Evaluation of Human Dental Plaque Lactic Acid Bacilli for Probiotic Potential and Functional Analysis in Relevance to Oral Health.

Members of the lactic acid bacillus group are well-known probiotics and primarily isolated from fermented food, dairy products, intestinal and gut environment of human. Since probiotics from the human source are preferred, there exists a huge repertoire of lactobacilli in the human oral cavity which could prove a much better niche to be exploited for these beneficial microorganisms. Therefore, in this study, four lactobacilli strains, including strain DISK7, reported earlier, isolated from dental plaque samples of a healthy humans were evaluated for their probiotic potential. Strains displayed 99.9% of 16S rRNA gene sequence identity with species of the genera Lactobacillus and Limosilactobacillus. All strains showed lactic acid production, tolerance to low pH and antibiotic sensitivity. Variations were observed among strains in their aggregation ability, biofilm formation, bile salt resistance and cholesterol degradation. Further, we analyzed the interaction of strains with other oral commensals and opportunistic pathogens in co-culture experiments. Isolates DISK7 and DISK26 exhibited high co-aggregation (> 70%) with secondary colonizers, Streptococcus pyogenes and Veillonella parvula, respectively, but their aggregation ability was decreased with opportunistic pathogens. Furthermore, strains showed a substantial increase in biofilm in co-culture with other Lactobacillus isolates, indicating their ability to proliferate commensal bacteria in the oral environment. These microbes continually evolve in terms of niche adaptation as evidenced in genome analysis. The highlight of the investigation is the isolation and evaluation of the probiotic lactobacilli from the human oral cavity, which could prove a much better niche to be exploited for the effective commercialization of these beneficial microbes. Taken together, probiotic properties and interaction with commensal bacteria, these isolates exhibit the huge potential to be developed as alternative bioresource agents for maintenance of oral health. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01108-2.

Lysobacter arvi sp. nov. Isolated from Farmland Soil.

A bacterial strain designated as UC was isolated from farmland soil. Strain UCT formed a pale yellow colony on nutrient agar. Cell morphology revealed it as the rod-shaped bacterium that stained Gram-negative. The 16S rRNA gene sequence analysis identified strain UCT as a member of the genus Lysobacter that showed high identity with L. soli DCY21T (99.5%), L. panacisoli CJ29T (98.7%), and L. tabacisoli C8-1T (97.9%). It formed a distinct cluster with these strains in the neighbor-joining phylogenetic tree. A similar tree topology was observed in TYGS-based phylogenomic analysis. However, genome sequence analyses of strain UCT showed 87.7% average nucleotide identity and 34.7% digital DNA-DNA hybridization similarity with the phylogenetically closest species, L. soli DCY21T. The similarity was much less with other closely related strains of the genus Lysobacter. The G + C content of strain UCT was 68.1%. Major cellular fatty acids observed were C14:0 iso (13.4%), C15:0 iso (13.6%), and C15:0 anteiso (14.8%). Quinone Q-8 was the major respiratory ubiquinone. Predominant polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylglycerol. Production of xanthomonadin pigment was observed. Based on phenotypic differences and phylogenomic analysis, strain UCT represents a novel species of the genus Lysobacter, for which the name Lysobacter arvi is proposed. The type strain of the novel species is UCT (= KCTC 92613T = JCM 23757T = MTCC 12824T).

An antimicrobial thiopeptide producing novel actinomycetes Streptomyces terrae sp. nov., isolated from subsurface soil of arable land.

An antimicrobial producing Gram-positive, aerobic, nonmotile, and filamentous actinobacterial strain SKN60T was isolated from soil The isolate exhibited 99.3% and 99.0% identity with Streptomyces laurentii ATCC 31255T and S. roseicoloratus TRM 44457T, respectively, in 16S rRNA gene sequence analysis. However, the genome sequence displayed maximum ANI (88.45%) and AAI (85.61%) with S. roseicoloratus TRM 44457T. Similarly, the dDDH showed 33.7% identity with S. roseicoloratus TRM 44457T. It formed a cluster with S. roseicoloratus TRM 44457T and S. laurentii ATCC 31255T in phylogenomic tree. Cell wall analysis revealed the presence of diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylcholine as major polar lipids and diaminopimelic acid as diagnostic diamino acid. Major fatty acids were iso-C15:0, anteiso-C15:0, and iso-C16:0. The G+C content was found to be 72.3 mol%. Genome sequence analysis using antiSMASH database showed occurrence of a thiopeptide biosynthesis gene cluster with 94% similarity to berninamycin from S. bernensis UC5144. The mass of 1146 Da is identical with berninamycin. But subtle differences observed in leader peptide sequence of thiopeptide and berninamycin. Notably, S. bernensis is not validly reported and thus SKN60T is the only strain containing berninamycin BGC as no other phylogenetic relative had it. Additionally, strain SKN60T differed in phenotypic and genetic characteristics with all phylogenetic relatives of the genus Streptomyces. Therefore, it is proposed as a novel species with the name Streptomyces terrae sp. nov. strain SKN60T (=MTCC 13163T; = JCM 35768T).

