Polyphasic Approach Research Articles

A polyphasic molecular approach to characterize a collection of grapevine endophytic bacteria with bioprotective potential.

The work presented here was conducted to characterize the biodiversity of a collection of bacterial isolates, mainly wood endophytes, as part of a research project focused on exploring their bioprotective potential for postharvest biological control of fruits. This work was the basis for the development of a tailored method combining 16S rDNA sequencing and Rep-PCR to differentiate the isolates and identify them to genus level or below. More than one hundred isolates obtained from wood and roots of different grapevine genotypes were cultured on appropriate growth media and then subjected to the specified multistep molecular identification. We have obtained good dereplication for grapevine-endophytic bacteria, together with reliable genetic identification. Both are essential prerequisites to properly characterize a biome bank and, at the same time, beneficial prerequisites to subsequently perform a correct bioprotection assessment.

Inacoccus terrestris sp. nov. (Chroococcaceae), a new cyanobacterium from soil of the European Russian Subarctic

ABSTRACT A strain of an Inacoccus species (Cyanobacteria, Chroococcaceae) was isolated from a terrestrial habitat in the Murmansk Region, Russia, and characterized using a ‘polyphasic approach’ that included molecular, morphological and ecological evidence. The 16S rRNA gene phylogeny supported the strong monophyly of the genus Inacoccus, with the separation of four lineages. One of them corresponded to the recently described species Inacoccus carmineus from Brazil, while two others belonged to previously published but still taxonomically undescribed Inacoccus strains from Romania and Portugal. The fourth lineage represented a new species, Inacoccus terrestris sp. nov., from the Murmansk Region (European Russian Subarctic). Inacoccus terrestris occurred in soil and, in culture, formed microscopic, amorphous colonies. The slime contained up to four blue-green or olive-green cells, and the mucilage was more or less clearly delimited and colourless. The species is characterized by the absence of nanocytes. Its 16S rRNA gene similarity with other Inacoccus strains (with the exception of BACA0595) is lower than 98.7%, and 16S–23S ITS ITS similarity is lower than 97%, which supports its delimitation as a separate new species. Inacoccus terrestris is further characterized by unique hypothetical secondary structures of D1-D1' and Box-B helices.

Diversity of Rock-Inhabiting Fungi in Tarragona Province, Spain.

Rock-inhabiting fungi (RIF) are usually extremely tolerant or extremophilic, as they can survive on natural and artificial rocks despite being exposed to stressful conditions. RIF have serious negative effects on the appearance and cohesion of rocky substrates, causing the alteration and decomposition of building materials, but also on human and animal health, as they can act as opportunistic pathogens. Their identification is therefore of great importance, especially in urban areas. In the present study, culturing techniques for isolating fungi, and a polyphasic taxonomic approach to their identification, were used to assess the diversity of micromycetes that darken the surfaces of buildings in various villages and cities in Tarragona Province (Spain). Sixty-four species of RIF belonging to forty-one genera were identified, including a new genus (Coccodomyces) and the following six new fungal species: Coccodomyces pleiosporus, Exophiala caementiphila, Exophiala multiformis, Neocatenulostroma spinulosum, Neodevriesia longicatenispora, and Paradevriesia holothallica. Thus, we have established that building materials are ecological niches where a high biodiversity of RIF can develop.

Clostridium lamae sp. nov., a novel bacterium isolated from the fresh feces of alpaca.

