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Antibiogram of ESBL and MBL Producing Pseudomonas International Journal of Systematic and Evolutionary Microbiology (2015), 65, 1347–1353 DOI 10.1099/ijs.0.000110 Flavirhabdus iliipiscaria gen. nov., sp. nov., isolated from intestine of flounder (Paralichthys olivaceus) and emended descriptions of the genera Flavivirga, Algibacter, Bizionia and Formosa Qismat Shakeela, Ahmed Shehzad, Yunhui Zhang, Kaihao Tang and Xiao-Hua Zhang Correspondence College of Marine Life Sciences, Ocean University of China, Qingdao, PR China Xiao-Hua Zhang [email protected] A Gram-stain-negative, orange-coloured, rod-shaped bacterium, designated strain Th68T, was isolated from the intestine of flounder (Paralichthys olivaceus). The isolate required sea salts for growth. Gliding motility was not observed. Flexirubin-type pigments were present. 16S rRNA gene sequence analysis indicated that strain Th68T represented a distinct phyletic line within the family Flavobacteriaceae with less than 96.1 % similarity to members of the recognized genera of the family. The DNA G+C content was 33.0 mol%. The major fatty acids were iso-C15 : 0, iso-C15 : 1 G, iso-C17 : 0 3-OH and iso-C15 : 0 3-OH. The major polar lipids were phosphatidy- lethanolamine, two unidentified aminolipids and two unidentified polar lipids. Menaquinone 6 (MK-6) was the only respiratory quinone. On the basis of the phenotypic, chemotaxonomic and phylogenetic data, strain Th68T represents a novel species of a new genus in the family Flavobacteriaceae, for which the name Flavirhabdus iliipiscaria gen. nov., sp. nov. is proposed. The type strain of Flavirhabdus iliipiscaria is Th68T (5JCM 18637T5KCTC 32141T). The family Flavobacteriaceae was derived from the isolated from the tissue homogenate by the plate dilution etymology of its type genus Flavobacterium, which means method on marine agar 2216 (MA; Becton Dickinson) at yellow-coloured bacterium (Bernardet & Nakagawa, 2006). 28 uC and purified by streaking three times on MA. One of the important characteristics of members of this Winogradskyella thalassocola LMG 22492T, Algibacter family is the presence of carotenoids or flexirubin-type pectinivorans JCM 17107T, Flaviramulus basaltis DSM pigments or both, although unpigmented flavobacteria also 18180T, Gaetbulibacter saemankumensis KCTC 12379T, occur (Bernardet et al., 1996, 2002). At the time of writing, Bizionia paragorgiae LMG 22571T, Formosa algae KCTC the bacterial family Flavobacteriaceae comprises 114 genera 12364T and Flavivirga jejuensis JCM 17113T were used as with validly published names (http://eztaxon-e.ezbiocloud. reference strains. For short-term preservation cultures of net). An additional yellow-pigmented flavobacterial strain, strain Th68T and the reference strains were maintained at Th68T, isolated from the intestine of flounder was studied 16 uC and for long-term cultures were preserved in sterile according to the minimal standards for describing new taxa 0.85 % (w/v) saline supplemented with 15 % (v/v) glycerol in the family Flavobacteriaceae (Bernardet et al., 2002). On at 280 uC. the basis of evidence derived from a polyphasic taxonomic The genomic DNA of strain Th68T was extracted and the approach, the isolate is considered to represent a novel 16S rRNA gene was obtained by PCR amplification with species of a new genus in the family Flavobacteriaceae. two universal primers (B8F, 59-AGAGTTTGATCCTG- Strain Th68T was isolated from the intestine of cultured GCTCAG-39; B1510, 59-GGTTACCTTGTTACGACTT-39) flounder (Paralichthys olivaceus) in 2010, from a fish farm (Weisburg et al., 1991). For cloning and sequencing of the in Shandong Province, China. The surface of the fish was 16S rRNA gene, the PCR product was purified using a cleaned with 75 % alcohol and the fish was then dissected TIANgel Midi Purification kit (TIANGEN Biotech), ligated and a piece of intestinal tissue was sampled aseptically for into the pUCm-T vector (TaKaRa) and sequenced at the the isolation of intestinal bacteria. Strain Th68T was Beijing Genomics Institute. The EzTaxon-e server was used for identification of phylogenetic neighbours and calcula- The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene tion of pairwise 16S rRNA gene sequence similarities sequence of Flavirhabdus iliipiscaria Th68T is JX412960. (http://eztaxon-e.ezbiocloud.net/; Kim et al., 2012). The Four supplementary figures are available with the online Supplementary 16S rRNA gene sequences of related strains were down- Material. loaded from the NCBI database and aligned using the Downloaded from www.microbiologyresearch.org by 000110 G 2015 IUMS Printed in Great Britain 1347 IP: 39.54.211.55 On: Fri, 14 Sep 2018 07:34:01 Q. Shakeela and others CLUSTAL X program (Thompson et al., 1997). Phylogenetic and S2, available in the online