Complete genome sequences of Lacticaseibacillus paracasei INIA P272 (CECT 8315) and Lacticaseibacillus rhamnosus INIA P344 (CECT 8316) isolated from breast-fed infants reveal probiotic determinants.

Lacticaseibacillus paracasei INIA P272 and Lacticaseibacillus rhamnosus INIA P344, isolated from breast-fed infants, are two promising bacterial strains for their use in functional foods according to their demonstrated probiotic and technological characteristics. To better understand their probiotic characteristics and evaluate their safety, here we report the draft genome sequences of both strains as well as the analysis of their genetical content. The draft genomes of L. paracasei INIA P272 and L. rhamnosus INIA P344 comprise 3.01 and 3.26 Mb, a total of 2994 and 3166 genes and a GC content of 46.27 % and 46.56 %, respectively. Genomic safety was assessed following the EFSA guidelines: the identification of both strains was confirmed through Average Nucleotide Identity, and the absence of virulence, pathogenic and antibiotic resistance genes was demonstrated. The genome stability analysis revealed the presence of plasmids and phage regions in both genomes, however, CRISPR sequences and other mechanisms to fight against phage infections were encoded. The probiotic abilities of both strains were supported by the presence of genes for the synthesis of SCFA, genes involved in resistance to acid and bile salts or a thiamine production cluster. Moreover, the encoded exopolysaccharide biosynthesis genes could provide additional protection against the deleterious gastrointestinal conditions, besides which, playing a key role in adherence and coaggregation of pathogenic bacteria together with the high number of adhesion proteins and domains encoded by both genomes. Additionally, the bacteriocin cluster genes found in both strains, could provide an advantageous ability to compete against pathogenic bacteria. This genomic study supports the probiotic characteristics described previously for these two strains and satisfies the safety requirements to be used in food products.

Rapid and improved identification of drinking water bacteria using the Drinking Water Library, a dedicated MALDI-TOF MS database.

According to the European Directives (UE) 2020/2184 and 2009/54/EC, which establishes the sanitary criteria for water intended for human consumption in Europe, water suitable for human consumption must be free of the bacterial indicators Escherichia coli, Clostridium perfringens and Enterococcus spp. Drinking water is also monitored for heterotrophic bacteria, which are not a human health risk, but can serve as an index of bacteriological water quality. Therefore, a rapid, accurate, and cost-effective method for the identification of these colonies would improve our understanding of the culturable bacteria of drinking water and facilitate the task of water management by treatment facilities. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is potentially such a method, although most of the currently available mass spectral libraries have been developed in a clinical setting and have limited environmental applicability. In this work, a MALDI-TOF MS drinking water library (DWL) was defined and developed by targeting bacteria present in water intended for human consumption. This database, made up of 319 different bacterial strains, can contribute to the routine microbiological control of either treated drinking water or mineral bottled water carried out by water treatment and distribution operators, offering a faster identification rate compared to a clinical sample-based library. The DWL, made up of 96 bacterial genera, 44 of which are not represented in the MALDI-TOF MS bacterial Bruker Daltonics (BDAL) database, was found to significantly improve the identification of bacteria present in drinking water.

Genome Sequence of Bifidobacterium breve INIA P734 (CECT 8178), a Strain Isolated from Human Breast Milk.

The draft genome sequence of Bifidobacterium breve INIA P734, a strain shared by mother and child, is reported. It consists of 50 contigs, with 2,391,925 bp, 2,099 genes, and a G+C content of 58.8%. The genome analysis revealed the absence of antibiotic resistance and pathogenicity-related genes.

Genomic Insights Into Five Strains of Lactobacillus plantarum With Biotechnological Potential Isolated From chicha, a Traditional Maize-Based Fermented Beverage From Northwestern Argentina.

