System. Appl. Microbiol. 23, 355-363 (2000) SYSTEI\IL4TIC AND © Urban & Fischer Verlag _htt-,-p_:llw_w_w_.ur_ba_n_fis_ch_er_.de--"/jo_u_rn_als_/s_am ____________ APPLIED MICROBIOLOGY Phylogenetic Evidence for Novel and Genetically Different Intestinal Spirochetes Resembling Brachyspira aalborgi in the Mucosa of the Human Colon as Revealed by 16S rONA Analysis BERTIL PETTERSSONl, MEl WANG2, CLAES FELLSTROM 3 , MATHIAS UHLEN\ GORAN MOLIN 2 , BENGT jEPPSSON\ and SIV AHRNE2 1 Department of Biotechnology, Royal Institute of Technology, Stockholm, Sweden 2 Laboratory of Food Hygiene, P.O. Box 124, Lund University, Lund, Sweden 3 Department of Medicine and Surgery, Swedish University of Agricultural Sciences, Faculty of Veterinary Medicine, Uppsala, Sweden 4 Department of Surgery, Lund University Hospital, Lund and Department of Surgery Malmo University Hospital, Malmo, Sweden Received May 31, 2000 Summary Intestinal spirochetes (Brachyspira spp.) are causative agents of intestinal disorders in animals and hu- mans. Phylogenetic analysis of cloned 16S rRNA genes from biopsies of the intestinal mucosa of the colon from two Swedish 60-years old adults without clinical symptoms revealed the presence of intesti- nal spirochetes. Seventeen clones from two individuals and 11 reference strains were analyzed and the intestinal spirochetes could be divided into two lineages, the Brachyspira aalborgi and the Brachyspira hyodysenteriae lineages. All of the clones grouped in the B. aalborgi lineage. Moreover, the B. aalborgi lineage could be divided into three distinct phylogenetic clusters as confirmed by bootstrap and signa- ture nucleotide analysis. The first cluster comprised 6 clones and the type strain B. aalborgi NCTC 11492 T • The cluster 1 showed a 16S rRNA gene similarity of 99.4-99.9%. This cluster also harbored the only other strain of B. aalborgi isolated so far, namely strain WI, which was subjected to phylogenetic analysis in this work. The second cluster harbored 9 clones with a 98.7 to 99.5% range of 16S rDNA similarity to the B. aalborgi cluster 1. Two clones branched distinct and early of the B. aalborgi line forming the third cluster and was found to be 98.7% similar to cluster 1 and 98.3-99.1 % to cluster 2. Interestingly, this shows that considerable variation of intestinal spirochetes can be found as constituents of the colonic micro biota in humans, genetically resembling B. aalborgi. The presented data aid signifi- cantly to the diagnostic and taxonomic work on these organisms. Key words: Brachyspira aalborgi - Human colon - intestinal spirochetes - micro biota - Phylogeny - 16S rDNA Introduction Gastrointestinal disorders such as chronic diarrhea and rectal bleeding, clinically defined as intestinal spiro- chetosis (IS), has been characterized for a variety of ani- mal species as reviewed by DUHAMEL, 1997; SWAYNE and McLAREN, 1997; TAYLOR and TROIT, 1997. Diagnosti- cally, the disease can be visualized by the appearance of a so-called "false brush border" originating from a mat formed by a large amount of spirochetes, which are at- tached by one end to the epithelial layer of the colonic mucosa (HARLAND and LEE, 1967). Spirochetes associat- ed with IS are known as Brachyspira pilosicoli, formerly classified in the genus Serpulina (TROIT et aI., 1996) and Brachyspira aalborgi (HOVIND-HoUGEN et aI., 1982). Evolutionary, these species have been shown to belong to a group of closely related organisms forming a distinct clade by bisecting the phylogenetic tree of the order Spirochaetales (PASTER et aI., 1991, PEITERSSON et a\., 1996). B. pilosicoli seem to be a frequently isolated or- ganism in association with IS in pigs, dogs, chickens and humans (TAYLOR et aI., 1980; TROIT et aI., 1997; McLAREN et aI., 1997; TRIVETT-MoORE et a\., 1998). Moreover, a marked genetic diversity has been found among B. pilosicoli isolates as recovered from humans and animals (ATyEO et aI., 1996; LEE et aI., 1994). On the other hand, B. aalborgi has only been isolated twice (HOVIND-HoUGEN et aI., 1982; KRAAZ et aI., 2000) and 0723-2020/00/23/03-355 $ 15.00/0