A novel Gram-positive, anaerobic, nonspore-forming, rod-shaped bacterium, designated strain NGMCC 1.200840T, was isolated from the alpacas fresh feces. The taxonomic position of the novel strain was determined using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed strain NGMCC 1.200840T was a member of the genus Clostridium and closely related to Clostridium tertium DSM 2485T (98.16% sequence similarity). Between strains NGMCC 1.200840T and C. tertium DSM 2485T, the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were 79.91% and 23.50%, respectively. Genomic DNA G + C content is 28.44mol%. The strain can utilise D-glucose, D-mannitol, D-lactose, D-saccharose, D-maltose, D-xylose, L-arabinose, D-cellobiose, D-mannose, D-melezitose, D-raffinose, D-sorbitol, L-rhamnose, D-trehalose, D-galactose and Arbutin to produce acid. The optimal growth pH was 7, the temperature was 37°C, and the salt concentration was 0-0.5% (w/v). The major cellular fatty acids (> 10%) included iso-C15:0, anteiso-C15:0 and iso-C17:0 3-OH. The polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, three unidentified phospholipids and two unidentified aminolipids. Based on phenotypic, phylogenetic and chemotaxonomic characteristics, NGMCC 1.200840T represents a novel species within the genus Clostridium, for which the named Clostridium lamae sp. nov. is proposed. The type strain is NGMCC 1.200840T (= CGMCC 1.18014T = JCM 35704T).

Amycolatopsis mongoliensis sp. nov., a novel actinobacterium with antifungal activity isolated from a coal mining site in Mongolia.

A novel filamentous actinobacterium designated strain 4-36T showing broad-spectrum antifungal activity was isolated from a coal mining site in Mongolia, and its taxonomic position was determined using polyphasic approach. Optimum growth occurred at 30 °C, pH 7.5 and in the absence of NaCl. Aerial and substrate mycelia were abundantly formed on agar media. The colour of aerial mycelium was white and diffusible pigments were not formed. Phylogenetic analyses based on 16S rRNA gene sequence showed that strain 4-36T formed a distinct clade within the genus Amycolatopsis. The 16S rRNA gene sequence similarity showed that the strain was mostly related to Amycolatopsis lexingtonensis DSM 44544T and Amycolatopsis rifamycinica DSM 46095T with 99.3 % sequence similarity. However, the highest digital DNA-DNA hybridization value to closest species was 44.1 %, and the highest average nucleotide identity value was 90.2 %, both of which were well below the species delineation thresholds. Chemotaxonomic properties were typical of the genus Amycolatopsis, as the major fatty acids were C15 : 0, iso-C16 : 0 and C16 : 0, the cell-wall diamino acid was meso-diaminopimelic acid, the quinone was MK-9(H4), and the main polar lipids were diphosphatidylglycerol, phosphatidylmethanolamine and phosphatidylethanolamine. The in silico prediction of chemotaxonomic markers was also carried out by phylogenetic analysis. The genome mining for biosynthetic gene clusters of secondary metabolites in strain 4-36T revealed the presence of 34 gene clusters involved in the production of polyketide synthase, nonribosomal peptide synthetase, ribosomally synthesized and post-translationally modified peptide, lanthipeptide, terpenes, siderophore and many other unknown clusters. Strain 4-36T showed broad antifungal activity against several filamentous fungi. The phenotypic, biochemical and chemotaxonomic properties indicated that the strain could be clearly distinguished from other species of Amycolatopsis, and thus the name Amycolatopsis mongoliensis sp. nov. is proposed accordingly (type strain, 4-36T=KCTC 39526T=JCM 30565T).

Microbacterium aquilitoris sp. nov. and Microbacterium gwkjiense sp. nov., isolated from beach.