Lactic acid bacteria (LAB) are indigenous microorganisms that have been involved in food fermentations throughout history to preserve food and also to give special characteristics to them. The traditional fermented foods that are still being elaborated in indigenous populations around the world are a potential source of LAB with important biotechnological properties and/or beneficial to health. In a previous work, LAB biodiversity associated with chicha, a traditional maize-based fermented beverage from Northwestern Argentina, was studied, both by culture dependent and independent methods. From that study, 392 isolates were recovered, mostly members of Lactobacillus and Leuconostoc. Biotechnological characterization of representative isolates led to the selection of five strains belonging to the species Lactobacillus plantarum for their ability to produce vitamin B2 (riboflavin) and vitamin B9 (folates), their antimicrobial properties and antibiotics susceptibility. In this work, we present the Whole Genome Sequences (WGS) of these five strains that have been deposited in the Spanish Type Culture Collection: M5MA1 (= CECT 8962), M9MM1 (= CECT 8963), M9MM4 (= CECT 8964), M9MG6 (= CECT 8965), and M9Y2 (= CECT 8966), and a detailed description of their characterization, through a genomic approach, analyzing the genes responsible for these biotechnological properties, making a comparative study of the five genomes and reporting the aspects related to food safety, in accordance with the recommendations of the European Food Safety Authority (EFSA FEEDAP Panel, 2018) aiming at their use in the design of functional foods. The analysis unveiled, for the five strains, the complete set of genes for folate and riboflavin biosynthesis, the absence of pathogenic factors, the presence of CRISPR and cas genes, phage sequences, insertion elements and an aminoglycosides resistance gene, aadA, whose resistance could not be proved phenotypically in any strain. Genomic comparisons showed that strain CECT 8962 was significantly different in terms of genetic content and allowed the identification of carbohydrates metabolism and membrane transport related genes as the main components of the unique and accessory genome.

Shimia thalassica sp. nov., and reclassification of Pseudopelagicola gijangensis as Shimia gijangensis comb. nov., and Thalassobius activus as Cognatishimia activa comb. nov.

Strain CECT 7735T, a marine Gram-reaction negative, aerobic, non-motile bacterium, was isolated from coastal seawater in Valencia, Spain. Strain CECT 7735T is chemoorganotrophic, mesophilic, slightly halophilic, grows at 15-28 °C but not at 4 or 37 °C, requires seawater for growth and grows up to 6 % salinity. The major cellular fatty acid is summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The G+C content of the genome is 55.7 mol%. Comparative analysis of the 16S rRNA gene sequence shows the strain is affiliated to the family Rhodobacteraceae, in the class Alphaproteobacteria, with highest similarities to Phaeobacter species (97.0-97.5 %), Shimia species (96.5-97.3 %) and Pseudopelagicola gijangensis (96.5 %). Further phylogenomic analysis through the up-to-date-bacterial core gene (UBCG) set showed P. gijangensis to be its closest relative. Average nucleotide identity and in silico DNA-DNA hybridization values are lower than 85 and 21 %, respectively, with its phylogenetic relatives, suggesting that strain CECT 7735T represents a new species. The average amino acid identity value was over 70 % with the genome of the type strain of P. gijangensis and with all those of Shimia species. These values, together with UBCG set trees, suggest that the new species and P. gijangensisbelong to the same genus and that Pseudopelagicola should be reclassified as a Shimia species. We conclude that strain CECT 7735T represents a new species in the genus Shimia, for which we propose the name Shimiathalassica sp. nov. In addition, Pseudopelagicola gijangensis is reclassified as Shimiagijangensis comb. nov. From the same phylogenomic study, it can be concluded that Thalassobius activus should be reclassified in the genus Cognatishimia as Cognatishimiaactiva comb. nov.

Ruegeria denitrificans sp. nov., a marine bacterium in the family Rhodobacteraceae with the potential ability for cyanophycin synthesis.

