A Gram-stain-negative, red pigment-producing, aerobic, and rod-shaped bacterial strain (A2-2T) was isolated from a bleached scleractinian coral (Porites lutea). Strain A2-2T grew with 1.0-7.0 % (w/v) NaCl (optimum, 3.0 %), at pH 6.0-11.0 (optimum, pH 8.0), and at 18-41 °C (optimum, 35 °C). Results of phylogenetic analysis based on 16S rRNA gene sequences suggested that strain A2-2T fell within the genus Spartinivicinus and was closely related to Spartinivicinus ruber S2-4-1HT (98.1 % sequence similarity) and Spartinivicinus marinus SM1973T (98.0 % sequence similarity). The predominant cellular fatty acids of strain A2-2T were C16 : 0 (31.0 %), summed feature 3 (29.0 %), summed feature 8 (11.7 %), C12 : 0 3-OH (6.4 %), and C10 : 0 3-OH (5.5 %), while the major respiratory quinone was Q-9. The polar lipids mainly comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and an unidentified phospholipid. The genome size of strain A2-2T was 6.8 Mb, with a G+C content of 40.2 mol%. The DNA-DNA hybridization value was 24.2 % between A2-2T and S. ruber S2-4-1HT and 36.9 % between A2-2T and S. marinus SM1973T, while the average nucleotide identity values were 80.1 and 88.8 %, respectively. Based on these findings, strain A2-2T could be recognized to represent a novel species of the genus Spartinivicinus, for which the name Spartinivicinus poritis sp. nov. is proposed. The type strain is A2-2T (=MCCC 1K08228T=KCTC 8323T).

A Gram-stain-negative, yellow-pigmented, and facultatively aerobic bacterium, designated strain GPA1T, was isolated from plastic waste landfill soil in the Republic of Korea. The cells were non-motile short rods exhibiting oxidase-negative and catalase-positive activities. Growth was observed at 15-40 °C (optimum, 30 °C), at pH 6.0-9.0 (optimum, pH 7.0-8.0) and in the presence of 0-2.5 % (w/v) NaCl (optimum, 0 %). Menaquinone-7 was the sole respiratory quinone, and iso-C15 : 0, C16 : 1  ω5c, and iso-C17 : 0 3-OH were the major cellular fatty acids (>10 % of the total fatty acids). Phosphatidylethanolamine was identified as a major polar lipid. Phylogenetic analyses based on 16S rRNA gene sequences and 120 concatenated marker protein sequences revealed that strain GPA1T formed a distinct lineage within the genus Chitinophaga. The genome of strain GPA1T was 6078 kb in size with 53.8 mol% G+C content. Strain GPA1T exhibited the highest similarity to Chitinophaga rhizosphaerae T16R-86T, with a 98.6 % 16S rRNA gene sequence similarity, but their average nucleotide identity and digital DNA-DNA hybridization values were 82.5 and 25.9 %, respectively. Based on its phenotypic, chemotaxonomic, and phylogenetic characteristics, strain GPA1T represents a novel species of the genus Chitinophaga, for which the name Chitinophaga pollutisoli sp. nov. is proposed. The type strain is GPA1T (=KACC 23415T=JCM 36644T).

Novel Gram-positive, catalase-negative, α-haemolytic cocci were isolated from breast milk samples of healthy mothers living in Hanoi, Vietnam. The 16S rRNA gene sequences of these strains varied by 0-2 nucleotide polymorphisms. The 16S rRNA gene sequence of one strain, designated as BME SL 6.1T, showed the highest similarity to those of Streptococcus salivarius NCTC 8618T (99.4 %), Streptococcus vestibularis ATCC 49124T (99.4 %), and Streptococcus thermophilus ATCC 19258T (99.3 %) in the salivarius group. Whole genome sequencing was performed on three selected strains. Phylogeny based on 631 core genes clustered the three strains into the salivarius group, and the strains were clearly distinct from the other species in this group. The average nucleotide identity (ANI) value of strain BME SL 6.1T exhibited the highest identity with S. salivarius NCTC 8618T (88.4 %), followed by S. vestibularis ATCC 49124T (88.3 %) and S. thermophilus ATCC 19258T (87.4 %). The ANI and digital DNA-DNA hybridization values between strain BME SL 6.1T and other species were below the cut-off value (95 and 70 %, respectively), indicating that it represents a novel species of the genus Streptococcus. The strains were able to produce α-galactosidase and acid from raffinose and melibiose. Therefore, we propose to assign the strains to a new species of the genus Streptococcus as Streptococcus raffinosi sp. nov. The type strain is BME SL 6.1T (=VTCC 12812T=NBRC 116368T).

