key: cord-0021560-2pfciro8 authors: Kim, Joon Ki; Choi, Chi-Hwan; Kim, Dae-Won; Kim, Su Yeon; Hwang, Kyu Jam; Lee, Woo-Kon; Shin, Min Kyoung; Jung, Myunghwan; Choi, Young Sill title: Cohnella cholangitidis sp. nov., a novel species of the genus Cohnella isolated from a clinical specimen in Korea date: 2021-09-25 journal: Arch Microbiol DOI: 10.1007/s00203-021-02565-3 sha: 087cd68d433c7aefa1be38a9fa92805f7516c64f doc_id: 21560 cord_uid: 2pfciro8 A Gram-positive, aerobic, rod-shaped bacterium, designated as strain 1605-214(T), was isolated from the blood sample of a patient with cholangitis. Based on its 16S rRNA gene sequence, the strain 1605-214(T) belonged to the genus Cohnella and exhibited 97.9% sequence identity with Cohnella luojiensis DSM 24270(T) (GQ214052). DNA–DNA hybridization, digital DNA–DNA hybridization, and average nucleotide identity values between the two species were 23% ± 1.9, 21.1%, and 77.2%, respectively. The cellular fatty acids of strain 1605-214(T) were mainly comprised of anteiso-C(15:0) (36.1%), iso-C(16:0) (16.5%), and C(16:0) (15.1%). The predominant quinone was menaquinone-7; predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and aminophospholipid-1. The cell wall peptidoglycan of strain 1605-214(T) contained meso-diaminopimelic acid. DNA G + C content of strain 1605-214(T) was 50.6 mol%. 5187 genes out of a total of 5413 (94.6%) were assigned putative functions using eggNOG v5.0. Based on genotypic characteristics and genomic sequence analysis results, strain 1605-214(T) was confirmed to represent a novel species of genus Cohnella, for which the name Cohnella cholangitidis sp. nov., was proposed. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s00203-021-02565-3. The first species of the genus Cohnella was described as Cohnella thermotolerans in a report by Kämpfer et al (2006) . Currently, the genus Cohnella comprises 37 species (LPSN: http:// www. bacte rio. net), including six that have not been validated. Most members of Cohnella were isolated from various environments such as soil (Cai et al. 2010; Kim et al. 2010 Kim et al. , 2011 , plants (Garcia-Fraile et al. 2008) , water (Shiratori et al. 2010) , and industrial materials (Kämpfer et al. 2006) . Cohnella cellulosilytica (Khianngam et al. 2012) and Cohnella faecalis (Zhu et al. 2019) were isolated from animal excrements. Cohnella hongkongensis (Kämpfer et al. 2006) and Cohnella massiliensis (Abou Abdallah et al. 2019) were isolated from clinical samples (Table S1 ). In the present study, we have described strain 1605-214 T as a novel species of the genus Cohnella. To our knowledge, this is the Strain 1605-214 T was isolated from the blood culture of a cholangitis patient at Gyeongsang National University Hospital in Jinju, South Korea (35°10′ 35.5′′ N, 128°05′ 44 .2′′ E). The strain was grown on a blood agar plate (BAP) (KisanBio, Korea) at 30 °C for 48 h and stored at − 70 °C in 10% glycerol. Initial attempts of identification were made using matrix-assisted laser desorption/ ionization-time-of-flight mass spectrometry (MALDI-TOF MS) with MALDI Biotyper software (Bruker Daltonik, Germany). The experiment was performed using C. luojiensis DSM 24270 T , C. suwonensis DSM 25950 T , and C. yongneupensis DSM 18998 T as reference strains for comparative analysis of species characteristics. The 16S rRNA gene sequence similarity was calculated by comparing its sequence with those on the EzTaxon server (http:// www. eztax on. org/) (Chun et al. 2007 ). 