Strain CECT 5091T, an aerobic, marine, Gram-reaction- and Gram-stain-negative, chemoheterotrophic bacterium was isolated from oysters harvested off the Spanish Mediterranean coast. Analysis of the 16S rRNA gene sequence placed the strain within the genus Ruegeria, in the family Rhodobacteraceae, with 16S rRNA gene similarities of 98.7, 98.7 and 98.4 % to Ruegeria conchae, Ruegeria atlanticaand Ruegeria arenilitoris, respectively. Average nucleotide identities (ANI) and in silico DNA-DNA hybridization (DDH) were determined, comparing the genome sequence of CECT 5091T with those of the type strains of 12 species of the genus Ruegeria: the values obtained were always below the thresholds (95-96 % ANI, 70 % in silico DDH) used to define genomic species, proving that CECT 5091T represents a novel species of the genus Ruegeria. The strain was slightly halophilic and mesophilic, with optimum growth at 26 °C, pH 7.0 and 3 % salinity, it required sodium and magnesium ions for growth and was able to reduce nitrate to dinitrogen. Carbon sources for growth include some carbohydrates (d-ribose, d-glucose, l-rhamnose, N-acetyl-d-glucosamine) and multiple organic acids and amino acids. The major cellular fatty acid was summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), representing 70 % of the total fatty acids. Carbon monoxide oxidation, cyanophycin synthetic ability and phosphatidylglycerol, diphosphatidylglycerol and phosphatidylcholine production are predicted from genome annotation, while bacteriochlorophyll a production was absent. The DNA G+C content of the genome was 56.7 mol%. We propose the name Ruegeriadenitrificans sp. nov. and strain CECT 5091T (=5OM10T=LMG 29896T) as the type strain for the novel species.

Vibrio palustris sp. nov. and Vibrio spartinae sp. nov., two novel members of the Gazogenes clade, isolated from salt-marsh plants (Arthrocnemum macrostachyum and Spartina maritima).

Two bacterial strains, EAod9T and SMJ21T, isolated from salt-marsh plants, were determined to be related to species of the genus Vibriofrom from 16S rRNA sequence comparisons. Their closest phylogenetic relatives are members of the Gazogenes clade, Vibrio mangrovi and Vibrio rhizosphaerae , which show the greatest similarity to the SMJ21TrRNA sequence (97.3 and 97.1 %, respectively), while EAod9T had less than 97.0 % similarity to any other species of the genus Vibrio. Both strains share the basic characteristics of the genus Vibrio, as they are Gram-stain negative, motile, slightly halophilic, facultatively anaerobic bacteria. In addition, they are oxidase-negative and unable to grow on TCBS Agar; they grow between 15 to 26 °C, pH 6 to 8 and in up to 10 % (w/v) total salinity. They produce indol, are positive in the Voges-Proskauer test and are negative for arginine dihydrolase, lysine and ornithine decarboxylases. Strain SMJ21T is aerogenic and red-pigmented, due to prodigiosin production, while strain EAod9T ferments glucose without gas and is not pigmented. The major cellular fatty acids of both novel strains were C16 : 1ω7c/C16 : 1ω6c and C16 : 0. WGSobtained for both strains, along with the other five members of the clade, allowed the determination of ANI indexes and in silico estimations of DDH values, which confirmed that the two strains represent two novel species of the genus Vibrio: Vibriopalustris sp. nov. (with EAod9T=CECT 9027T=LMG 29724T as the proposed type strain) and Vibrio spartinae sp. nov. (with SMJ21T=CECT 9026T=LMG 29723T as the proposed type strain).

Whole genome sequences reveal Vibrio hemicentroti Kim et al. 2013 as a later heterotypic synonym of Vibrio splendidus (Beijerinck 1900) Baumann et al. 1981.

The synonymy between Vibrio hemicentrotiKim et al. 2013 and Vibrio splendidus(Beijerinck 1900) Baumann et al. 1981 was suggested after a recent multilocus sequence analysis of the Splendidus clade, which included the type strains of both species. To clarify their status, we have determined genomic indexes from whole genome sequences of strains V. hemicentroti CECT 8714T and V. splendidus NCCB 53037T. Average Nucleotide Identities of 96.0-96.7 % and an in silico DNA-DNA hybridization value of 70.2 %, as well as similarity levels of selected housekeeping gene sequences support the consideration of V.hemicentroti as a later heterotypic synonym of V. splendidus.

Comparative Genomics of Thalassobius Including the Description of Thalassobius activus sp. nov., and Thalassobius autumnalis sp. nov.

