Four New Members of The Family Cytophagaceae: Cryseosolum Histidinii gen. nov., sp. nov., Cryseosolum Indiensis gen. nov., sp. nov., Reichenbachia Cretensis, gen. nov., sp. nov., and Reichenbachia Soli, gen. nov., sp. nov. Isolated From Diverse Habitat

Four strains isolated, PWU4T, PWU20T, PWU5T and PWU37T were from both of soil in Germany, India and a faces sheep collected in Crete Island, respectively. Cells were Gram-negative, strictly aerobic, rod shaped, grew optimally between 28oC and 34oC, pH between 7.0 and 8.0 without the addition of NaCl. Catalase and oxidase-negative and grew on most mono- and disaccharides, a few polysaccharides and organic acid. The predominant menaquinone was MK-7. Major fatty acid was c16:1 ω7c (PWU4T and PWU20T) and c16:1 ω5c (PWU5T and PWU37T). The DNA G+C content of them were 50.2 mol %; 51.6 mol %; 39.8 mol % and 53.8 mol %, respectively. The 16S rRNA gene sequence analysis revealed that the closest relatives of them are less than 93.8% compared to Ohtaekwangia koreensis 3B-2T and Ohtaekwangia kribbensis 10AOT. It classified in two groups, where PWU4T, PWU20T shared 93.0% and PWU5T, PWU37T shared 97.5% sequence similarity. However, in both groups represent different species on the low average nucleotide identity (ANI) of their genomes, 69.7% and 83.8%, respectively. We proposed that the four strains represent four novel species of two new genera in the family Cytophagaceae. The type species of the novel genus Cryseosolum are Cryseosolum histdinii gen. nov., sp. nov. strain PWU4T (=DSM 111594T=NCCB 100798T), Cryseosolum indiensis sp. nov. strain PWU20T (=DSM 111597T=NCCB 100800T). The type species of the novel genus Reichenbachia are Reichenbachia cretensis gen. nov., sp. nov. strain PWU5T (=DSM 111596T=NCCB 100799T), Reichenbachia soli sp. nov. strain PWU37T (=DSM 111595T=NCCB 100801T).

Jeotgalibacillus Auranticolor sp. nov., Isolated from Freshwater Collected from Baiyangdian Lake Lake, China

A novel Gram-positive, strictly aerobic, rod-shaped, orange-pigmented bacterial strain, designated R-1-5s-1T, was isolated from Baiyangdian Lake, China. Strain R-1-5s-1T grew at 15-37℃ (optimum 37℃) and pH 7-11 (optimum pH 8) in Luria-Bertani medium. Based on 16S rRNA gene sequence analysis, strain R-1-5s-1T was assigned to the genus Jeotgalibacillus and showed the closest relationships with Jeotgalibacillus salarius ASL-1T (97.69%), Jeotgalibacillus alkaliphilus JC303T (97.29%), Jeotgalibacillus marinus DSM 1297T (97.15%), Jeotgalibacillus campisalis SF-57T (97.01%), and Jeotgalibacillus spp. (≤ 97%). The predominant polar lipids were phosphatidylglycerol and diphosphatidylglycerol; the major cellular fatty acids were iso-C15:0, anteiso-C15:0, iso-C17:0, and anteiso-C17:0; and the major respiratory quinones were MK-7 and MK-8. The peptidoglycan type of the cell wall was A1a linked via L-lysine as the diamino acid. The G+C content was 43.6%, and the draft genome size of strain R-1-5s-1T was 3.4 Mbp. Between strain R-1-5s-1T and the related strain J. salarius ASL-1T, the ANI and dDDH relatedness values were 78.9% and 20.8%, respectively. Phylogenetic, chemotaxonomic, and genotypic analyses revealed that strain R-1-5s-1T is a novel species in the genus Jeotgalibacillus, for which the name Jeotgalibacillus auranticolor sp. nov. is proposed. The type strain is R-1-5s-1T (=CGMCC 1.13567T=KCTC 43038T).