Three Gram-reaction-positive bacterial strains, designated KSW-18T, KSW2-22, and KSW4-11T, were isolated from seawater, and two dried seaweed samples collected at Gwakji Beach in Jeju, Republic of Korea, respectively, and their taxonomic positions were examined by a polyphasic approach. The 16S rRNA gene phylogeny showed that strain KSW4-11T was tightly associated with Microbacterium oleivorans NBRC 103075T, while strains KSW-18T and KSW2-22 formed a distinctive subline at the base of a clade including the above two strains. The three isolates showed high sequence similarity with one another (99.7-99.9%; 1-4 nt differences) and Microbacterium oleivorans (99.8-99.9%; 1-3 nt differences). The chemotaxonomic features were typical for the genus Microbacterium; Lysine as the diagnostic diamino acid and N-glycolylated muramic acid of the peptidoglycans, the predominant menaquinones of MK-11, MK-10 and MK-12, the major fatty acids of anteiso-C15:0 and anteiso-C17:0, and the major polar lipids including diphosphatidylglycerol, phosphatidylglycerol, and two or three unidentified glycolipids. In core genome-based phylogenetic tree, strains KSW-18T and KSW2-22 were closely associated with Microbacterium oleivorans NBRC 103075T, while strain KSW4-11T formed a distinctive subline at the base of a clade including the above three strains, in contrast to the 16S rRNA gene tree. Strains KSW-18T and KSW2-22 shared an OrthoANIu of 98.6% and a digital DNA-DNA hybridization of 87.6% with each other, representing that they were strains of a species, while the OrthoANIu and digital DNA-DNA hybridization values between strains KSW-18T and KSW4-11T, and between both of these isolates and all members of the genus Microbacterium were ≤86.5% and ≤30.7%, respectively. The analyses of overall genomic relatedness indices and phenotypic distinctness support that the three isolates represent two new species of the genus Microbacterium. Based on the results obtained here, Microbacterium aquilitoris sp. nov. (type strain KSW-18T = KCTC 49623T = NBRC 115222T) and Microbacterium gwkjiense sp. nov. (type strain KSW4-11T = KACC 23321T = DSM 116380T) are proposed.

Igniting taxonomic curiosity: The amazing story of Amazonocrinis with the description of a new genus Ahomia gen. nov. and novel species of Ahomia, Amazonocrinis, and Dendronalium from the biodiversity-rich northeast region of India.

Five cyanobacterial strains exhibiting Nostoc-like morphology were sampled from the biodiversity hotspots of the northeast region of India and characterized using a polyphasic approach. Molecular and phylogenetic analysis using the 16S rRNA gene indicated that the strains belonged to the genera Amazonocrinis and Dendronalium. In the present investigation, the 16S rRNA gene phylogeny clearly demarcated two separate clades of Amazonocrinis. The strain MEG8-PS clustered along with Amazonocrinis nigriterrae CENA67, which is the type strain of the genus. The other three strains ASM11-PS, RAN-4C-PS, and NP-KLS-5A-PS clustered in a different clade that was phylogenetically distinct from the Amazonocrinis sensu stricto clade. Interestingly, while the 16S rRNA gene phylogeny exhibited two separate clusters, the 16S-23S ITS region analysis did not provide strong support for the phylogenetic observation. Subsequent analyses raised questions regarding the resolving power of the 16S-23S ITS region at the genera level and the associated complexities in cyanobacterial taxonomy. Through this study, we describe a novel genus Ahomia to accommodate the members clustering outside the Amazonocrinis sensu stricto clade. In addition, we describe five novel species, Ahomia kamrupensis, Ahomia purpurea, Ahomia soli, Amazonocrinis meghalayensis, and Dendronalium spirale, in accordance with the International Code of Nomenclature for algae, fungi, and plants (ICN). Apart from further enriching the genera Amazonocrinis and Dendronalium, the current study helps to resolve the taxonomic complexities revolving around the genus Amazonocrinis and aims to attract researchers to the continued exploration of the tropical and subtropical cyanobacteria for interesting taxa and lineages.

Antimicrobial potential of Streptomyces coeruleofuscus SCJ isolated from microbiologically unexplored garden soil in Northwest Morocco

Research on microorganisms in various biotopes is required to identify new, natural potent molecules. These molecules are essential to control the development of multi-drug resistance (MDR). In the present study, a Streptomyces sp., namely SCJ, was isolated from a soil sample collected from a Moroccan garden. SCJ isolate was identified on the basis of a polyphasic approach, which included cultural, micro-morphological, biochemical, and physiological characteristics. The sequence of the 16S rRNA gene of the SCJ strain showed 99.78% similarity to strains of Streptomyces coeruleofuscus YR-T (KY753282.1). The preliminary screening indicated that the SCJ isolate exhibited activity against Candida albicans ATCC 60,193, Escherichia coli ATCC 25,922, Staphylococcus aureus CECT 976, Staphylococcus aureus ATCC 25,923, Bacillus cereus ATCC 14,579, Pseudomonas aeruginosa ATCC 27,853, as well as various other clinical MDR bacteria and five phytopathogenic fungi. The ethyl acetate extract of the isolated strain demonstrated highly significant (p < 0.05) antimicrobial activity against multi-resistant bacteria and phytopathogenic fungi. The absorption spectral analysis of the ethyl acetate extract of the SCJ isolate obtained showed no absorption peaks characteristic of polyene molecules. Moreover, no hemolytic activity against erythrocytes was observed in this extract. GC–MS analysis of the ethyl acetate extract of the SCJ isolate revealed the presence of 9 volatile compounds including 3,5-Dimethylpyrazole, and pyrrolizidine derivatives (Pyrrolo[1,2-a]pyrazine 1,4-dione, hexahydro-3-(2-methylpropyl)), which could potentially explain the antimicrobial activity demonstrated in this study.

