International Journal of Systematic and Evolutionary Microbiology (2016), 66, 1323–1329 DOI 10.1099/ijsem.0.000882 Apibacter adventoris gen. nov., sp. nov., a member of the phylum Bacteroidetes isolated from honey bees Waldan K. Kwong1,2 and Nancy A. Moran2 Correspondence 1Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT, USA Waldan K. Kwong 2Department of Integrative Biology, University of Texas, Austin, TX, USA [email protected] Honey bees and bumble bees harbour a small, defined set of gut bacterial associates. Strains matching sequences from 16S rRNA gene surveys of bee gut microbiotas were isolated from two honey bee species from East Asia. These isolates were mesophlic, non-pigmented, catalase-positive and oxidase-negative. The major fatty acids were iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 0 and C16 : 0 3-OH. The DNA G+C content was 29–31 mol%. They had ,87 % 16S rRNA gene sequence identity to the closest relatives described. Phylogenetic reconstruction using 20 protein-coding genes showed that these bee-derived strains formed a highly supported monophyletic clade, sister to the clade containing species of the genera Chryseobacterium and Elizabethkingia within the family Flavobacteriaceae of the phylum Bacteroidetes. On the basis of phenotypic and genotypic characteristics, we propose placing these strains in a novel genus and species: Apibacter adventoris gen. nov., sp. nov. The type strain of Apibacter adventoris is wkB301T (5NRRL B-65307T5NCIMB 14986T). Bacteria from the phylum Bacteroidetes are often constitu- In July and August of 2014, samples of the Asian honey bee ents of animal microbiotas. In humans, members of the (Apis cerana) and the giant honey bee (Apis dorsata) were genera Prevotella and Bacteroides are among the most collected from Singapore and Kuala Lumpur, Malaysia numerically abundant gut symbionts, and appear to (Table S1, available in the online Supplementary Material). anchor different states of stability (enterotypes) within The entire intestinal tract was removed and crushed in the community (Arumugam et al. 2011). Honey bees and 19 % (v/v) glycerol and frozen. Bacteria were recovered bumble bees have also been found to harbour members by plating out the frozen samples onto heart infusion of the phylum Bacteroidetes: culture-independent 16S agar (Hardy Diagnostics) supplemented with 5 % defibri- rRNA gene surveys of their gut communities have ident- nated sheep blood and incubating at 35 8Cin5%CO2. ified sequences falling within the order Flavobacteriales, After 2–3 days, small (,0.5 mm) white, non-haemolytic with .10 % divergence from described species. Although colonies appeared. Three isolates were chosen for further their prevalence appears sporadic compared with the core characterization: wkB180, wkB309 and wkB301T. bee gut microbiota (Babendreier et al., 2007; Koch & Strains wkB180, wkB309 and wkB301T grew on heart infusion Schmid-Hempel, 2011; Ahn et al., 2012; Moran et al., agar, brain heart infusion agar, trypticase soy agar, Columbia 2012; Lim et al., 2015), the bee Flavobacteriales sequences agar and lysogeny broth (LB) agar, but exhibited poor or no form a monophyletic clade, suggesting they are specific to growth on nutrient agar, MacConkey agar, Lactobacillus bee guts, or to other environments (e.g. plants, nectar, MRS agar or R2A agar (all media from BD Difco). No hive material) where they are prone to be ingested by growth was observed under anaerobic conditions (90 % N , bees. Here, we provide the first description of members 2 5% CO,5%H) or in ambient air. In liquid culture, of this novel group. 2 2 growth was observed in Insectagro DS2 (Corning), Columbia broth, trypticase soy broth, and LB (without NaCl), but not in Lactobacillus MRS. On solid media, the strains formed The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA smooth, round, pale/semi-translucent colonies. Strain T gene sequences of strains wkB301 , wkB309 and wkB180 are wkB309 exhibited rapidly spreading margins of growth KT149221, KT149220 and KT149222 respectively. Protein-coding extending from colony edges, consistent with gliding motility. genes from these strains are deposited under the accession numbers KT149223– KT149282. DNA was extracted as described by Powell et al. (2014) Four supplementary tables and one supplementary figure are available with the following modifications. RNase A was added with the online Supplementary Material. directly to cetyltrimethylammonium bromide (CTAB) Downloaded from www.microbiologyresearch.org by 000882 G 2016 IUMS Printed in Great Britain 1323 IP: 54.70.40.11 On: Sun, 04 Nov 2018 04:06:39 W. K. Kwong and N. A. Moran buffer prior to bead-beating, a final concentration of 0.5 % Ornithobacterium rhinotracheale, Chryseobacterium gleum 