Investigating Bacterial and Free-Living Protozoa Diversity in Biofilms of Hot Water Pipes of Apartment Buildings in the City of Riga (Latvia)

Background: Biofilms, when formed on the surfaces of water pipes, can be responsible for a wide range of water quality and operational problems. We sought to assess the bacterial and free-living protozoa (FLP) diversity, in relation to the presence of Legionnaire's disease-causing bacteria Legionella pneumophila (L. pneumophila) in 45 biofilms of hot water distribution system pipes of apartment buildings in Riga, the capital city of Latvia.Results: 16S rRNA amplicon sequencing (metataxonomics) revealed that each biofilm contained 224 rather evenly distributed bacterial genera and that most common and most abundant were two genera, completely opposites in terms of their oxygen requirements: the obligately anaerobic Thermodesulfovibrio and the strictly aerobic Phenylobacterium. Water temperature and north-south axis (i.e., different primary water sources) displayed the most significant effect on the inter-sample variations, allowing us to re-construct three sub-networks (modules) of co-occurring genera, one involving (potentially FLP-derived) Legionella spp. Pangenome-based functional profile predictions suggested that all three may be dominated by pathways related to the development and maintenance of biofilms, including quorum sensing and nutrient transport, as well as the utilization of various energy sources, such as carbon and nitrogen. In our 18S rRNA amplicon sequencing data, potential hosts of L. pneumophila were detected in 11 out of 12 biofilm samples analyzed, however, in many cases, their relative abundance was very low (<1%). By validating our findings using culture-based methods, we detected L. pneumophila (serogroups 2, 3, 6 and 9) in nine (20%) biofilms, whereas FLP (mostly Acanthamoeba, Vahlkampfidae and Vermamoeba spp.) were present in six (~13%) biofilms. In two biofilms, L. pneumophila and its potential hosts were detected simultaneously, using culture-based methods.Conclusions: Overall, our study sheds light on the community diversity of hot water biofilms and predicts how several environmental factors, such as water temperature and source might shape it.

Aliidiomarina indica sp. nov., isolated from deep seawater

A Gram-stain-negative, rod-shaped bacterial strain, designated SW123T, was isolated from a deep-sea water sample collected from the Indian Ocean. Strain SW123T was strictly aerobic, catalase- and oxidase-positive. The predominant cellular fatty acids were iso-C15 : 0, iso-C17 : 0 and summed feature 9 (comprising C16 : 0-methyl or iso-C17 : 1 ω9c). Ubiquinone-8 was the sole respiratory quinone. The major polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The genomic DNA G+C content was 49.4 mol%. 16S rRNA gene sequence analysis showed that strain SW123T was closely related to Aliidiomarina shirensis AIST (96.7 % sequence similarity), Aliidiomarina iranensis GBPy7T (96.3%), Aliidiomarina haloalkalitolerans AK5T (96.0%) and Aliidiomarina celeris F3105T (95.9%). Phylogenetic trees based on 16S rRNA gene sequences showed that strain SW123T represented a novel member of the genus Aliidiomarina , forming a distinct cluster with A. celeris F3105T. On the basis of phylogenetic inference and phenotypic characteristics, we propose that strain SW123T represents a novel species of the genus Aliidiomarina , with the name Aliidiomarina indica sp. nov. The type strain is SW123T (=CGMCC1.16169T=KCTC 82234T).

