid was produced from glucose and maltose. Lysine and ornithine decarboxylases and arginine dihydrolase were negative. Indole was produced and urease was positive using Christensen's urea broth. Nitrate reduction was negative and nitrite reduction was mostly negative. Gelatinase production varied from weak to strong and was in general faster and stronger for genomovar 1 isolates. Casein hydrolysis was negative or very weak. Growth did not occur at 42 °C. Assimilation and alkalinization of acetate, aspartate, L-alanine and L-serine were positive on Simmons' agar base, whereas citrate and most of the other organic salts were negative. Isolates were variable for galacturonate. Aesculin hydrolysis was variable. Alkaline phosphatase, trypsin, pyrrolidonyl aminopeptidase, 
-glucosidase and N-acetyl-glucosaminidase were positive, whereas -galactosidase, -mannosidase, -fucosidase and 
-xylosidase were negative. -galactosidase (ONPG) was variable. On API 50 CH strips, only maltose, glycerol and starch were assimilated within 3 days. Glucose was assimilated by some of the isolates. Table 2 summarizes some of the biochemical characteristics that differ between the two genomovars; aesculin, ONPG and galacturonate yielded characteristic reactions that separated the two genomovars, but exceptions occurred and isolate NF 1138, belonging to genomovar 2, even had the biochemical profile of genomovar 1. Empedobacter brevis differed from all Wautersiella falsenii isolates in being urease-negative and having strong casein hydrolysis activity (Table 2). All CDC II-h isolates were urease-negative, in contrast to the Wautersiella falsenii isolates.
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; Kämpfer et al., 2003). The most abundant fatty acid was 15 : 0 iso, followed by summed feature 4 (15 : 0 iso 2-OH and/or 16 : 1
7ct), 17 : 0 iso 3-OH and 15 : 0 iso 3-OH. The fatty acid profiles of strains of the two genomovars were different from those of Empedobacter brevis and Chryseobacterium species (Table 3).
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, of the type strain of genomovar 1 and the reference strain of genomovar 2 were 33.8±0.4 and 34.4±0.2 %, respectively, in agreement with values obtained for the closely related genera Chryseobacterium and Empedobacter (Bernardet et al., 2002).
For DNA–DNA hybridization, a microplate method was used, modified after Lind & Ursing (1986), and as described by Ziemke et al. (1998). Reciprocal DNA–DNA hybridizations, i.e. with one of the two strains labelled first and the other labelled subsequently, were carried out between the two reference strains of Wautersiella falsenii and Empedobacter brevis LMG 4011T. Values obtained were 29.4 % (respectively 25.5 % reciprocal) for Wautersiella falsenii genomovar 1 strain NF 993T versus Empedobacter brevis LMG 4011T and 33.0 % (respectively 35.8 % reciprocal) for Wautersiella falsenii genomovar 2 strain NF 770 versus Empedobacter brevis LMG 4011T, indicating that the reference strains of the two genomovars do not belong to Empedobacter brevis.
DNA–DNA hybridization values of 104.6 % (respectively reciprocal 103.0 %) were obtained for genomovar 1 strains NF 993T and NF 835, 86.6 % (respectively reciprocal 93.9 %) for strains NF 993T and NF 869 and 88.2 % and 93.5 % for genomovar 2 reference strain NF 770 with, respectively, isolates NF 1084 and NF 1138. A value of 63.6 % was obtained for strain NF 993T (labelled) versus strain NF 770, indicating that the two strains represent separate genomovars and borderline the same species.
Although two separate groups are clearly present within the genus Wautersiella, at present it is not possible to describe them as separate species, because of the lack of a phenotypic characteristic that can unambiguously differentiate between the two groups. Therefore it was decided to describe the two groups as genomovars.
Description of Wautersiella gen. nov.
Wautersiella (Wau.ter'si.ella. N.L. fem. dim. n. Wautersiella named after Georges Wauters, a contemporary Belgian microbiologist, who first recognized this group of organisms as a separate entity and to honour him for his lifelong contribution to bacterial taxonomy).
Non-motile, Gram-negative rods, 2–3 µm in length, growing aerobically at 20, 30 and 37 °C on standard media such as tryptic soy agar or blood agar. Colonies are approximately 2 mm in diameter after growth on blood agar for 48 h at 30 °C. Main cellular fatty acids are 15 : 0 iso, 17 : 0 iso 3-OH, summed feature 4 and 15 : 0 iso 3-OH. The G+C content of the DNA is 33.8–34.4 mol%. The type and only species is Wautersiella falsenii.
Description of Wautersiella falsenii sp. nov.
Wautersiella falsenii (fal.sen'i.i. N.L. gen. n. falsenii of Falsen, to honour the contemporary Norwegian microbiologist, Enevold Falsen, for his lifelong interest in bacterial taxonomy and for his systematic characterization of bacteria at the CCUG, Göteborg, Sweden).
Oxidative acidification of glucose and maltose. Positive for urease and for indole production. Nitrates are not reduced, but nitrite reduction is variable. Lysine and ornithine decarboxylases and arginine dihydrolase are not present. Citrate is negative. Alkaline phosphatase, trypsin (benzyl-arginine arylamidase) and pyrrolidonyl aminopeptidase are positive. Gelatin hydrolysis is positive or weakly and delayed positive. Casein hydrolysis is negative or weakly and delayed positive. Comprises two genomovars that differ in 16S rRNA gene sequence and in tDNA-PCR pattern. All genomovar 1 isolates (n=14) rapidly hydrolyse gelatin and aesculin, are -galactosidase (ONPG)-negative and all but one alkalinize galacturonate. All genomovar 2 isolates (n=12) but one are aesculin- and galacturonate-negative. Most isolates are ONPG-positive and slightly gelatin-positive. Fatty acids are as described for the genus, with only minor differences between the two genomovars.
The type strain of the species and reference strain for genomovar 1 is NF 993T (=CCUG 51536T=CIP 108861T), which was isolated from a surgical wound. The reference strain for genomovar 2, NF 770 (=CCUG 51537=CIP 108860), was isolated from blood.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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