Solibacillus palustris sp. nov., isolated from wetland soil of ecology park.

A Gram-staining-positive, motile, aerobic and rod-shaped bacterium, designated strain MA9T was isolated from wetland soil of ecology park, in Seoul, Republic of Korea. This bacterium was characterized to determine its taxonomic position by using the polyphasic approach. Strain MA9T grew at 10-37 °C and at pH 6.0-9.5 on TSB. Menaquinone MK-7 was the predominant respiratory quinone and iso-C15 : 0, iso-C16 : 0 and C16 : 1 ω7c alcohol were the major fatty acids. The main polar lipids were phosphatidylethanolamine (PE), phosphatidylserine (PS), diphosphatidylglycerol (DPG) and phosphatidylglycerol (PG). The peptidoglycan type of the cell wall was A4α l-Lys-d-Glu. Based on 16S rRNA gene sequencing, strain MA9T clustered with species of the genus Solibacillus and appeared closely related to S. silvestris DSM 12223T (97.8 % sequence similarity), S. cecembensis DSM 21993T (97.6 %), S. isronensis DSM 21046T (97.6 %) and S. kalamii DSM 101595T (96.6 %). The G+C content of the genomic DNA was 37.0 mol%. Digital DNA-DNA hybridization between strain MA9T and type strains of S. silvestris, S. isronensis, S. cecembensis and S. kalamii resulted in values below 70 %. Strain MA9T could be differentiated genotypically and phenotypically from the recognized species of the genus Solibacillus. The isolate therefore represents a novel species, for which the name Solibacillus palustris sp. nov. is proposed, with the type strain MA9T (=KACC 22212T = LMG 32188T).

Myceligenerans pegani sp. nov., an endophytic actinomycete isolated from Peganum harmala L. in Xinjiang, PR China.

An endophytic actinomycete designated TRM65318T, was isolated from the root of Peganum harmala L. Its taxonomic status was determined using a polyphasic approach. Comparative 16S rRNA gene sequence analysis indicated that strain TRM65318T is phylogenetically most closely related to Myceligenerans salitolerans XHU 5031T (98.15 %) and Myceligenerans xiligouense DSM 15700T (97.78 %). The peptidoglycan belonged to type A4α. The polar lipids were phosphatidylinositol, phosphatidylglycerol, diphosphatidylglycerol, two unknown lipids and three glycolipids. The predominant menaquinones were MK-9(H4) and MK-9(H6) and the whole-cell sugars contained glucose, mannose and galactose. Major fatty acids were anteiso-C15 : 0, iso-C15 : 0 and C16 : 0. Strain TRM65318T had a genome size of 5881012 bp with a genome G+C content of 71.79 mol%. The average nucleotide identity and DNA-DNA hybridization values between strain TRM65318T and the most closely related species were much lower than the thresholds commonly used to define species. At the same time, differences in phenotypic and genotypic data showed that strain TRM65318T could be clearly distinguished from M. salitolerans XHU 5031T. Therefore, it is concluded that strain TRM65318T represents a novel species of the genus of Myceligenerans. The proposed name for this organism is Myceligenerans pegani sp. nov., with type strain TRM65318T (=CCTCC AA 2019057T=LMG 31679T).