Infective Endocarditis in a Patient due to Roseomonas Gilardii in a Tertiary Care Hospital

Roseomonas gilardiii a species of Gramnegative, strictly aerobic, coccobacilli-shaped, pink-pigmented bacterium pathogenic for humans, causing rare cause of bacteremia and other infections especially who are immunosuppressed .These bacterias are detected after several days growth in culture, and typical pink, mucoid colonies are found.In this case infective endocarditis in a patient due to roseomonas gilardii and the clinical course with isolation of the organism in Bangabandhu Sheikh Mujib Medical University (BSMMU)is reported. Bangladesh J Medicine July 2022; 33(1) : 109-113

Microbacterium paulum sp. nov., isolated from microfiltered milk

A novel Gram-stain-positive, strictly aerobic, short rod-shaped bacterium, designated 2CT, was isolated from freshly packaged microfiltered milk. This strain was able to grow within the NaCl concentration range of 0–5 % (w/v), temperature range of 8–37 °C (optimally at 30 °C) and at pH 6.0–10.0. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 2CT was closely related to species of the genus Microbacterium , with the highest sequence similarity (99.2 %) to Microbacterium lacticum DSM 20427T as well as Microbacterium flavum DSM 18909T (=YM18-098T). The phylogenetic tree based on 16S rRNA genes showed that strain 2CT clustered with M. flavum DSM 18909T. However, the phylogenetic tree based on concatenated 16S rRNA and four housekeeping genes showed that strain 2CT clustered with M. lacticum DSM 20427T. Furthermore, the phylogenomic tree showed that strain 2CT clustered with M. lacticum DSM 20427T and M. flavum DSM 18909T. The major respiratory quinones were MK-10, MK-11 and MK-12. The predominant cellular fatty acids were anteiso-C15 : 0, iso-C16 : 0 and anteiso-C17 : 0. The polar lipid composition of strain 2CT consisted of diphosphatidylglycerol, phosphatidylglycerol, three unidentified glycolipids and two unidentified lipids. The cell-wall peptidoglycan type was a variant of B1α {Gly} [l-Lys] d-Glu-l-Lys, with the amino acids lysine, glycine, alanine and glutamic acid. The whole-cell sugars consisted of galactose, glucose, ribose and minor amounts of rhamnose. In addition, strain 2CT showed a glycolyl-type cell wall. The genomic DNA G+C content was 69.8mol%, while the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values with the closely related Microbacterium species were below the recognized thresholds of 95–96 % ANI and 70 % DDH for species definition. Based on the phenotypic and genotypic data, strain 2CT (=LMG 32277T=CECT 30329T) is considered to represent a new species, for which the name Microbacterium paulum sp. nov. is proposed.

Roseomonas oleicola sp. nov., isolated from an oil production mixture in Yumen Oilfield, and emended description of Roseomonas frigidaquae

A pink, ovoid-shaped, Gram-stain-negative, strictly aerobic and motile bacterial strain, designated ROY-5-3T, was isolated from an oil production mixture from Yumen Oilfield in PR China. The strain grew at 4–42 °C (optimum, 30 °C), at pH 5–10 (optimum, 7) and with 0–5 % (w/v) NaCl (optimum, 0%). The results of phylogenetic analysis based on 16S rRNA gene sequences indicated that ROY-5-3T belongs to the genus Roseomonas and shared the highest pairwise similarities with Roseomonas frigidaquae CW67T (98.1%), Roseomonas selenitidurans BU-1T (97.8%), Roseomonas tokyonensis K-20T (97.7%) and Roseomonas stagni HS-69T (97.3%). The average nucleotide identity and digital DNA–DNA hybridization values between ROY-5-3T and other related type strains of Roseomonas species were less than 84.08 and 28.60 %, respectively, both below the species delineation threshold. Pan-genomic analysis showed that the novel isolate ROY-5-3T shared 3265 core gene families with the four closely related type strains in Roseomonas , and the number of strain-specific gene families was 513. The major fatty acids were identified as summed feature 8 (C18 : 1 ω6c/C18 : 1 ω7c), summed feature 3 (C16 : 1 ω6c/C16 : 1 ω7c) and C16 : 0. Strain ROY-5-3T contained Q-10 as the main ubiquinone and the genomic DNA G+C content was 69.8 mol%. The major polar lipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol. Based on the phylogenetic, morphological, physiological, chemotaxonomic and genome analyses, strain ROY-5-3T represents a novel species of the genus Roseomonas for which the name Roseomonas oleicola sp. nov. is proposed. The type strain is ROY-5-3T (=CGMCC 1.13459T =KCTC 82484T).

