Downloaded from www.microbiologyresearch.org by IP: 54.211.150.184 On: Fri, 09 Sep 2016 07:01:34 Kaistia hirudinis sp. nov., isolated from the skin of Hirudo verbana Stefanie P. Glaeser, 1 Harald Galatis, 2 Karin Martin 3 and Peter Ka ¨ mpfer 1 Correspondence Peter Ka ¨ mpfer peter.kaempfer @umwelt.uni-giessen.de 1 Institut fu ¨ r Angewandte Mikrobiologie, Justus-Liebig-Universita ¨ t Giessen, D-35392 Giessen, Germany 2 Biebertaler Blutegelzucht, D-35444 Biebertal, Germany 3 Leibniz-Institut fu ¨ r Naturstoff-Forschung und Infektionsbiologie e. V., Hans-Kno ¨ ll-Institut, D-07745 Jena, Germany A Gram-negative, rod-shaped bacterium was isolated from the skin of the medical leech Hirudo verbana and studied for its taxonomic allocation. 16S rRNA gene sequence similarities to other strains showed that the strain was closely related to species of the genus Kaistia. Kaistia geumhonensis was shown to be the most closely related species (96.8 %), followed by Kaistia soli (96.6 %) and Kaistia dalseonensis (96.2 %). All other species of the genus Kaistia showed 16S rRNA gene sequence similarities ,96 %. Chemotaxonomic data for strain E94 T (major ubiquinone: Q-10; major polar lipids: diphosphatidylglycerol, phosphatidylglycerol, phosphatidyl- ethanolamine, hydroxyphosphatidylethanolamine, phosphatidylserine, unknown phospholipids, aminolipids and aminophospholipids; and major fatty acids: C 18 : 1 v7c,C 19 : 0 v8c cylco, C 16 : 0 and C 18 : 0 ) supported the affiliation of the isolate to the genus Kaistia. Phenotypic differentiation of strain E94 T from all species of the genus Kaistia was possible using different physiological characters. Strain E94 T represents a novel species of the genus Kaistia, for which the name Kaistia hirudinis sp. nov. is proposed, with the type strain E94 T (5LMG 26925 T 5CIP 110381 T 5CCM 8401 T ). The genus Kaistia was proposed by Im et al. (2004) and constitutes on the basis of 16S rRNA gene sequence studies a lineage within the family Rhizobiaceae in the class Alphaproteobacteria. At the time of writing, six species with validly published names of the genus Kaistia have been described and a further species has been proposed. Type strains of all species were isolated from different envir- onmental sources. Kaistia adipata KCTC 12095 T , the first recognized member of the genus was isolated from a soil sample (Im et al., 2004), Kaistia granuli KCTC 12575 T was isolated from anaerobic sludge (Lee et al., 2007), Kaistia soli KACC 12605 T was found in a peat layer (Weon et al., 2008), Kaistia terrae KACC 12910 T from a wetland environment (Kim et al., 2010), and Kaistia geumhonensis DSM 18799 T , Kaistia dalseonensis DSM 18800 T (Jin et al., 2011) and Kaistia defluvii B6-12 T (Jin et al., 2012) from sediment samples. Strain E94 T was isolated from the skin of the medical leech Hirudo verbana from a Leech farm in Biebertal (Germany). The strain was isolated and maintained on R2A agar (Oxoid) after incubation at 25 u C for 48h. Cultivation for further analyses was done on tryptone soy agar (TSA; Oxoid) at 28 u C for 48 h. The Gram-staining of cells from this strain was performed by the modified Hucker method according to the protocol of Gerhardt et al. (1994). Catalase activity was tested by bubble formation after covering a colony with 3 % H 2 O 2 . Cytochrome oxidase activity was determined with the Microbiology Bactident oxidase test (Merck). Urease activity was tested on urea agar base (Merck) according to the instructions of the manufacturer. In addition, cell morpho- logy was observed under a Zeiss light microscope at 61000 magnification, with cells grown for 3 days at 28 u C on TSA. For phylogenetic analysis, genomic DNA was extracted with the GenElute Plant Genomic DNA kit (Sigma). The nearly full-length 16S rRNA gene (1308 nt) was PCR- amplified with the universal primers 27f and 1492r (Lane, 1991), purified with a PCR-purification kit (Qiagen) and sequenced by the Sanger method with the same primers. Detailed phylogenetic analyses were performed in ARB release 5.2 (Ludwig et al., 2004) using the ‘All-Species Living Tree’ Project (LTP; Yarza et al., 2008) database release LTPs106 (August 2011). Sequences not included in the LTP database were aligned with SINA (v1.2.9) according to the SILVA seed alignment [http://www.arb-silva.de; The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Kaistia hirudinis E94 T is KC254734. International Journal of Systematic and Evolutionary Microbiology (2013), 63, 3209–3213 DOI 10.1099/ijs.0.049619-0 049619 G 2013 IUMS Printed in Great Britain 3209