Rambellisea gigliensis and Rambellisea halocynthiae, gen. et spp. nov. (Lulworthiaceae) from the Marine Tunicate Halocynthia papillosa.

In this study, 15 Lulworthiales strains isolated from the marine tunicate Halocynthia papillosa collected in the central Tyrrhenian Sea were characterized using a polyphasic approach (morpho-physiological, molecular, and phylogenetic analyses). Based on multi-locus phylogenetic inference and morphological characters, a new genus, Rambellisea, and two new species, R. halocynthiae and R. gigliensis (Lulworthiales), were proposed. Multi-locus phylogenetic analyses using the nuclear ribosomal regions of DNA (nrITS1-nr5.8S-nrITS2, nrLSU, and nrSSU) sequence data strongly supported the new taxa. Phylogenetic inference, estimated using Maximum Likelihood and Bayesian Inference, clearly indicates that Rambellisea gen. nov. forms a distinct clade within the order Lulworthiales. Moreover, the two new species were separated into distinct subclades, solidly supported by the analyses. This is the first report of Lulworthiales species isolated from animals.

Description of Chryseobacterium fluminis sp. nov., a keratinolytic bacterium isolated from a freshwater river.

A Gram-stain-negative, aerobic, rod-shaped bacterial strain, designated MMS21-Ot14T, was isolated from a freshwater river, and shown to represent a novel species of the genus Chryseobacterium on the basis of the results from a polyphasic approach. The 16S rRNA gene sequence analysis revealed that MMS21-Ot14T represented a member of the genus Chryseobacterium of the family Weeksellaceae and was closely related to Chryseobacterium hagamense RHA2-9T (97.52 % sequence similarity), Chryseobacterium gwangjuense THG A18T (97.46 %) and Chryseobacterium gregarium P 461/12T (97.27 %). The optimal growth of MMS21-Ot14T occurred at 25-30 °C, pH 6.0-7.0 and in the absence of NaCl. MMS21-Ot14T was capable of hydrolysing casein, starch, DNA, Tween 20 and tyrosine. The strain also showed keratinolytic activity with keratin azure and decolourising activity with remazol brilliant blue R (RBBR), which indicated potential ability to degrade keratin and lignin. The main polar lipids of MMS21-Ot14T were phosphatidylethanolamine, unidentified aminophospholipids, unidentified aminolipids, an unidentified phospholipid and several unidentified lipids. The predominant fatty acids of MMS21-Ot14T were iso-C15 : 0 and iso-C17 : 0 3-OH, and the major isoprenoid quinone was menaquinone 6 (MK-6). The whole genome of MMS21-Ot14T was 5 062 016 bp in length with a DNA G+C content of 37.7 %. The average nucleotide identity and digital DNA-DNA hybridisation values between MMS21-Ot14T and phylogenetically related members of the genus Chryseobacterium were well below the threshold values for species delineation. It is evident from the results of this study that MMS21-Ot14T should be classified as representing a novel species of the genus Chryseobacterium, for which the name Chryseobacterium fluminis sp. nov. (type strain, MMS21-Ot14T = KCTC 92255T = LMG 32529T) is proposed.

Polyphasic molecular approach to the characterization of methanogens in the saliva of Tunisian adults

ObjectivesMethanogenic archaea are a minor component of human oral microbiota. Due to their relatively low abundance, the detection of these neglected microorganisms is challenging. This study concerns the presence of methanogens in salivary samples collected from Tunisian adults to evaluate their prevalence and burden using a polyphasic molecular approach. MethodsA total of 43 saliva samples were included. Metagenomic and standard 16S rRNA sequencing were performed as an initial screening to detect the presence of methanogens in the oral microbiota of Tunisian adults. Further investigations were performed using specific quantitative real-time PCR targeting Methanobrevibacter oralis and Methanobrevibacter smithii. ResultsMethanobrevibacter was detected in 5/43 (11.62 %) saliva samples after metagenomic 16S rRNA data analysis. The presence of M. oralis was confirmed in 6/43 samples by standard 16S rRNA sequencing. Using real-time PCR, methanogens were detected in 35/43 (81.39 %) samples, including 62.79 % positive for M. oralis and 76.74 % positive for M. smithii. These findings reflect the high prevalence of both methanogens, revealed by the high sensitivity of the real-time PCR approach. Interestingly, we also noted a significant statistical association between the detection of M. smithii and poor adherence to a Mediterranean diet, indicating the impact of diet on M. smithii prevalence. ConclusionOur study showed the presence of methanogens in the oral microbiota of Tunisian adults with an unprecedented relatively high prevalence. Choice of methodology is also central to picturing the real prevalence and diversity of such minor taxa in the oral microbiota.