Limosilactobacillus walteri sp. nov., a novel probiotic antimicrobial lipopeptide-producing bacterium.

A Gram-positive facultative anaerobe, nonspore forming, and nonmotile bacterial strain M31 was isolated from faecal contaminated soil. The strain is previously reported to produce a novel antimicrobial lipopeptide and displayed probiotic properties. The strain M31 is catalase negative and fermented d-galactose, d-glucose, esculin, d-maltose, d-lactose, d-melibiose, d-raffinose, d-saccharose (weak reaction), d-xylose (weak reaction), d-ribose (weak reaction), and l-arabinose (weak reaction). The majority of fatty acids were C16:0 (53.9%), C18:0 (26.9%), and C19:0 cyclo ω8c (19.1%). The genome is 2 234 040 bp long with 38.81% guanine-cytosine (GC) content. The pairwise ortho average nucleotide identity and digital DNA-DNA hybridization values of strain M31 with its closest relative species from Limosilactobacillus reuteri clade and Lm. rudii is below the recommended cut-off of 95% and 70%, respectively. Herein, we propose Lm. walteri sp. nov. as a novel species of the genus Limosilactobacillus with M31 = MTCC 12838 = JCM 32759 = KCTC 25569.

Taxonomic and genomic characterization of Sporosarcina cyprini sp. nov., moderately tolerant of Cr+6 and Cd+2 isolated from the gut of invasive fish Cyprinus carpio var. communis (Linn., 1758).

A Gram-stain-positive, motile, and rod-shaped bacterium, designated as strain MB25T, was isolated from the gut of Cyprinus carpio from the highly polluted river Yamuna, India. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain MB25T belonged to the genus Sporosarcina, sharing the highest sequence similarity with S. luteola Y1T (98.98%) and S. koreensis S-K12T (98.91%). Digital DNA-DNA hybridization and average nucleotide identity values of strain MB25T with strain Y1T and S-K12T were 18.9, 77.69, and 18.2, 76.80 respectively. Genome analysis of strain MB25T revealed its biotechnological properties such as tolerance to potent heavy metals, genes for the production of carbohydrate-active enzymes, antimicrobial compounds, and also degradation of aromatic compounds. The G + C content of strain MB25T genome was 45%. Growth observed at 10-40 °C (optimum, 28-30 °C), pH 6.0-8.5 (optimum pH 7.5-8.0); NaCl concentrations up to 6.0% (w/v). The dominant respiratory quinone was MK-7, cell wall peptidoglycan is of the A-4 type containing amino acids Lys-Glu and the major fatty acids are anteiso-C11:0 and iso-C15: 0. The major polar lipids of strain MB25T are diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. On the basis of phenotypic, chemotaxonomic, phylogenetic, and phylogenomic data, strain MB25T represents a novel species of the genus Sporosarcina, for which the name Sporosarcina cyprini sp. nov. is proposed. The type strain is MB25T (= MCC 4366 T = JCM 34521 T = CCM 9113 T).

Lacrimispora defluvii PI-S10-B5AT sp. nov., an Obligate Anaerobe, Isolated from an Industrial Waste and Reclassification of Hungatella xylanolytica as Lacrimispora xylanolytica and Clostridium indicum as Lacrimispora indica Comb. nov.

A bacterial strain was isolated from the waste slurry of an industrial effluent treatment plant near Patancheru, Hyderabad, India, and designated as PI-S10-B5AT. It was an obligately anaerobic, spore-forming, rod-shaped, motile bacterium that stained Gram-positive. The strain revealed high 16S rRNA gene sequence identity with Hungatella xylanolytica DSM 3808T (99.4%) followed by members of the genus Lacrimispora (98.8-93.3%). However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization of genome sequence exhibited similarity in the range of 94.3-68.7% and 57.4-18.8%, respectively, with all closely related strains. A multi-gene phylogenetic analysis of strain PI-S10-B5AT was performed to investigate the taxonomic affiliation, which revealed formation of a coherent cluster with the members of the genus Lacrimispora. The DNA G + C content was 41.8 mol%. Major polar lipids were glyco- and phospholipids. The fatty acids analysis showed C16:0 to be the major fatty acid. The predominant respiratory quinone was menaquinone-7 (MK-7). Based on phenotypic, chemotaxonomic, and whole-genome phylogenetic analysis, strain PI-S10-B5AT is assigned as a novel species of the genus Lacrimispora, for which the name Lacrimispora defluvii is proposed. The type strain of the novel species is PI-S10-B5AT (= MTCC 12280T; = DSM 24980T) isolated from waste slurry of effluent treatment plant. The genomic analysis of type strains of C. indicum PI-S10-A1BT and H. xylanolytica DSM 3808T showed ANI and AAI values consistent with members of the genus Lacrimispora. Therefore, these strains are ascertained to the genus Lacrimispora and reclassified as Lacrimispora indica and Lacrimispora xylanolytica comb. nov.