16S rRNA gene sequencing was performed using universal primers 27F (3′ -AGA GTT TGATCMTGG CTC AG-5′) and 1492R (5′ -TAC GGY TAC CTT GTT ACG ACTT-3′) (Lane 1991) . Gram staining was performed using Gram Stain Kits (BD), and a catalase test was performed by adding 3% hydrogen peroxide solution to bacteria smeared on slides. The growth conditions for strain 1605-214 T were determined at different pH values (4-10, at pH intervals of 0.5 unit) on BAP. For analysis of its biochemical and enzymatic characteristics, VITEK 2 GP (bioMérieux, France) was used according to the manufacturer's instructions. To analyze its isoprenoid quinones, the cell biomass of strain 1605-214 T was obtained from cultures grown on BAP for 2 days at 30 °C. Quinones were extracted using the chloroform/methanol method [(C:M, 2:1, v/v)]. The extracted quinones were vacuum-evaporated and re-extracted using n-hexane-water (1:1, v/v). The purified quinones were analyzed using a reverse-phase HPLC system (Younglin, Korea), as described by Hiraishi et al. (1992) . The polar lipid composition of strain 1605-214 T was determined as described previously (Minnikin et al. 1980 ). The polar lipid composition was analyzed by two-dimensional thin-layer chromatography (2D-TLC) on TLC Kiesel gel 60F254 (Merck, Germany) plates (10 × 10 cm). The cellular fatty acid composition of the isolated strain was analyzed according to Miller's method (Miller 1982) . Agilent Technologies 6890 Gas Chromatography was performed to analyze the prepared samples, and an A30 m × 0.320 mm × 0.25 μm crosslinked methyl siloxane column (HP-1) was used as a separation column. The profile was analyzed using Sherlock MIS Software. Peak identification, retention time, peak area, and area ratio were determined by comparison with the standard calibration solution. The diaminopimelic acid in the cell wall was analyzed using a previously described method (Hasegawa et al. 1983 ). DNA-DNA hybridization was performed using the fluorometric microwell method (Ezaki et al. 1989 ). Genomic DNA was extracted by digestion of the bacteria with proteinase K in 10% SDS, followed by purification using the phenol extraction and ethanol precipitation methods. The primary sequencing library was prepared according to the protocol of the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences, USA). The secondary sequencing library was prepared according to the protocol of the Ion Xpress Plus Fragment Library kit (Thermo Fisher Scientific, USA). The genome was sequenced using PacBio RS II (Pacific Biosciences, USA) and Ion S5 (Thermo Fisher Scientific, USA) sequencing platforms. SPAdes Genome Assembler (v3.1) was adopted for de novo assembly sequence reads generated by NGS platforms PacBio RS II and Ion S5, and produced contigs and scaffold sequences. SSPACE program was used for scaffolding contigs and scaffold sequences, and the remaining sequencing errors including gaps and low-quality region were corrected using Proovread (v2.14.0). The genome of strain 1605-214 T was initially annotated using the PROKKA (Seemann 2014) software package. The NCBI Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al. 2016 ) software package was used to generate the final annotation. The predicted protein sequences were classified into functional groups in Clusters of Orthologous Groups (COG) using eggNOG 5.0 (Huerta-Cepas et al. 2019). The resistance genes and virulence factors were identified using AMRFinderPlus (Feldgarden et al. 2019 ) and VFdb (Liu et al. 2018) , respectively. An initial genomic distance calculation was conducted by searching for the genetically closest strains in EzTaxon Server (Chun et al. 2007 ) and Type Strain Genome Server (TYGS) (Meier-Kolthoff and Göker 2019). The 16S rRNA sequences of 37 type strains belonging to the Cohnella genus were downloaded from the list of prokaryotic names with standing in nomenclature (LPSN) (Parte 2018). The multiple sequence alignment was processed using MAFFT (Katoh and Standley 2013) . Phylogenetic trees were constructed with 1000 bootstrap replicates using the neighborjoining (NJ) method by MEGA7 (Kumar et al. 2016 ) and the maximum-likelihood (ML) method by RAxML (Stamatakis 2014). Figtree software was used to visualize the trees (http:// tree. bio. ed. ac. uk/ softw are/ figtr ee). Genomic sequence similarity comparison was conducted using the available genomes of the five closest Cohnella species. OrthoANI (Lee et al. 2016 ) and digital DNA-DNA hybridization (dDDH) (Meier-Kolthoff et al. 2013 ) were used to compare genome similarities. To calculate the average genomic identity of orthologous