Polyphasic characterization and genomic insights into Nocardioides turkmenicus sp. nov. isolated from a desert soil.

Strain KC13T, a novel desert-adapted, non-motile, Gram-stain-positive, rod-shaped, aerobic bacterium, was isolated from a soil sample collected from the Karakum Desert, Turkmenistan and characterised by a polyphasic approach. Phylogenetic analysis based on 16S rRNA sequences revealed that strain KC13T was a member of the genus Nocardioides, and formed a distinct cluster with Nocardioides luteus DSM 43366T (99.3% sequence identity), Nocardioides albus DSM 43109T (98.9%), Nocardioides panzhihuensis DSM 26487T (98.3%) and Nocardioides albertanoniae DSM 25218T (97.9%). The orthologous average nucleotide identity and digital DNA-DNA hybridization values were in the range of 85.8-91.0% and 30.2-35.9%, respectively, with the type strains of closely related species. The genome size of strain KC13T was 5.3Mb with a DNA G + C content of 69.7%. Comprehensive genome analyses showed that strain KC13T, unlike its close relatives, had many genes associated with environmental adaptation. Strain KC13T was found to have chemotaxonomic and phenotypic characteristics of members of the genus Nocardioides and some differences from phylogenetic neighbours. Based on the chemotaxonomic, genomic, phenotypic and phylogenetic data, strain KC13T represents a novel species of the genus Nocardioides, for which the name Nocardioides turkmenicus sp. nov. is proposed, and the type strain is KC13T (= JCM 33525T = CGMCC 4.7619T).

Pelagibacterium flavum sp. nov., Isolated from Soil Sample.

A Gram-stain-negative, yellow, moist and circular, aerobic, motile, and rod-shaped bacterium, designated YIM 151497T, was isolated from soil sample collected from Blue-Bridge, Weizhou Island, Guangxi province, China. Classification using a polyphasic approach suggested that strain YIM 151497T belonged to the genus Pelagibacterium, and was closely relevant to Pelagibacterium nitratireducens JLT2005T (98.8%), Pelagibacterium halotolerans CGMCC 1.7692T (98.7%), Pelagibacterium lixinzhangensis H64T (98.1%), and Pelagibacterium luteolum CGMCC 1.10267T (97.1%). The growth ranges of temperature, pH, and NaCl were 4-40℃, pH 4.0-10.0, and 0-7% NaCl, respectively. It was positive for catalase and oxidase. The primary respiratory quinone was Q-10. The elemental fatty acids were Summed Feature 8 (constituting C18:1ω7c and/or C18:1ω6c), C19:0 cyclo ω8c, C16:0, and C18:1ω7c 11-methyl. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, and three unidentified glycolipids. The DNA G+C content based on the complete genome sequence was 60.7mol%. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between strain YIM 151497T and species of Pelagibacterium were in the ranges of 73.9-86.3% and 19.7-31.3%, respectively. The Average Amino Acid Identity (AAI) between strain YIM 151497T and species of Pelagibacterium were in the ranges of 68.8-88.8%. On the basis of these data, strain YIM 151497T is considered to represent a novel species of the genus Pelagibacterium with the name of Pelagibacterium flavum sp. nov. Type strain is strain YIM 151497T (= KCTC 49826T = CGMCC 1.61521T = MCCC 1K08053T).

Polyphasic Characterization of Brucella spp. in Livestock Slaughtered from Abattoirs in Eastern Cape, South Africa.