Saccharobesus litoralis gen. nov., sp. nov., a novel alginate-degrading bacterium isolated from the surface of intertidal algal turf

A novel rod-shaped, Gram-stain-negative, strictly aerobic and alginate-degrading marine bacterium, designated CCB-QB4T, was isolated from a surface of algal turf collected from a coastal area of Penang, Malaysia. The cells showed motility by a lateral flagellum. The rod-shaped cells formed long chains end-to-end. Phylogenetic analysis based on the 16S rRNA gene sequence of strain CCB-QB4T showed 94.07, 92.69, 91.52 and 90.90 % sequence similarity to Algibacillus agarilyticus RQJ05T, Catenovulum maritimum Q1T, Catenovulum agarivorans YM01T and Catenovulum sediminis D2T, respectively. Strain CCB-QB4T formed a cluster with A. agarilyticus RQJ05T. Strain CCB-QB4T was catalase-negative, oxidase-positive, and degraded agar, alginate, and starch. Cell growth was observed at 15–40 °C, at pH 7.0–10.0 and in the presence of 1–6 % (w/v) NaCl and glucose. The major fatty acids were summed feature 3 (C16 : 1 ω7c/iso-C15 : 0 2-OH), C16 : 0 and C18 : 1 ω7c. The polar lipids were phosphatidylethanolamine, two unidentified aminolipids, two unidentified glycolipids, an unidentified phospholipid and unidentified lipid. The major respiratory quinone was ubiquinone-8. The genomic DNA G+C content was 46.7 mol%. Based on the phenotypic, chemotaxonomic and phylogenetic data, strain CCB-BQ4T represents a novel species in a new genus, for which the name Saccharobesus litoralis gen. nov., sp. nov. is proposed. The type strain is CCB-QB4T (=JCM 33513T=CCB-MBL 5008T).

First Report of Xanthomonas euvesicatoria Causing Bacterial Leaf Spot of Pepper (Capsicum annuum) in Montenegro

