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1 Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019, USA
2 School of Food Biosciences, University of Reading, Reading RG6 6AP, UK
3 Culture Collection, Department of Clinical Bacteriology, University of Göteborg, SE-413 46 Göteborg, Sweden
Correspondence
Ralph S. Tanner
rtanner{at}ou.edu
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The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CCUG 46293T is AJ575430.
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). Members of this group are common in activated sludge and other parts of wastewater-treatment plants (Benedict & Carlson, 1971; Güde, 1980). By using probes specific for the Cytophaga–Flavobacterium group, researchers have found that this group constitutes a significant portion (11–24 %) of the microbial community of activated sludge from wastewater plants that employ enhanced biological phosphate removal (Wagner et al., 1994; Liu et al., 2005), and that its members contribute directly to phosphate removal in activated sludge (Van Ommen Kloeke & Geesey, 1999). Three isolates of a numerically dominant, heterotrophic, Gram-negative bacterium were recovered during a study of the impact of the discharge of treated wastewater into the Canadian River at Norman, OK, USA. Biochemical and phenotypic analysis showed that the organisms possessed traits similar to those of species belonging to the Cytophaga–Flavobacterium–Bacteroides group of organisms (Paster et al., 1985; Segers et al., 1993). Studies involving 16S rRNA gene sequence analysis indicated that the isolates represented a new subline within the Bergeyella–Chryseobacterium–Riemerella branch of the Flavobacteriaceae (Vandamme et al., 1994; Bernardet et al., 1996). A most probable number (MPN)-PCR approach was developed to detect (and enumerate) this organism from the wastewater influent and from human stool samples, a possible source of the bacterium. It is therefore proposed that a novel genus and species be created to accommodate the novel organism recovered from sewage.
Isolates NRS1T, NRS30 and NRS32 were isolated from untreated wastewater from a water-treatment plant located at Norman, OK, USA. Strain NRS1T was isolated as a most numerous culturable heterotroph from an MPN dilution series in 0.5x tryptic soy broth (Becton Dickinson). Strains NRS30 and NRS32 were isolated by direct plating of untreated wastewater from the same source on nutrient agar. Tryptic soy broth or tryptic soy agar (Becton Dickinson) was used for routine culture unless otherwise stated. The strains were characterized biochemically by using a combination of conventional tests performed as described previously (Smibert & Krieg, 1991), and by using the API Rapid ID 32S, API Rapid ID 32A and API ZYM test systems according to the manufacturer's instructions (bioMérieux). Hydrolysis of casein, DNA and urea and reduction of nitrate were assessed by inoculation on the appropriate media (Becton Dickinson). Citrate utilization was assessed by using Simmons' citrate agar (Becton Dickinson). Hydrolysis of xanthine, hypoxanthine and uric acid was investigated by using the method of Bowman et al. (1996). Hydrolysis of alginate and chitin was investigated by using the method of West & Colwell (1984) and pectin hydrolysis was assessed by using the method of Hildebrand (1971). Resistance to antibiotics was investigated by using the method of Bauer et al. (1966). The fermentation products of glucose were determined by using ion-exclusion HPLC with an Aminex HPX-87H column (Bio-Rad) and a mobile phase consisting of 0.0025 M H2SO4. The pH range and optimum was determined by incubation on 1.5 % tryptone broth supplemented with one of the following (at 30 g l–1): HOMOPIPES [homopiperazine-N,N'-bis-2-(ethanesulfonic acid)] at pH 4 and 5, MES [2-(N-morpholino)ethanesulfonic acid] at pH 6, TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid] at pH 7 and 8 or CAPSO [3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid] at pH 9.