gene sequences (AGIOS) (Ramasamy et al. 2014 ) between genomes, the sets of orthologous proteins were first obtained using BLASTP, with the reciprocal-best-BLAST-hits (RBH) approach (minimal coverage of 50%, amino acid identity of 30%). The mean percentages of nucleotide sequence identity between the orthologous genes were then calculated. Strain 1605-214 T has been deposited in two microbial culture collections: the National Culture Collection for Three attempts to identify strain 1605-214 T by MALDI-TOF MS failed. Phylogenetic analysis, based on 16S rRNA gene sequences (Table S2 ) revealed that strain 1605-214 T belonged to the genus Cohnella and was closely related to Cohnella luojiensis DSM 24270 T (97.9%) (Fig. 1 , Figure S1 ). The optimal conditions of strain 1605-214 T for growth were a temperature of 30 °C and a pH of 7 (Table 1) . Based on VITEK 2 GP results, all four Cohnella spp. strains were positive for beta-galactosidase, beta galactopyranosidase, and alpha-galactosidase. In addition, strain 1605-214 T was positive for alpha-glucosidase and negative for D-trehalose, whereas Cohnella luojiensis, which is considered a genetically close species, was negative and positive, respectively, for the above-mentioned enzymes. The major lipid classes of strain 1605-214 T were identified as diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), and aminophospholipid-1 (APL1). The major quinone present in the strain was identified as MK-7. The cell wall peptidoglycan of strain 1605-214 T contained meso-diaminopimelic acid. The G + C content of strain 1605-214 T was 50.6 mol%, and the major fatty acids were anteiso-C 15:0 (36.1%), iso-C 16:0 (16.5%), and C 16:0 (15.1%) ( Table 2 ). The relatedness of DNA between strain 1605-214 T and C. luojiensis DSM 24270 T was 23.0% ± 1.9. Based on phenotypic and genotypic distinctness and DNA-DNA hybridization results, strain 1605-214 T was confirmed to be a novel pathogenic species similar to C. luojiensis. The complete genome of strain 1605-214 T is 6,408,853 bp in length with a GC content of 51.2%. Out of the 5867 predicted genes, 5481 genes code for proteins and 95 code for RNA (8 genes are 5S rRNA genes; 8 genes are 16S rRNA; 8 genes are 23S rRNA genes; 67 genes are tRNA genes; 4 genes are ncRNAs genes) (Table S3) . From the analysis of Clusters of Orthologous Groups of proteins (COGs), a total of 5,187 genes (94.6%) were assigned putative functions (Table S4 ). The strain 1605-214 T contained 896 genes (16.3%) for information storage and processing, 1224 genes (22.3%) for cellular processes and signaling, and 2108 genes (38.5%) for metabolism. An in silico search for the resistome of this strain revealed that the clbC gene (90.4% identity) (Hansen et al. 2012 ) confers resistance to PhLOPSa (phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A) antibiotics and was identified by NCBI AMRFinder program (Feldgarden et al. 2019 ). An in silico search for virulence factors revealed eight proteins with high identity percentages conferring potential pathogenicity. These proteins were LPS biosynthesis protein PseA-like (79.2% identity), chaperonin GroEL (75.3%), translation elongation factor Tu (73.6%), UTP-glucose-1-phosphate uridylyltransferase gtaB (71.8%), imidazole glycerol phosphate synthase subunit HisF (71.3%), ATP-dependent Clp protease proteolytic subunit clpP (71.3%), enolase eno (70.3%), and glucose-1-phosphate thymidyl transferase rmlA (70.2%). At the time of manuscript preparation, the 16S rRNA sequences of the type strains were analyzed as mentioned above; however, a comparison at the whole-genome level was not possible. Therefore, the Cohnella cholangitidis 1605-214 T was further compared to four type strains, including C. luojiensis (Table 3 ). The four strains were selected based on the results of the dDDH analysis from TYGS. Additionally, average nucleotide identity (ANI) analysis was also performed for the strains. The dDDH and ANI results for the assessed strains were, respectively, as follows: C. luojiensis ( The