Streptococcus sciuri sp. nov., Staphylococcus marylandisciuri sp. nov. and Staphylococcus americanisciuri sp. nov., isolated from faeces of eastern grey squirrel (Sciurus carolinensis).

One novel Streptococcus strain (SQ9-PEAT) and two novel Staphylococcus strains (SQ8-PEAT and GRT3T) were isolated from faeces of a wild eastern grey squirrel. The strains were non-spore-forming, non-motile Gram-positive cocci, facultative anaerobes. The genomes for these strains were sequenced. The 16S rRNA gene and core-genome-based phylogenetic analyses showed that strain SQ9-PEAT was closely related to Streptococcus hyointestinalis, strain SQ8-PEAT to Staphylococcus pettenkoferi and Staphylococcus argensis, and strain GRT3T to Staphylococcus rostri, Staphylococcus muscae and Staphylococcus microti. Average nucleotide identity and pairwise digital DNA-DNA hybridization values calculated for these novel strains compared to type strain genomes of phylogenetically related species within the genera Streptococcus and Staphylococcus clearly revealed that strain SQ9-PEAT represents a novel species of the genus Streptococcus and strains SQ8-PEAT and GRT3T represent two novel species of the genus Staphylococcus. Phenotypical features of these novel type strains differed from the features of the type strains of other phylogenetically related species. MALDI-TOF mass spectrometry supported identification of these novel species. Based on these data, we propose one novel species of the genus Streptococcus, for which the name Streptococcus sciuri sp. nov. with the type strain SQ9-PEAT (=DSM 114656T=CCUG 76426T=NCTC 14727T) is proposed, and two novel species of the genus Staphylococcus, for which the names Staphylococcus marylandisciuri sp. nov. with the type strain SQ8-PEAT (=DSM 114685T=CCUG 76423T=NCTC 14723T) and Staphylococcus americanisciuri sp. nov. with the type strain GRT3T (=DSM 114696T=CCUG 76427T=NCTC 14722T) are proposed. The genome G+C contents are 38.29, 36.49 and 37.26 mol% and complete draft genome sizes are 1 692 266, 2 371 088 and 2 237 001 bp for strains SQ9-PEAT, SQ8-PEAT and GRT3T, respectively.

Paracholeplasma manati sp. nov. isolated from Florida manatees (Trichechus manatus latirostris).

Four novel strains of a member of the genus Paracholeplasma (OakleyT, Holly, Lorelei and Ariel) were isolated from skin of Florida manatees (Trichechus manatus latirostris). These strains were phenotypically and genetically characterized and compared with the known species of the genera Acholeplasma (A.), Alteracholeplasma (Al.), Haploplasma (H.), Paracholeplasma (P.) and Mariniplasma (M.). All the strains produced acid from glucose but did not hydrolyze arginine or urea. All were propagated in ambient air supplemented with 5±1 % CO2 at 35-37 °C using SP4-Z, Columbia and brain-heart infusion medium. Colonies on solid medium showed a typical fried-egg appearance and transmission electron microscopy revealed a typical mycoplasma-like cellular morphology. The results of phylogenetic analyses based on partial 16S rRNA, rpoB, gyrB and parE gene sequences and the whole proteome data indicated that the novel species is a unique species but phylogenetically closely related to Paracholeplasma vituli, Paracholeplasma morum and 'Paracholeplasma brassicae'. The average nucleotide identity and digital DNA-DNA hybridization values between strain OakleyT and the closely related species were significantly lower than the accepted thresholds for describing novel prokaryotic species at the genomic level. On the basis of the genomic, phenotypic and phylogenetic properties, the novel strains represent a novel species of the genus Paracholeplasma, for which the name Paracholeplasma manati sp. nov. with the type strain OakleyT (=NCTC 14352T =DSM 110686T) is proposed. The genomic DNA G+C content and complete draft genome size for the type strain are 38.35 % and 1 873 856 bp, respectively.

Neisseria montereyensis sp. nov., Isolated from Oropharynx of California Sea Lion (Zalophus californianus): Genomic, Phylogenetic, and Phenotypic Study.

A novel Neisseria strain, designated CSL10203-ORH2T, was isolated from the oropharynx of a wild California sea lion (Zalophus californianus) that was admitted to The Marine Mammal Center in California, USA. The strain was originally cultured from an oropharyngeal swab on BD Phenylethyl Alcohol (PEA) agar with 5% sheep blood under aerobic conditions. Phylogenetic analyses based on 16S rRNA, rplF, and rpoB gene sequences and the core genome sequences indicated that the strain was most closely related to only N. zalophi CSL 7565T. The average nucleotide identity and digital DNA-DNA hybridization values between strain CSL10203-ORH2T and the closely related species N. zalophi CSL 7565T were 89.84 and 39.70%, respectively, which were significantly lower than the accepted species-defined thresholds for describing novel prokaryotic species at the genomic level. Both type strains were phenotypically similar but can be easily and unambiguously distinguished between each other by the analysis of their housekeeping genes, e.g., rpoB, gyrB, or argF. The major fatty acids in both type strains were C12:0, C16:0, C16:1-c9, and C18:1-c11. Based on the genomic, phenotypic, and phylogenetic properties, the novel strain represents a novel species of the genus Neisseria, for which the name Neisseria montereyensis sp. nov. with the type strain CSL10203-ORH2T (= DSM 114706T = CCUG 76428T = NCTC 14721T) is proposed. The genome G + C content is 45.84% and the complete draft genome size is 2,310,535 bp.