Fatty acid methyl ester analysis was performed on overnight cultures grown on tryptic soy agar at 37 °C by Microcheck, Inc. (Northfield, VT, USA) according to methods described previously (Miller, 1982; Sassar, 1990). For the detection of flexirubin-type pigments, pigmented cells were subjected to the KOH test as previously described for preliminary analysis (Hirsch & Reichenbach, 1981). For further characterization of pigments, cells were grown on tryptic soy agar and the pigments were extracted with acetone. The cell debris was then pelleted and the crude extract was analysed by using a DU 640 spectrophotometer (Beckman Coulter). Isoprenoid quinones were extracted as described by Collins et al. (1977) and analysed by HPLC as described by Groth et al. (1997). DNA was isolated by the method of Lawson et al. (1989). The DNA G+C content (mol%) was determined by HPLC according to Mesbah et al. (1989). The 16S rRNA genes of the isolates were amplified by PCRs using universal primers pA (positions 8–28, Escherichia coli numbering) and pH (positions 1542–1522) (Hutson et al., 1993). The amplified products were purified by using a QIAquick PCR purification kit (Qiagen) and directly sequenced with primers directed towards conserved positions of the rRNA gene and the dRhodamine terminator cycle sequencing kit (ABI) using an automatic DNA sequencer. The closest known relatives of the novel isolates were determined by performing database searches using the program FASTA (Pearson & Lipman, 1985). These sequences and those of other related strains were retrieved from GenBank and aligned with the newly determined sequences by using the program SEQtools (Rasmussen, 2002). The resulting multiple sequence alignment was corrected manually using the program GeneDoc (Nicholas et al., 1997) and a phylogenetic tree was constructed according to the neighbour-joining method (Saitou & Nei, 1987) with the programs SEQtools and TREEVIEW (Page, 1996). The stability of the groupings was estimated by bootstrap analysis (1000 replications) using the same programs. For the MPN-PCR analysis, a pair of oligonucleotide primers, Cloac-001f (5'-TATTGTTTCTTCGGAAATGA) and Cloac-001r (5'-ATGGCAGTTCTATCGTTAAGC), were designed (using Primer3 software; Rozen & Skaletsky, 2000) that were specific to the 16S rRNA gene of the newly isolated organism. DNA was isolated from 10 human stool samples (200 mg each) by using the QIAamp DNA Stool Mini kit (Qiagen) according to the manufacturer's instructions. Cells were harvested from wastewater influent and effluent samples by centrifugation and were then washed with a saline buffer. Washed whole cells were then added to the PCR mixture after a freeze–thaw cycle to achieve lysis. MPN-PCR (Picard et al., 1992) was performed by means of serial 10-fold dilutions in triplicate and visualization of the PCR product by agarose gel electrophoresis. The MPN was calculated according to the tables of Cochran (1950).
All strains stained Gram-negative and displayed pleomorphic cell morphologies depending on the medium used. In broth cultures, most cells were long (up to 27 µm); when cultured on agar plates, most cells were much shorter (5–9 µm). Similar pleomorphic cell morphologies have been described for members of the Flavobacteriaceae (Simon & White, 1971). A battery of phenotypic and biochemical tests, including API Rapid ID 32S, API Rapid ID 32A and API ZYM, were performed on all strains, the results of which appear in the genus and species descriptions and in Table 1. Sensitivities to the following antibiotics were tested: ampicillin (10 µg), carbenicillin (100 µg), cefaclor (30 µg), ceftriaxone (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), doxycycline (30 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), oxytetracycline (30 µg), streptomycin (10 µg), sulfathiazole (0.25 mg), tetracycline (30 µg) and trimethoprim (5 µg). All three strains were resistant to erythromycin and kanamycin; strain NRS1T was also resistant to streptomycin and strain NRS32 was resistant to nalidixic acid. The KOH test for flexirubin-type pigments was negative for all strains. The absorbance spectra of crude pigment extract from all strains exhibited a triple-peak signature (418, 451 and 483 nm) characteristic of carotenoid-type pigments (Schmidt et al., 1994). The fatty acid profiles of the newly isolated strains consisted mainly of branched-chain fatty acids, with iso-C13 : 0 (8–9 %), iso-C15 : 0 (40–46 %), iso-C15 : 1 (7–10 %) and iso-C17 : 0 3-OH (5–9 %) predominating. The detailed fatty acid composition is shown in Table 2.
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, confirmed that the unknown bacterium (as exemplified by strain NRS1T) is a member of the Bergeyella–Chryseobacterium–Riemerella cluster of the Flavobacteriaceae. In particular, the unknown bacterium forms an association with a cluster of organisms that includes Riemerella anatipestifer (88.8 % sequence similarity), Riemerella columbina (94.6 % sequence similarity), Bergeyella zoohelcum (94.4 % sequence similarity) and an as-yet uncultured bacterium (99.2 % sequence similarity) recovered from uranium mining waste (Fig. 1).
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On the basis of tree-topology considerations and sequence-divergence values of 5 % or more with respect to the aforementioned taxa, the unidentified bacterium was only distantly related to these taxa and merits classification at a similar taxonomic rank (i.e. genus). Therefore, on the basis of both the phenotypic and the phylogenetic findings, we consider that the unknown Gram-negative rod isolated from raw sewage merits classification in a novel genus and a novel species, for which the name Cloacibacterium normanense gen. nov., sp. nov. is proposed. Characteristics that are useful in distinguishing Cloacibacterium normanense from its closest phylogenetic relatives are shown in Table 1
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Description of Cloacibacterium gen. nov.