2-mercaptoethanol was used, and the lysate was incubated and Flavobacterium aquatile, respectively (Table S1). How- at 56 8C for 1 h instead of overnight. Samples were sub- ever, our strains could not be confidently placed in relation mitted to the Genome Sequencing and Analysis Facility to the clades encompassing the genera Empedobacter and at the University of Texas at Austin for Illumina MiSeq Chryseobacterium (clades E and C, respectively) using 16S 26300 bp library preparation and whole genome sequen- rRNA gene sequences alone (Fig. 1). To obtain better cing. Sequence reads were assembled with CLC Genomics phylogenetic resolution, we extracted and aligned 20 pro- Workbench 5.5 (CLC bio). Genome sizes and DNA tein sequences from the strains’ genomes and that of G+C content were inferred from draft assemblies. sequenced relatives to produce a tree based on 9746 total residues (Fig. 2). This tree strongly suggested that the bee Phylogenetic analysis based on 16S rRNA sequences gut strains are sister to clade C, and not to clade E. All phy- (1250 bp) showed that strains wkB180, wkB309 and logenetic analysis was done in MEGA 6 (Tamura et al. 2013). wkB301T clustered together and were only distantly related to previously described species (Fig. 1). Strain wkB301T We conducted further assays to characterize the phenotypic had 87.6, 87.5, 87.2 and 85.5 % 16S rRNA gene sequence traits of our strains. Scanning electron micrographs identity to the type strains of Empedobacter brevis, were taken to determine cell size and morphology. LMG 18212T (AJ271010) 97 Chryseobacterium joosteii T 61 Chryseobacterium ureilyticum F-Fue-04IIIaaaa (AM232806) 92 T Chryseobacterium vrystaatense R-23566 (AJ871397) T 39 Chryseobacterium gleum ATCC 35910 (ACKQ01000057) T 0.2 Chryseobacterium hominis NF802 (AM261868) 100 H9T (EF204446) 22 Chryseobacterium bovis T 100 Chryseobacterium daecheongense CPW406 (AJ457206) T Chryseobacterium wanjuense R2A10-2 (DQ256729) 39 LMG 23089T (AM040439) 96 Chryseobacterium piscium 50 T Chryseobacterium indoltheticum LMG 4025 (AY468448) 51 T Chryseobacterium aquaticum 10-46 (AM748690) T Clade C 71 Chryseobacterium xinjiangense TSBY 67 (DQ166169) T 69 100 Epilithonimonas tenax EP105 (AF493696) T Epilithonimonas lactis H1 (NR_115989) T 100 Cloacibacterium normanense CCUG 46293 (AJ575430) T Cloacibacterium rupense R2A-16 (EU581834) T 77 88 Bergeyella zoohelcum ATCC 43767 (AGYA01000006) T 78 Riemerella anatipestifer DSM 15868 (NR_074429) Flavobacteriaceae 90 8151T (NR_117970) Riemerella columbipharyngis Bacteroidetes 100 T Soonwooa buanensis HM0024 (NR_116776) 51 T Elizabethkingia meningoseptica ATCC 13253 (ASAN01000081) 100 T 47 Elizabethkingia anophelis R26 (NR_116021) T Cruoricaptor ignavus IMMIB L-12475 (NR_108875) T Ornithobacterium rhinotracheale DSM 15997 (CP003283) 40 T Moheibacter sediminis M0116 (KF694750) T Weeksella virosa DSM 16922 (CP002455) 80 85 T (KF694751) 51 100 Chishuiella changwenlii BY4 Clade E T 88 Empedobacter falsenii NF 993 (AM084341) T Empedobacter brevis LMG 4011 (AM177497) 100 Apibacter adventoris wkB180 (KT149222) Apis dorsata Apibacter adventoris wkB301T (KT149221) 100 100 uncultured clone HBG A2V3-2 (DQ837638) Apis mellifera uncultured clone HBG A2V3-4 (DQ837639) 100 sp. B1 (KJ741206) 100 Bacteroidetes 100 Apis cerana Bee-associated Apibacter adventoris wkB309 (KT149220) 85 uncultured clone BJ07179A4 (HM215037) 100 uncultured clone D08062F1 (HM215038) Bumble bee uncultured clone D08033D2 (HM215036) 97 uncultured clone Bt231.55A2 (JQ388908) Flavobacterium aquatile LMG 4008T (JRHH01000003) Prevotella melaninogenica ATCC 25845T (AY323525) Bacteroides fragilis NCTC 9343T (CR626927) Escherichia coli MG1655 (NC_000913) Fig. 1. 16S rRNA gene phylogeny of strains wkB301T, wkB309, wkB180 and close relatives. Hosts of origin for bee-derived sequences or strains are highlighted. The tree was built with a maximum-likelihood algorithm using the Generalized Time Reversible substitution model with gamma-distributed rates and invariant sites. Escherichia coli MG1655 is the outgroup. Bootstrap percentages (based on 1000 replicates) are shown at nodes. Bar, 0.2 substitutions per site. Downloaded from www.microbiologyresearch.org by 1324 International Journal of Systematic and Evolutionary Microbiology 66 IP: 54.70.40.11 On: Sun, 04 Nov 2018 04:06:39 Apibacter adventoris gen. nov., sp. nov. T 100 Chryseobacterium indologenes NBRC 14944 (NZ_BAVL00000000) 99 T 0.2 Chryseobacterium gleum ATCC 35910 (NZ_ACKQ00000000) 100 Chryseobacterium taiwanense TPW19 (NZ_JWTA00000000) 99 Chryseobacterium vrystaatense LMG 22846T (NZ_JPRI00000000) 100 Epilithonimonas lactis H1T (NZ_JPLY00000000) Chryseobacterium haifense DSM 19056T (NZ_JASZ00000000) 73 100 Chryseobacterium antarcticum LMB 24720 (NZ_JPEP00000000) Clade