A taxogenomic study was conducted to describe two new Thalassobius species and to analyze the internal consistency of the genus Thalassobius along with Shimia and Thalassococcus. Strains CECT 5113T, CECT 5114, CECT 5118T, and CECT 5120 were isolated from coastal Mediterranean seawater, Spain. Cells were Gram-negative, non- motile coccobacilli, aerobic chemoorganotrophs, with an optimum temperature of 26°C and salinity of 3.5-5%. Major cellular fatty acids of strains CECT 5113T and CECT 5114 were C18 : 1 ω7c/ω6c and C10 : 0 3OH, G+C content was 54.4-54.5 mol% and were able to utilize propionate, L-threonine, L- arginine, and L-aspartate as carbon sources. They exhibited 98.3% 16S rRNA gene sequence similarity, 75.0-75.1 ANIb and 19.5-20.9 digital DDH to type strain of their closest species, Thalassobius maritimus. Based on these data, strains CECT 5113T and CECT 5114 are recognized as a new species, for which the name Thalassobius activus is proposed, with strain CECT 5113T (=LMG 29900T) as type strain. Strains CECT 5118T and CECT 5120 were found to constitute another new species, with major cellular fatty acids C18 : 1 ω7c/ω6c and C18 : 1 ω7c 11-methyl and a G+C content of 59.8 mol%; they were not able to utilize propionate, L-threonine, L- arginine or L-aspartate. Their closest species was Thalassobius mediterraneus, with values of 99.6% 16S rRNA gene sequence similarity, 79.1% ANIb and 23.2% digital DDH compared to the type strain, CECT 5383T. The name Thalassobius autumnalis is proposed for this second new species, with strain CECT 5118T (=LMG 29904T) as type strain. To better determine the phylogenetic relationship of the two new species, we submitted 12 genomes representing species of Thalassobius, Shimia, and Thalassoccocus, to a phylogenomic analysis based on 54 single protein-encoding genes (BCG54). The resulting phylogenomic tree did not agree with the current genera classification, as Thalassobius was divided in three clades, Thalassobius sensu stricto (T. mediterraneus, T. autumnalis sp. nov., and T. gelatinovorus), Thalassobius aestuarii plus the three Shimia spp (S. marina, S. haliotis, and Shimia sp. SK013) and finally, Thalasobius maritimus plus T. activus sp. nov. Thalassococcus halodurans remained apart from the two genera. Phenotypic inferences from explored genomes are presented.

Grimontia celer sp. nov., from sea water.

Strain 96-237T, a Gram-reaction-negative, curved- to spiral-shaped motile bacterium, isolated from coastal marine water, was found to be related to species of the genus Grimontia by 16S rRNA gene sequence comparison, sharing 98.3 % similarity to Grimontia marina CECT 8713Tand 98.8 % to 'Grimontiaindica' AK16. Phenotypic analysis revealed that strain 96-237T is slightly halophilic, mesophilic and facultatively anaerobic, fermenting d-glucose, d-ribose, d-mannose, d-mannitol, maltose and sucrose. It was positive for oxidase and indole production and negative for arginine dihydrolase and lysine and ornithine decarboxylases. Its major fatty acids were C16 : 1ω7c/C16 : 1ω6c (SF3), C18 : 1ω7c and C16 : 0. Its DNA G+C content was 48.4 mol%. The strain was different at the species level from all other species of the genusGrimontia, with average nucleotide identity indices of 79.6 % to Grimontia. hollisae CECT 8713T, 87.8 % to G. marina CECT 5069T and 89.1 % to 'G. indica' AK16 genomes. Thus, the strain represents a novel species for which we propose the name Grimontia celer and 96-237T (=CECT 9029T =KCTC 42960T =LMG 29238T) as the type strain.

Draft genome of Leisingera aquaemixtae CECT 8399(T), a member of the Roseobacter clade isolated from a junction of fresh and ocean water in Jeju Island, South Korea.

We report the draft genome sequence and annotation of Leisingera aquaemixtae CECT 8399(T) (DDBJ/EMBL/GenBank accession number CYSR00000000) which comprises 4,614,060 bp, 4313 protein coding genes, 54 tRNA coding genes and 7 rRNA coding genes. General findings of the annotated genome, such as pigment indigoidine operon, phenylacetate oxidation genes or predictable number of replicons, are commented in comparison to other Leisingera species. Average Nucleotide Identity between available genomes of type strains of species of Leisingera and Phaeobacter genera has been calculated to evaluate its current classification.

Draft genome sequence of Thalassobius mediterraneus CECT 5383(T), a poly-beta-hydroxybutyrate producer.