In livestock, brucellosis is mainly an asymptomatic disease except when abortion occurs; therefore, two serological tests are used for diagnosis as no single test is suitable. Abattoir samples enable a combination of culture, molecular, and serological tests to detect brucellosis. This study assessed Brucella-specific PCR (ITS-PCR) to detect brucellosis and to conduct a molecular characterization of Brucella spp. isolated from PCR-positive livestock (n = 565) slaughtered at abattoirs and the appropriate sample tissue(s). ITS-PCR detected Brucella DNA in 33.6% of cattle, 14.5% of sheep, and 4.7% of pig tissues. Impure Brucella cultures from PCR-positive tissues were 43.6% (44/94) of cattle, 51.7% (15/29) of sheep, and 50% (2/4) of pigs with predominantly B. abortus identification with AMOS-PCR and low isolation of mixed B. abortus and B. melitensis in all species. In cattle, 33% of isolates were from lymph nodes, while in sheep 38.0% were from the liver and kidney and only from tonsils in pigs (2/4). Brucella infections identified with AMOS-PCR were present in seropositive and mainly seronegative (75.6-100%) livestock with the potential to cause brucellosis during pregnancy or breeding. This study demonstrated the value of the polyphasic approach, especially with chronic infections and the potential risk of these asymptomatic animals.

Aquimarina aquimarini sp. nov. and Aquimarina spinulae sp. nov., novel bacterial species with versatile natural product biosynthesis potential isolated from marine sponges.

This study describes two Gram-negative, flexirubin-producing, biofilm-forming, motile-by-gliding and rod-shaped bacteria, isolated from the marine sponges Ircinia variabilis and Sarcotragus spinosulus collected off the coast of Algarve, Portugal. Both strains, designated Aq135T and Aq349T, were classified into the genus Aquimarina by means of 16S rRNA gene sequencing. We then performed phylogenetic, phylogenomic and biochemical analyses to determine whether these strains represent novel Aquimarina species. Whereas the closest 16S rRNA gene relatives to strain Aq135T were Aquimarina macrocephali JAMB N27T (97.8 %) and Aquimarina sediminis w01T (97.1 %), strain Aq349T was more closely related to Aquimarina megaterium XH134T (99.2 %) and Aquimarina atlantica 22II-S11-z7T (98.1 %). Both strains showed genome-wide average nucleotide identity scores below the species level cut-off (95 %) with all Aquimarina type strains with publicly available genomes, including their closest relatives. Digital DNA-DNA hybridization further suggested a novel species status for both strains since values lower than 70 % hybridization level with other Aquimarina type strains were obtained. Strains Aq135T and Aq349T grew from 4 to 30°C and with between 1-5 % (w/v) NaCl in marine broth. The most abundant fatty acids were iso-C17 : 03-OH and iso-C15 : 0 and the only respiratory quinone was MK-6. Strain Aq135T was catalase-positive and β-galactosidase-negative, while Aq349T was catalase-negative and β-galactosidase-positive. These strains hold unique sets of secondary metabolite biosynthetic gene clusters and are known to produce the peptide antibiotics aquimarins (Aq135T) and the trans-AT polyketide cuniculene (Aq349T), respectively. Based on the polyphasic approach employed in this study, we propose the novel species names Aquimarina aquimarini sp. nov. (type strain Aq135T=DSM 115833T=UCCCB 169T=ATCC TSD-360T) and Aquimarina spinulae sp. nov. (type strain Aq349T=DSM 115834T=UCCCB 170T=ATCC TSD-361T).

Syncephalastrum massiliense sp. nov. and Syncephalastrum timoneanum sp. nov. Isolated from Clinical Samples.