Four rod-shaped, non-motile, non-spore-forming, facultative anaerobic, Gram-stain-positive lactic acid bacteria, designated as EB0058T, SCR0080, LD0937T and SCR0063T, were isolated from different corn and grass silage samples. The isolated strains were characterized using a polyphasic approach and EB0058T and SCR0080 were identified as Lacticaseibacillus zeae by 16S rRNA gene sequence analysis. Based on whole-genome sequence-based characterization, EB0058T and SCR0080 were separated into a distinct clade from Lacticaseibacillus zeae DSM 20178T, together with CECT9104 and UD2202, whose genomic sequences are available from NCBI GenBank. The average nucleotide identity (ANI) values within the new subgroup are 99.9 % and the digital DNA-DNA hybridization (dDDH) values are 99.3-99.9 %, respectively. In contrast, comparison of the new subgroup with publicly available genomic sequences of L. zeae strains, including the type strain DSM 20178T, revealed dDDH values of 70.2-72.5 % and ANI values of 96.2-96.6 %. Based on their chemotaxonomic, phenotypic and phylogenetic characteristics, EB0058T and SCR0080 represent a new subspecies of L. zeae. The name Lacticaseibacillus zeae subsp. silagei subsp. nov. is proposed with the type strain EB0058T (=DSM 116376T=NCIMB 15474T). According to the results of 16S rRNA gene sequencing, LD0937T and SCR0063T are members of the Lacticaseibacillus group. The dDDH value between the isolates LD0937T and SCR0063T was 67.6 %, which is below the species threshold of 70 %, clearly showing that these two isolates belong to different species. For both strains, whole genome-sequencing revealed that the closest relatives within the Lacticaseibacillus group were Lacticaseibacillus huelsenbergensis DSM 115425 (dDDH 66.5 and 65.9 %) and Lacticaseibacillus casei DSM 20011T (dDDH 64.1 and 64.9 %). Based on the genomic, chemotaxonomic and morphological data obtained in this study, two novel species, Lacticaseibacillus parahuelsenbergensis sp. nov. and Lacticaseibacillus styriensis sp. nov. are proposed and the type strains are LD0937T (=DSM 116105T=NCIMB 15471T) and SCR0063T (=DSM 116297T=NCIMB 15473T), respectively.

Two Gram-staining-negative, facultative anaerobic, rod-shaped and phosphate-solubilizing strains designated SG2303T and SG2305, were isolated from paddy soil in China. Phylogenetic analysis based on 16 S rRNA gene sequences indicated that SG2303T and SG2305 represented a member of the genus Crenobacter within the family Neisseriaceae of the phylum Pseudomonadota. Strain SG2303T displayed higher 16 S rRNA gene sequence similarities with members of the genus Crenobacter ranging from 93.5 to 94.0%. Strains C. luteus YIM 78141T and C. cavernae K1W11S-77T were closest related to the isolated strains and were considered as type strains. Growth of strain SG2303T occurred at 10-55 °C (optimum 37 °C), pH 5.0-9.0 (optimum pH 6.0-7.0) and 0-1% (w/v) NaCl (optimum 0%). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain SG2303T and its closely related taxa were 76.1-78.2% and 20.5-22.1%, respectively. The genomic DNA G + C content was 62.2%. The quinone of strain SG2303T was Q-8. The major fatty acids (> 10%) of strain SG2303T were C16:0 (30.6%), summed feature 3 (C16:1ω7c and/or C16:1ω6c) (26.0%) and C12:0 3OH (12.1%). The polar lipids were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phospholipids (PL), glycolipid (GL) and unidentified lipids (UL). Based on the results of the phylogenetic, physiological, biochemical, and morphological analysis, strain SG2303T is recognized as a novel species of the genus Crenobacter, for which the name Crenobacter oryzisoli sp. nov. is proposed. The type strain is SG2303T (= GDMCC 1.3970T = JCM 36468T). In addition, SG2303T was also able of phosphorus solubilization and promoting the growth of rice seeds. Strain SG2303T exhibited a relatively high dissolvable phosphorus content of 2.52 µg·mL- 1.