Thalassobius mediterraneus is the type species of the genus Thalassobius and a member of the Roseobacter clade, an abundant representative of marine bacteria. T. mediterraneus XSM19(T) (= CECT 5383(T)) was isolated from the Western Mediterranean coast near Valencia (Spain) in 1989. We present here the draft genome sequence and annotation of this strain (ENA/DDBJ/NCBI accession number CYSF00000000), which is comprised of 3,431,658 bp distributed in 19 contigs and encodes 10 rRNA genes, 51 tRNA genes and 3276 protein coding genes. Relevant findings are commented, including the complete set of genes required for poly-beta-hydroxybutyrate (PHB) synthesis and genes related to degradation of aromatic compounds.

Draft genomic sequence of Nereida ignava CECT 5292(T), a marine bacterium of the family Rhodobacteraceae.

Nereida ignava strain 2SM4(T) (= CECT 5292(T) = DSM 16309(T) = CIP 108404(T) = CCUG 49433(T)) is a marine bacterium belonging to the Roseobacter group of the family Rhodobacteraceae within the class Alphaproteobacteria. The strain was isolated from sea water surrounding cultivated oysters 2-3 miles off the Mediterranean coast near Valencia (Spain) and was phylogenetically related to uncultured clones of gall symbiont bacteria of some species of Prionitis alga. Here we describe the genome sequence and annotation of this organism, the type strain of the single species of this genus. The genome comprised 2,888,349 bp, 2,872 protein-coding genes and 52 RNA genes. The annotation revealed the capacity to produce bacteriocins, vitamins and auxins. Besides, it contained sulfur cycling related genes.

Draft genome sequence of Shimia marina CECT 7688(T).

Shimia marina is a member of the family Rhodobacteraceae described in 2006. Strain CL-TA03(T) (=CECT 7688(T)) was isolated from a biofilm formed on an acrylic slide submerged in surface water in a coastal fish farm in Tongyeong, Korea. Here we report the draft genome sequence and annotation of S. marina CECT 7688(T) which is composed by 4,001,860bp arranged in 45 scaffolds with a G+C content of 57.4%, 3878 protein coding genes, 40 tRNA genes, 4 rRNA genes and 1 repeat region. An overview of annotated genes revealed diverse genes encoding for exopolysaccharide and capsular biosynthesis enzymes, secondary metabolite biosynthesis enzymes, multiple antibiotic and metal resistance and the ability for degrading aromatic compounds.

Draft genomes of Nautella italica strains CECT 7645(T) and CECT 7321: Two roseobacters with potential pathogenic and biotechnological traits.

Nautella italica is a member of the family Rhodobacteraceae described in 2009. Strain LMG 24365(T) (=CECT 7645(T), =DSM 26436(T), =CCUG 55857(T)) was isolated from a marine electroactive biofilm growing in a stainless steel cathode exposed to natural water in Genoa, Italy. Strain AD 41 (=CECT 7321) was isolated from water surrounding cultivated gilthead seabream larvae in Cádiz, Spain. The genomes of strains CECT 7645(T) and CECT 7321 were sequenced, assembled, annotated and compared. Here we describe the most relevant findings: biofilm formation, quorum sensing, resistance to multiple drugs, heavy metals and oxidative stress, cytotoxins, and poly-ß-hydroxybutyrate (PHB) production genes. These genomes were also compared to current available genomes in NCBI Genome Database from members of the genus Nautella, Nautella sp. R11 and Nautella sp. ECSMB14104. The comparison showed a higher similarity between strains CECT 7645(T) and R11 compared to strain CECT 7321 and strain ECSMB14104. The genome similarity indexes allowed confirming and assigning strains CECT 7321, R11 and ECSMB14104 to the species N. italica.

Draft Genome Sequences of Vibrio renopiscarius Strains CECT 8603T and CECT 8604, Two Marine Gammaproteobacteria Isolated from Cultured Gilthead Sea Bream (Sparus aurata).

Vibrio renopiscarius DCR 1-4-2(T) (CECT 8603(T)) and DCR 1-4-12 (CECT 8604) were isolated from healthy gilthead sea bream (Sparus aurata) from Mediterranean fish farms (Castellón, Spain). Their draft genome sequences (30 and 44 contigs, respectively) have 4.3 Mbp and a G+C content of 45.2 mol% and contain almost 3,700 protein-encoding genes.