Characterization and Antimicrobial Studies of Iturin-Like and Bogorol-Like Lipopeptides From Brevibacillus spp. Strains GI9 and SKDU10.

Accession numbers for whole-genome sequence of Brevibacillus sp. strain GI9 and SKDU10 are CAGD01000001 to CAGD01000061 and LSSO00000000, respectively. Members of the genus Brevibacillus have been demonstrated to produce a variety of bioactive compounds including polyketides, lipopeptides and bacteriocins. Lipopeptides are non-ribosomally synthesized surface-active compounds with antimicrobial, antitumor, and immune-stimulatory activities. They usually exhibit strong antifungal and antibacterial activities and are considered as promising compounds in controlling fungal diseases. In this study, we have characterized two lipopeptides from Brevibacillus sp. strains GI9 and SKDU10. The corresponding lipopeptides were purified by reverse-phase high-performance liquid chromatography. Mass analysis and characterization by MALDI-TOF-MS (Matrix-assisted laser desorption ionization time-of-flight mass spectrometry) analysis revealed production of an iturin-like lipopeptide by strain GI9 and bogorol-like lipopeptide by strain SKDU10. Both lipopeptides exhibited broad spectrum antibacterial activity and inhibited the growth of various fungi. They showed minimum inhibitory concentration (MIC) values between 90 and 300 µg/ml against indicator strains of bacteria and drug-resistant Candida indicator strains. The lipopeptides did not show phytotoxic effect in seed germination experiments but caused hemolysis. Further, both lipopeptides inhibited the growth of fungi on fruits and vegetables in in vitro experiments, thereby exhibited potential use in biotechnological industry as effective biocontrol agents.

Paraclostridium dentum, a novel species with pathogenic features isolated from human dental plaque sample.

A strictly anaerobic bacterial strain designated as SKVG24 was isolated from subgingival dental plaque samples of patients suffering from periodontitis. Cells were stained Gram-positive, rod shaped with endospore. The strain showed negative reaction to catalase and oxidase enzymes, but positive for gelatinase activity. Optimal growth was observed at 37 °C temperature and 7.0 pH. The 16S rRNA gene sequence BLAST analysis assigned strain SKVG24 to the genus Paraclostridium as it displayed 99.93% identity with P. benzoelyticum JC272T followed by P. bifermentans ATCC 638T (99.79%). However, average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) of the whole genome sequence showed <97% and <70% identity, respectively, with type strains of all closely related species. The G + C content of the DNA was 28.7 mol%. Total lipids profile showed presence of glycolipids as major lipids. Pathogenic features like hemolysis, gelatin hydrolysis and production of volatile sulfur compounds exhibited by strain SKVG24T were analogous to those observed in the established oral pathogenic strains. Further, whole genome sequence analysis confirmed the presence of genes encoding virulence factors and provided genomic insights on adaptation of the strain in oral environment. Based on the phenotypic and genetic differences with phylogenetic relatives, strain SKVG24T is proposed to represent a new species of the genus Paraclostridium with potential pathogenic ability, for which the name Paraclostridium dentum sp. nov., is suggested. The proposed type strain is SKVG24T (MTCC 12836T; = JCM 32760T).

Salinicoccus cyprini sp. nov., isolated from the gut of mirror carp, Cyprinus carpio var. specularis.

A novel orange to pink coloured bacterial strain designated as CT19T was isolated from the gastrointestinal tract of mirror carp, Cyprinus carpio var. specularis (Lacepède, 1803) collected from the Gobind Sagar reservoir at village Lathiani, Una, Himachal Pradesh, India. Cells of the strain were found to be aerobic, Gram-stain-positive, non-motile and non-spore-forming coccoids. Based on the 16S rRNA gene sequence, the strain was closely related to Salinicoccus hispanicus J-82T (=DSM 5352T; 97.4 %), followed by S. sesuvii CC-SPL15-2T (=DSM 23267T; 96.4 %), S. amylolyticus JC304T (=KCTC 33661T; 95.6 %) and S. roseus DSM 5351T (95.4 %). Identity with all other members of the genus were <94.5 %. The draft genome of strain CT19T was assembled to 2.4 Mbp with a G+C content of 47.9 mol%. Average nucleotide identity and digital DNA-DNA hybridization values between strain CT19T and S. hispanicus J-82T were found to be 85.9 and 31.3% respectively which is far below the threshold for species delineation. Iso-C15 : 0, anteiso-C15 : 0, iso-C17 : 0, C16 : 0 and anteiso-C17 : 0 were the major cellular fatty acids of strain CT19T. Major polar lipids were diphosphatidylglycerol, phosphatidylgylcerol and an unidentified glycolipid. Respiratory quinone system was composed of menaquinone-6 and major cell wall amino acid was l-lysine. Based on phylogenomic, physiological and biochemical characteristics, strain CT19T represents a novel species of the genus Salinicoccus for which the name Salinicoccus cyprini sp. nov. is proposed. The type strain is CT19T (=KCTC 43022T =CCM 8886T=MCC 3834T).