Mucormycosis is known to be a rare opportunistic infection caused by fungal organisms belonging to the Mucorales order, which includes the Syncephalastrum species. These moulds are rarely involved in clinical diseases and are generally seen as contaminants in clinical laboratories. However, in recent years, case reports of human infections due to Syncephalastrum have increased, especially in immunocompromised hosts. In this study, we described two new Syncephalastrum species, which were isolated from human nails and sputum samples from two different patients. We used several methods for genomic and phenotypic characterisation. The phenotypic analysis relied on the morphological features, analysed both by optical and scanning electron microscopy. We used matrix-assisted laser desorption-ionization time-of-flight mass spectrometry, energy-dispersive X-ray spectroscopy, and BiologTM technology to characterise the proteomic, chemical mapping, and carbon source assimilation profiles, respectively. The genomic analysis relied on a multilocus DNA sequence analysis of the rRNA internal transcribed spacers and D1/D2 large subunit domains, fragments of the translation elongation factor-1 alpha, and the β-tubulin genes. The two novel species in the genus Syncephalastrum, namely S. massiliense PMMF0073 and S. timoneanum PMMF0107, presented a similar morphology: irregular branched and aseptate hyphae with ribbon-like aspects and terminal vesicles at the apices all surrounded by cylindrical merosporangia. However, each species displayed distinct phenotypic and genotypic features. For example, S. timoneanum PMMF0107 was able to assimilate more carbon sources than S. massiliense PMMF0073, such as adonitol, α-methyl-D-glucoside, trehalose, turanose, succinic acid mono-methyl ester, and alaninamide. The polyphasic approach, combining the results of complementary phenotypic and genomic assays, was instrumental for describing and characterising these two new Syncephalastrum species.

Tardiphaga alba sp. nov., a heavy-metal-tolerant bacterium isolated from garden soil.

A previously undescribed, heavy-metal-tolerant, motile, Gram-negative bacterium, designated strain SK50-23T, was characterized using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain SK50-23T was closely related to Tardiphaga robiniae LMG 26467T and the non-phototrophic 'Rhodopseudomonas boonkerdii' NS23T (98.1 and 97.3 % 16S rRNA gene sequence similarity, respectively). Strain SK50-23T possessed a circular genome of 5.86 Mb, with a DNA G+C content of 61.9 mol%. Digital DNA-DNA hybridization showed 20.8-21.6 % similarity between strain SK50-23T and related species. In addition, the whole-genome average nucleotide identity values between strain SK50-23T and related species ranged from 75.1 to 83.5 %. The major cellular fatty acid identified in strain SK50-23T was C18 : 1ω7c, and the main isoprenoid quinone present was ubiquinone Q-10. Strain SK50-23T could be assigned to the genus Tardiphaga with the species name Tardiphaga alba sp. nov. based on morphological, chemotaxonomic and genome-based taxonomic characteristics, and 16S rRNA gene-based phylogenetic characteristics. The type strain of the proposed novel species is SK50-23T (=NBRC 108825T=CGMCC No. 1.12037T).

Gansulinema gen. nov. and Komarkovaeasiopsis gen. nov.: Novel Oculatellacean genera (Cyanobacteria) isolated from desert soils and hot spring.

To increase the understanding of simple thin filamentous cyanobacteria in harsh environmental areas, we previously isolated and identified four strains (XN101, XN102, GS121, NX122) from desert soils and hot spring in China. As a result, two new Oculatellacean genera of these four strains, Gansulinema gen. nov. and Komarkovaeasiopsis gen. nov., are described based on a polyphasic approach. The ultrastructure of these strains showed a similar arrangement of peripheral thylakoids with three to four parallel layers, indicating that they belonged to the orders Nodosilineales, Oculatellales, or Leptolyngbyales. In the 16S rRNA gene phylogeny, two sequences of the Gansulinema strains and the two sequences of the Komarkovaeasiopsis strains formed two independent and robust clusters, within the order Oculatellales. The 16S rRNA gene sequences of strains of Komarkovaeasiopsis and Gansulinema showed low identity to each other (≤93.2%) and to other sequences of the Oculatellacean genera (≤94.5% and ≤93.3%, respectively). Furthermore, the 16S-23S internal transcribed spacer rRNA region secondary structures of strains of Komarkovaeasiopsis and Gansulinema were not consistent with all existing descriptions of Oculatellacean taxa. These data suggest that cyanobacterial communities are rich sources of new taxa in under-exploited areas, such as desert soils and hot spring in China.