Supplementary Material), trees were reconstructed using the neighbour-joining, the isolate formed a distinct phylogenetic branch within the maximum-likelihood and maximum-parsimony methods family Flavobacteriaceae. Thus, based on the 16S rRNA gene with Kimura two-state parameter model analyses (Kimura, sequence analysis, strain Th68T appeared to represent a novel 1980) implemented in the program MEGA version 5 species of a new genus in the family Flavobacteriaceae. (Tamura et al., 2011). In each case, bootstrap values were Gram-staining and flagellum staining were investigated calculated based on 1000 replicates. using standard methods (Beveridge et al., 2007). Cell Pairwise alignment according to the nearly complete 16S morphology was determined by transmission electron rRNA gene sequence (1441 bp) of strain Th68T showed microscopy (JEM-1200EX; JEOL) after cells had been 96.1 % sequence similarity to the type strain of Pontirhabdus negatively stained with 1 % (w/v) phosphotungstic acid. pectinivorans (Yi et al., 2011), which was later reclassified as Gliding motility was observed by the hanging-drop Algibacter pectinivorans by Park et al. (2013), followed by technique and production of flexirubin-type pigments was members of the genera Olleya (96.1–95.3 %), Flavivirga (96.1– estimated by a colour shift following exposure to 20 % (w/v) 95.1 %), Lacinutrix (96.0–95.0 %), Formosa (96.0–94.1 %), KOH (Bernardet et al., 2002). To test for anaerobic growth, Arenitalea (95.4 %), Winogradskyella (95.4–93.4 %), Bizionia bacterial strains were cultured on MA with resazurin as (95.1–94.1 %), Flaviramulus (95.0 %) and Gaetbulibacter an indicator of anaerobic conditions in an anaerobic jar (95.0–94.4 %). In the neighbour-joining (Fig. 1), max- filled with nitrogen and a packet of AaneroPack-Anaero imum-likelihood and maximum-parsimony trees (Figs S1 (Mitsibishi Gas Chemical) at 28 uC for 1 month. Salinity 100 Lacinutrix algicola AKS293T (DQ167238) 71 Lacinutrix mariniflava AKS432T (DQ167239) 0.02 100 Lacinutrix copepodicola DJ3T (AY69001) Lacinutrix himadriensis E4-9aT (FN317744) CAM030T (JN175350) 93 Olleya marilimosa Olleya aquimaris L-4T (FJ886713) Olleya namhaensis WT-MY15T (JQ327134) Flavirhabdus iliipiscaria Th68T (JX412960) 98 Winogradskyella damuponensis F081-2T (HQ336488) T 99 Winogradskyella thalassocola KMM 3907 (AY521223) T 97 Winogradskyella echinorum KMM 6211 (EU727254) Winogradskyella ulvae KMM 6390T (HQ456127) Algibacter miyuki WS-MY6T (KC662118) 89 Algibacter pectinivorans JC2675T (HM475134) Algibacter agarivorans KYW560T (JN864025) 75 T 83 Flaviramulus basaltis H35 (DQ361033) Th78T (JX412985) 96 Flaviramulus ichthyoenteri SMK-12T (AY883937) 86 Gaetbulibacter saemankumensis 96 Gaetbulibacter lutimaris D1-y4T (JF739861) Gaetbulibacter marinus IMCC 1914T (EF108219) 100 Bizionia echini KMM 6177T (FJ716799) Bizionia hallyeonensis T-y7T (JN885199) Bizionia paragorgiae KMM 6029T (AY651070) Formosa spongicola A2T (FJ348469) 99 Formosa algae KMM 3553T (AY228461) Formosa arctica IMCC 9485T (KF148059) T 92 Flavivirga amylovorans JC2681 (HM475138) Flavivirga jejuensis JC2682T (HM475139) Brumimicrobium glaciale IC156T (AF521195) Fig. 1. Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of strain Th68T and representatives of some other related members of the family Flavobacteriaceae. Percentage bootstrap values above 70 (1000 replicates) are shown at branch nodes. Filled circles indicate that the corresponding nodes were also recovered in trees generated with the maximum-parsimony and maximum-likelihood algorithms. Brumimicrobium glaciale IC156T (GenBank accession no. AF521195) was used as an outgroup. Bar, 0.02 substitutions per nucleotide position. Downloaded from www.microbiologyresearch.org by 1348 International Journal of Systematic and Evolutionary Microbiology 65 IP: 39.54.211.55 On: Fri, 14 Sep 2018 07:34:01 Flavirhabdus iliipiscaria gen. nov., sp. nov. and pH range supporting growth were investigated in 96- version 6.0), and identified by the TSBA6.0 database of the well microplates by measuring the optical densities (wave- Microbial Identification System (Sasser, 1990). For analysis length 590 nm). Growth in synthetic ZoBell broth was tested of respiratory quinones and polar lipids, cells were harvested in the presence of 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 10, 12, 15 and 20 % from MB after incubation at 28 uC for 72 h and freeze-dried. (w/v) NaCl or sea salts (Sigma) to determine salinity Polar lipids were extracted according to the procedures tolerance of the bacterium. The temperature range for described by Minnikin et al. (1984), and separated by two- growth was evaluated at 0, 4, 10, 16, 28, 30, 37 and 42 uCon dimensional TLC on silica gel 60 F254 plates (Merck) using MA and at pH 2.0–11.0 in marine broth
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