Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Wed, 02 Jan 2019 23:15:56 Cloacibacterium normanense gen. nov., sp. nov., a novel bacterium in the family Flavobacteriaceae isolated from municipal wastewater Toby D. Allen, 1 Paul A. Lawson, 1 Matthew D. Collins, 2 Enevold Falsen 3 and Ralph S. Tanner 1 Correspondence Ralph S. Tanner rtanner@ou.edu 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 Go ¨ teborg, SE-413 46 Go ¨ teborg, Sweden Phenotypic and phylogenetic studies were performed on three isolates of an unknown Gram-negative, facultatively anaerobic, non-motile, yellow-pigmented, rod-shaped organism isolated from raw sewage. 16S rRNA gene sequence analysis indicated that these strains were members of the Bergeyella–Chryseobacterium–Riemerella branch of the family Flavobacteriaceae. The unknown bacterium was readily distinguished from reference strains by 16S rRNA gene sequencing and biochemical tests. The organism contained menaquinone MK-6 as the predominant respiratory quinone and had a DNA G+C content of 31 mol%. A most probable number-PCR approach was developed to detect, and estimate the numbers of, this organism. Untreated wastewater from one plant yielded an estimated count of 1?4610 5 cells ml ”1 , and untreated wastewater from a second plant yielded an estimated count of 1?4610 4 cells ml ”1 . Signal was not detected from treated effluent or from human stool specimens. On the basis of the results of the study presented, it is proposed that the unknown bacterium be classified in a novel genus Cloacibacterium, as Cloacibacterium normanense gen. nov., sp. nov., which is also the type species. The type strain of Cloacibacterium normanense is strain NRS1 T (=CCUG 46293 T =CIP 108613 T =ATCC BAA-825 T =DSM 15886 T ). Members of the family Flavobacteriaceae are ubiquitous in aquatic habitats, where they are generally thought to play a role in the breakdown of complex organic matter (Bernardet et al., 2002). Members of this group are common in activated sludge and other parts of wastewater-treatment plants (Benedict & Carlson, 1971; Gu ¨de, 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, hetero- trophic, 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 Flavobacteri- aceae (Vandamme et al., 1994; Bernardet et al., 1996). A most probable number (MPN)-PCR approach was devel- oped 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 NRS1 T , NRS30 and NRS32 were isolated from untreated wastewater from a water-treatment plant located at Norman, OK, USA. Strain NRS1 T was isolated as a most numerous culturable heterotroph from an MPN dilution series in 0?56 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 Abbreviation: MPN, most probable number. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain CCUG 46293 T is AJ575430. 64218 G 2006 IUMS Printed in Great Britain 1311 International Journal of Systematic and Evolutionary Microbiology (2006), 56, 1311–1316 DOI 10.1099/ijs.0.64218-0