Leaf spot of pepper was observed on different pepper cultivars in central Montenegro during summer and early autumn in three consecutive growing seasons (2017 - 2019). Necrotic spots were numerous, varying in size, irregular in shape, brown, and surrounded by a weak halo. The most intensive symptoms were observed on lower leaves. In conditions conducive or the infection, the lesions merged resulting in the leaf drop. Symptoms were not observed on pepper stems and fruits. A total of seventeen bacterial strains were isolated from infected pepper leaves collected in seven different localities in the seasons of 2017-19. They formed yellow, convex, and mucoid colonies on yeast extract–dextrose–CaCO3 (YDC) medium and induced hypersensitive reaction in tobacco leaves. They were Gram negative, strictly aerobic, oxidase negative, catalase-positive, hydrolyzed gelatine and esculin and did not reduce nitrate, nor grew on 0.1% TTC and at 37°C. Out of tested 17 strains, eight hydrolyzed starch and three showed pectolytic activity, thus differing in these biochemical traits from Xanthomonas euvesicatoria (Xe) the reference strain KFB 1 (Obradović et al., 2004) used in all tests as a positive control. PCR analysis, with primer pair XeF/XeR, produced a single characteristic band of 173 bp in all 17 strains (Koenraadt et al. 2009). Additionally, the BOX-PCR profile of all the strains produced with the BOX A1R primer (Schaad et al. 2001) showed 100% homology with KFB 1. Based on the locality and year of isolation, nine strains were selected for amplification and partial sequencing of the gyrB gene using sets of primers described by Parkinson et al. (2007). Obtained partial DNA sequences showed that all nine strains (GenBank nos. MZ569011, MZ574079, MZ574080, MZ574081, MZ574082, MZ574083, MZ574084, MZ574085, and MZ574086) share 99.86 to 100% identity of gyrB sequence with Xe type strain ICPM:109 as well as 98.71 to 100 % of gyrB sequence identity with Xe strain LMG930 isolated from pepper in The United States. Pathogenicity of all strains was confirmed by spraying young pepper plants (cv. Slonovo uvo) using a handheld sprayer with the bacterial suspension (108 CFU/ml of sterile tap water), in three replicates. Sterile distilled water and reference Xe strain (KFB 1) were used as negative and positive controls, respectively. The inoculated plants were incubated under plastic bags in the greenhouse providing high humidity conditions for 48h. Symptoms were monitored for two weeks after inoculation. Lesions surrounded by a halo appeared on leaves of all inoculated plants within 10 to 15 days after inoculation, while plants inoculated with SDW remained symptomless. Koch’s postulates were confirmed by reisolation of the pathogen from necrotic tissue and identity check by PCR using primer set of Koenraadt et al. (2009). The pathogen race was determined according to the reaction of cv. Early Calwonder (ECW) and its isogenic lines (ECW-10R, ECW-20R, ECW-30R) (Stall et al. 2009). Obtained results indicated that all tested strains and reference strain Xe (KFB 1) belong to the pepper race P8. Based on pathogenic, biochemical, and molecular characteristics, the strains isolated from pepper leaves in Montenegro were identified as X. euvesicatoria. Pepper production is particularly significant for small farmers in Montenegro. Favorable climate, use of noncertified seed and lack of crop rotation contributes to the disease occurrence and severity. The disease has probably been around for years but the etiology was not confirmed so far. This is the first report of X. euvesicatoria affecting pepper in this country.

Aquibacillus kalidii sp. nov., an indole acetic acid-producing endophyte from a shoot of Kalidium cuspidatum, and reclassification of Virgibacillus campisalis Lee et al. 2012 as a later heterotypic synonym of Virgibacillus alimentarius Kim et al. 2011

A Gram-stain-positive, strictly aerobic, motile, endospore-forming, milk-white, indole acetic acid-producing, rod-shaped bacterial strain, designated as HU2P27T, was isolated from a shoot of Kalidium cuspidatum collected in Tumd Right Banner, Inner Mongolia, PR China. Strain grew at 10–40 °C (optimum, 30 °C), at pH 6.0–9.0 (optimum, pH 7.0) and with 0–14.0 % NaCl (optimum, 5.0–8.0 %). The strain tested positive for oxidase, catalase and nitrate reductase. The phylogenetic trees based on the 16S rRNA gene sequence and the core genome both showed that strain HU2P27T clustered with Aquibacillus koreensis BH30097T, sharing 97.7 % and <97.0 % of 16S rRNA gene similarity with A. koreensis BH30097T and any other type strain. Strain HU2P27T contained MK-7 as the major respiratory quinone. Its major fatty acids were anteiso-C15 : 0 and iso-C15 : 0, and the major polar lipids were phosphatidylglycerol, diphosphatidylglycerol and four unidentified phospholipids. The genomic DNA G+C content was 36.0 mol%. The average nucleotide identity, amino acid identity and digital DNA–DNA hybridization values of strain HU2P27T with A. koreensis BH30097T were 71.7, 69.2 and 19.4%, respectively. The phylogenetic, physiological and phenotypic results allowed the discrimination of strain HU2P27T from its phylogenetic relatives. The name Aquibacillus kalidii sp. nov. is therefore proposed. The type strain is strain HU2P27T (=CGMCC 1.18646T=KCTC 43248T). Based on the results of 16S rRNA gene and genome analyses, we propose the reclassification of Virgibacillus campisalis Lee et al. 2012 as a later heterotypic synonym of Virgibacillus alimentarius Kim et al. 2011.