Cloacibacterium (Clo.a'ci.bac.te'ri.um. L. fem. n. cloaca a sewer, canal; L. neut. n. bacterium a small rod; N.L. neut. n. Cloacibacterium a sewer rod).
Cells are Gram-negative, non-motile and pleomorphic rod-shaped. Pigment is not of the flexirubin-type. Yellow to orange carotenoid-type pigments are produced. Facultatively anaerobic. Catalase- and oxidase-positive. The major end product of glucose fermentation under both aerobic and anaerobic conditions is pyruvate. Fatty acids consist mainly of branched-chain fatty acids, with iso-C13 : 0, iso-C15 : 0, iso-C15 : 1 and iso-C17 : 0 3-OH predominating. The predominant respiratory quinone is MK-6. The DNA G+C content of the type strain of the type species is 31 mol%. The type species is Cloacibacterium normanense.
Description of Cloacibacterium normanense sp. nov.
Cloacibacterium normanense (nor.man.en'se. N.L. neut. adj. normanense pertaining to the city of Norman, OK, USA, where the organism was first isolated).
Displays the following features in addition to those given in the genus description. In broth cultures, cells are long (up to 27 µm); cells from agar plate cultures are much shorter (5–9 µm). After 48 h, colonies grown on tryptic soy agar are 1.0–1.5 mm in diameter, round, entire and very waxy. Growth occurs between 18 and 36 °C. No growth occurs at 4 °C or at 40 °C or above. No growth occurs on MacConkey agar. Optimum temperature for growth is 30 °C. All strains grow at pH 7 and 8; some strains also grow at pH 6 and 9. The pH optimum for all strains is 7. Indole-positive. Starch, aesculin, gelatin and casein are hydrolysed and DNA is hydrolysed weakly. Urea, chitin, pectin, alginate, uric acid, xanthine and hypoxanthine are not hydrolysed. Methyl red- and Voges–Proskauer-negative. Nitrate is not reduced. Cellulose and agar are not degraded. Most isolates tested are resistant to erythromycin (15 µg) and kanamycin (30 µg). All isolates tested are sensitive to carbenicillin (100 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), doxycycline (30 µg), gentamicin (10 µg), oxytetracycline (30 µg), sulfathiazole (0.25 mg) and tetracycline (30 µg). With the API kits, acid is produced with 
-cyclodextrin and mannose. Acid is not produced from alanine, ribose, mannitol, sorbitol, lactose, trehalose, raffinose, hippurate, glycogen, melibiose, melezitose, sucrose, L-arabinose, D-arabitol, tagatose or raffinose. Enzyme activity is detected for alkaline phosphatase, acid phosphatase, alanine arylamidase, arginine dihydrolase (weak reaction), arginine arylamidase, chymotrypsin, esterase C-4 (weak reaction), ester lipase C8, -glucosidase, 
-glucosidase, glycine arylamidase, glycyl tryptophan arylamidase, proline arylamidase, leucine arylamidase, leucyl glycine arylamidase, maltose, phenylalanine arylamidase, alanine phenylalanine proline arylamidase, leucine arylamidase, naphthol-AS-BI-phosphohydrolase, pyroglutamic acid arylamidase, tyrosine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase, serine arylamidase and valine arylamidase. Enzyme activity is not detected for N-acetyl--glucosaminidase, -arabinosidase, -fucosidase, -galactosidase, -galactosidase, -glucuronidase, glutamic acid arylamidase, -mannosidase, -mannosidase, methyl -D-glucopyranoside, pyroglutamic acid arylamidase, lipase C14 or trypsin. Fatty acids iso-C13 : 0 (8–9 %), iso-C15 : 0 (40–46 %), iso-C15 : 1 (7–10 %) and iso-C17 : 0 3-OH (5–9 %) predominate. The DNA G+C content is 31 mol%.
The type strain, NRS1T (=CCUG 46293T=CIP 108613T=ATCC BAA-825T=DSM 15886T), was isolated from untreated human wastewater. Additional strains of the species, strains NRS30 (=CCUG 48043) and NRS32 (=CCUG 48044), were also isolated from wastewater.
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ACKNOWLEDGEMENTS |
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