phenotypic, morphological, and biochemical characterizations, genome perspectives, and comparative genome analyses suggested that strain 1605-214 T represents a novel species of the genus Cohnella for which the name C. cholangitidis is proposed. Cohnella cholangitidis (chol.an.gi'ti.dis. N.L. gen. n. cholangitidis of cholangitis, derived from the disease of the patient from which this strain was isolated). Gram-positive, rod-shaped, catalase-positive, oxidasepositive, and facultative anaerobic. Colonies are grayishwhite in color and 0.5 mm in size on BAP. The optimal growth conditions are 30 °C and pH 7, although growth is also observed at 15-42 °C and pH 6-8. Positive for alphagalactosidase, beta-galactosidase, alpha-glucosidase, and beta-galactopyranosidase. The G + C content is 50.6 mol% and the major fatty acids are anteiso-C15:0 (36.1%), iso-C16:0 (16.5%), and C16:0 (15.1%). The major lipids are diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), and aminophospholipid-1 (APL1). The major quinone is MK-7. The cell wall contains meso-diaminopimelic acid. Strain 1605-214 T (= NCCP 16833 T , = DSM 112742 T ) was isolated from a clinical specimen at the Gyeongsang National University Hospital in Jinju, Gyeongsangnam-do, South Korea. The online version contains supplementary material available at https:// doi. org/ 10. 1007/ s00203-021-02565-3. Draft genome and description of Cohnella massiliensis sp. nov. a new bacterial species isolated from the blood culture of a hemodialysis patient Cohnella luojiensis sp. nov., isolated from soil of a Euphrates poplar forest EzTaxon: a web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains Validating the AMRFinder tool and resistance gene database by using antimicrobial resistance genotype-phenotype correlations in a collection of isolates Cohnella phaseoli sp. nov., isolated from root nodules of Phaseolus coccineus in Spain, and emended description of the genus Cohnella The order Bacillales hosts functional homologs of the worrisome cfr antibiotic resistance gene A rapid analysis for chemical grouping of aerobic actinomycetes Rapid profiling of bacterial quinones by two dimensional thin-layer chromatography eggNOG 5.0: a hierarchical functionally and phylogenetically annotated orthology resource based on 5090 organisms and 2502 viruses Cohnella thermotolerans gen. nov., sp. nov., and classification of 'Paenibacillus hongkongensis' as Cohnella hongkongensis sp. nov MAFFT multiple sequence alignment software version 7: improvements in performance and usability Cohnella cellulosilytica sp. nov., isolated from buffalo faeces Cohnella yongneupensis sp. nov. and Cohnella ginsengisoli sp. nov., isolated from two different soils Cohnella soli sp. nov. and Cohnella suwonensis sp. nov. isolated from soil samples in Korea MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets 16S/23S rRNA sequencing OrthoANI: an improved algorithm and software for calculating average nucleotide identity VFDB 2019: a comparative pathogenomic platform with an interactive web interface TYGS is an automated highthroughput platform for state-of-the-art genome-based taxonomy Genome sequence-based species delimitation with confidence intervals and improved distance functions Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters including hydroxy acid Thin layer chromatography of methanolysates of mycolic acid containing bacteria LPSN -List of prokaryotic names with standing in nomenclature (bacterio.net), 20 years on A polyphasic strategy incorporating genomic data for the taxonomic description of novel bacterial species Prokka: rapid prokaryotic genome annotation Cohnella fontinalis sp. nov., a xylanolytic bacterium isolated from fresh water RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies NCBI prokaryotic genome annotation pipeline Cohnella faecalis sp. nov., isolated from animal faeces in a karst cave Acknowledgements This research was supported and funded by Korea National Institute of Health (2017-NG45004-00). We would like to thank Editage for English language editing.