Two novel strain pairs (HM61T/HM23 and S-34T/S-58) were isolated from soil and the faeces of Tibetan antelope (Pantholops hodgsonii) collected at the Qinghai-Tibet Plateau of PR China. All four new isolates were aerobic, non-motile, Gram-stain-positive, catalase-positive, oxidase-negative, and short rod-shaped bacteria. The results of phylogenetic analysis based on the full-length 16S rRNA genes and 283 core genomic genes indicated that the four strains were separated into two independent branches belonging to the genus Nocardioides. Strains HM61T and HM23 were most closely related to Nocardioides pelophilus THG T63T (98.58 and 98.65 % 16S rRNA gene sequence similarity). Strains S-34T and S-58 were most closely related to Nocardioides okcheonensis MMS20-HV4-12T (98.89 and 98.89 % 16S rRNA gene sequence similarity). The G+C contents of the genomic DNA of strains HM61T and S-34T were 70.6 and 72.5 mol%, respectively. Strains HM61T, S-34T and the type strains of closely related species in the analysis had average nucleotide identity values of 75.4-90.5 % as well as digital DNA-DNA hybridization values between 20.1 and 40.8 %, which clearly indicated that the four isolates represent two novel species within the genus Nocardioides. The chemotaxonomic characteristics of strains HM61T and S-34T were consistent with the genus Nocardioides. The major fatty acids of all four strains were iso-C16 : 0, C17 : 1  ω8c or C18 : 1  ω9c. For strains HM61T and S-34T, MK-8(H4) was the predominant respiratory quinone, ll-2,6-diaminopimelic acid was the diagnostic diamino acid in the cell-wall peptidoglycan, and the polar lipids profiles were composed of diphosphatidylglycerol and phosphatidylglycerol. Based on phylogenetic, phenotypic, and chemotaxonomic data, we propose that strains HM61T and S-34T represent two novel species of the genus Nocardioides, respectively, with the names Nocardioides bizhenqiangii sp. nov. and Nocardioides renjunii sp. nov. The type strains are HM61T (=GDMCC 4.343T=JCM 36399T) and S-34T (=CGMCC 4.7664T=JCM 33792T).

A signal amplification electrochemical biosensor chip was developed to integrate loop-mediated isothermal amplification (LAMP) based on in situ nucleic acid amplification and methyl blue (MB) serving as the hybridization redox indicator for sensitive and selective foodborne pathogen detection without a washing step. The electrochemical biosensor chip was designed by a screen-printed carbon electrode modified with gold nanoparticles (Au NPs) and covered with polydimethylsiloxane membrane to form a microcell. The primers of the target were immobilized on the Au NPs by covalent attachment for in situ amplification. The electroactive MB was used as the electrochemical signal reporter and embedded into the double-stranded DNA (dsDNA) amplicons generated by LAMP. Differential pulse voltammetry was introduced to survey the dsDNA hybridization with MB, which differentiates the specifically electrode-unbound and -bound labels without a washing step. Pyrene as the back-filling agent can further improve response signaling by reducing non-specific adsorption. This method is operationally simple, specific, and effective. The biosensor showed a detection linear range of 102-107 CFU mL-1 with the limit of detection of 17.7 CFU mL-1 within 40 min. This method showed promise for on-site testing of foodborne pathogens and could be integrated into an all-in-one device.

A Gram-stain-negative, aerobic, rod-shaped and motile strain HL-JVS1T, was isolated from the gastric tract of a juvenile Pacific white shrimp. Molecular phylogenetic analysis based on 16S rRNA gene sequences of strain HL-JVS1T revealed its affiliation with the genus Pleionea, with close relatives including Pleionea mediterranea MOLA115T (97.5%) and Pleionea sediminis S1-5-21T (96.2%). The complete genome of strain HL-JVS1T consisted of a circular 4.4 Mb chromosome and two circular plasmids (6.6 and 35.0 kb) with a G + C content of 43.1%. The average nucleotide identity and digital DNA-DNA hybridization values between strain HL-JVS1T and the type strains of described Pleionea species were 69.7-70.4% and 18.3-18.6%, respectively. Strain HL-JVS1T grew at 10-40 °C (optimum, 30 °C) in the presence of 0.5 - 9.0% (w/v) sea salts (optimum, 2.0 - 2.5%), and at pH range of 5.5 - 10.0 (optimum, pH 6.5). The major fatty acids (> 10%) were summed feature 9 (iso-C17:1 ω9c and/or C16:0 10-methyl) (23.3%), iso-C16:0 (14.5%), iso-C11:0 3-OH (13.8%) and iso-C15:0 (11.0%). The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid, two unidentified aminolipids, and two unidentified lipids. The respiratory quinone was ubiquinone-8. The comprehensive phylogenetic, phylogenomic, phenotypic and chemotaxonomic results showed that strain HL-JVS1T is distinct from other Pleionea species. Hence, we propose strain HL-JVS1T as a novel species belonging to the genus Pleionea, for which the name Pleionea litopenaei sp. nov. is proposed with HL-JVS1T (= KCCM 90514T = JCM 36490T) as the type strain.

A Gram-negative, aerobic, rod-shaped, non-motile bacterium, designated as FTW29T, was isolated from surface seawater sampled in Futian district, Shenzhen, China. Growth of strain FTW29T was observed at 15-42 ℃ (optimum, 28-30 ℃), pH 4.0-9.0 (optimum, pH 5.5-7.5) and in the presence of 0.5-10% NaCl (optimum, 3.0% NaCl). Strain FTW29T showed 95.0-96.8% 16 S rRNA gene sequence similarity to various type strains of the genera Thioclava, Sinirhodobacter, Rhodobacter, Haematobacter and Frigidibacter of the family Paracoccaceae, and its most closely related strains were Thioclava pacifica DSM 10,166T (96.8%) and Thioclava marina 11.10-0-13T (96.7%). The phylogenomic tree constructed on the bac120 gene set showed that strain FTW29T formed a clade with the genus Thioclava, with a bootstrap value of 100%. The evolutionary distance values between FTW29T and type strains of the genus Thioclava were 0.17-0.19, which are below the recommended standard (0.21-0.23) for defining a novel genus in the family Paracoccaceae. In strain FTW29T, the major fatty acids identified were summed feature 8 (C18:1ω7c) and C16:0, and the predominant respiratory quinones were ubiquinone-10 and ubiquinone-9. The composition of polar lipids in strain FTW29T included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminolipid, two unidentified glycolipids and an unidentified lipid. The genome of strain FTW29T comprised one circle chromosome and six plasmids, with a G + C content of 61.4%. The average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values between strain FTW29T and seven type strains of the genus Thioclava were 76.6-78.4%, 53.2-56.4% and 19.3-20.4%, respectively. Altogether, the phenotypic, phylogenetic and chemotaxonomic evidence illustrated in this study suggested that strain FTW29T represents a novel species of the genus Thioclava, with the proposed name Thioclava litoralis sp. nov. The type strain is FTW29T (= KCTC 82,841T = MCCC 1K08523T).

The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by probe hybridization in the presence of carrier DNA. It is based on the transfer of a known amount of DNA onto an inert solid support, such as a nylon membrane, utilizing the dot-blot apparatus and without electrophoretic separation. Nylon membranes have the advantage of high nucleic acid binding capacity (400 µg/cm2), high strength, and are positively or neutrally charged. The probe used is a highly specific ssDNA fragment of 18 to 20 bases long labeled with digoxigenin (DIG). The probe will conjugate with the Leptospira DNA. Once the probe has hybridized with the target DNA, it is detected by an anti-digoxigenin antibody, allowing its easy detection through its emissions revealed in an X-ray film. The dots with an emission will correspond to the DNA fragments of interest. This method employs the non-isotopic labeling of the probe, which may have a very long half-life. The drawback of this standard immuno-label is a lower sensitivity than isotopic probes. Nevertheless, it is mitigated by coupling polymerase chain reaction (PCR) and dot-blot assays. This approach enables the enrichment of the target sequence and its detection. Additionally, it may be used as a quantitative application when compared against a serial dilution of a well-known standard. A dot-blot application to detect Leptospira from the three main clades in water samples is presented here. This methodology can be applied to large amounts of water once they have been concentrated by centrifugation to provide evidence of the presence of Leptospiral DNA. This is a valuable and satisfactory tool for general screening purposes, and may be used for other non-culturable bacteria that may be present in water, enhancing the comprehension of the ecosystem.