Mycoplasma miroungirhinis sp. nov. and Mycoplasma miroungigenitalium sp. nov., isolated from northern elephant seals (Mirounga angustirostris), Mycoplasma phocoenae sp. nov., isolated from harbour porpoise (Phocoena phocoena), and Mycoplasma phocoeninasale sp. nov., isolated from harbour porpoise and California sea lions (Zalophus californianus).

Seven novel independent strains of Mycoplasma species were isolated from northern elephant seals (ES2806-NAST, ES2806-GENT, ES3157-GEN-MYC and ES3225-GEN-MYC), a harbour porpoise (C264-GENT and C264-NAST), and a California sea lion (CSL7498). These strains were phenotypically and genetically characterized and compared to the known Mycoplasma species. Four strains (C264-GENT, C264-NAST, CSL7498 and ES2806-NAST) hydrolysed arginine but not urea and did not produce acid from carbohydrates. Strains ES2806-GENT, ES3157-GEN-MYC and ES3225-GEN-MYC did not produced acid from carbohydrates and did not hydrolyse arginine or urea; hence, it is assumed that organic acids are used as the energy source for them. All were isolated and propagated in ambient air supplemented with 5±1 % CO2 at +35-37 °C using either SP4 or PPLO medium. Colonies on solid medium showed a typical fried-egg appearance and transmission electron microscopy revealed a typical mycoplasma cellular morphology. The complete genomes were sequenced for all type strains. Average nucleotide and amino acid identity analyses showed that these novel strains were distant from the phylogenetically closely related Mycoplasma species. Based on these data, we propose four novel species of the genus Mycoplasma, for which the name Mycoplasma miroungirhinis sp. nov. is proposed with the type strain ES2806-NAST (=NCTC 14430T=DSM 110945T), Mycoplasma miroungigenitalium sp. nov. is proposed with the type strain ES2806-GENT (=NCTC 14429T=DSM 110944T) and representative strains ES3157-GEN-MYC and ES3225-GEN-MYC, Mycoplasma phocoenae sp. nov. is proposed with the type strain C264-GENT (=NCTC 14344T=DSM 110687T) and Mycoplasma phocoeninasale sp. nov. is proposed with the type strain C264-NAST (=NCTC 14343T=DSM 110688T) and representative strain CSL7498. The genome G+C contents are 24.06, 30.09, 28.49 and 29.05% and the complete genome sizes are 779 550, 815 486, 693 115, and 776 009 bp for strains ES2806-NAST, ES2806-GENT, C264-GENT and C264-NAST, respectively.

Streptococcus vicugnae sp. nov., isolated from faeces of alpacas (Vicugna pacos) and cattle (Bos taurus), Streptococcus zalophi sp. nov., and Streptococcus pacificus sp. nov., isolated from respiratory tract of California sea lions (Zalophus californianus).

Four novel independent strains of Streptococcus spp. were isolated from faeces of alpaca (SL1232T), cattle (KCJ4950), and from respiratory tract of wild California sea lions (CSL7508T, CSL7591T). The strains were indole-, oxidase- and catalase-negative, non-spore-forming, non-motile Gram-positive cocci in short and long chains, facultative anaerobes. The 16S rRNA gene of SL1232T and KCJ4950 shared 99.40-99.60% nucleotide similarity to strains of S. equinus, S. lutetiensis, S. infantarius, and the 16S rRNA gene of CSL7508T and CSL7591T demonstrated 98.72 and 98.92% similarity, respectively, to S. marimammalium. All other known Streptococcus species had the 16S rRNA gene sequence similarities of ≤95%. The genomes were sequenced for the novel strains. Average nucleotide identity (ANI) analysis for strains SL1232T and KCJ4950, showed the highest similarity to S. equinus, S. lutetiensis, and S. infantarius with 85.21, 87.17, 88.47, 85.54, 87.47 and 88.89%, respectively, and strains CSL7508T and CSL7591T to S. marimammalium with 87.16 and 83.97%, respectively. Results of ANI were confirmed by pairwise digital DNA-DNA hybridization and phylogeny, which also revealed that the strains belong to three novel species of the genus Streptococcus. Phenotypical features of the novel species were in congruence with closely related members of the genus Streptococcus and gave negative reactions with the tested Lancefield serological groups (A-D, F and G). MALDI-TOF mass spectrometry supported identification of the species. Based on these data, we propose three novel species of the genus Streptococcus, for which the name Streptococcus vicugnae sp. nov. is proposed with the type strain SL1232T (=NCTC 14341T=DSM 110741T=CCUG 74371T), Streptococcus zalophi sp. nov. is proposed with the type strain CSL7508T (=NCTC 14410T=DSM 110742T=CCUG 74374T) and Streptococcus pacificus sp. nov. is proposed with the type strain CSL7591T (=NCTC 14455T=DSM 111148T=CCUG 74655T). The genome G+C content is 36.89, 34.85, and 35.34 % and draft genome sizes are 1906993, 1581094 and 1656080 bp for strains SL1232T, CSL7508T, and CSL7591T, respectively.

Use of tangential flow filtration for improving detection of viral adventitious agents in cell substrates.

In this study, we assessed the feasibility of tangential flow filtration (TFF) for primary concentration of viral adventitious agents (AAs) from large volumes of cell substrate-derived samples, such as cell-free Chinese hamster ovary (CHO) culture supernatants (500 mL) and CHO cell lysates (50 mL), prior to virus detection in them by nucleic acid-based methods (i.e., qPCR and massively parallel sequencing (MPS). The study was conducted using the samples spiked with four model DNA viruses (bovine herpesvirus type 4, human adenovirus type 5, simian polyomavirus SV-40, and bovine parvovirus). The results showed that the combined TFF/MPS approach enables reliable detection of as low as 1000 genome equivalents (GE) of each of the four viruses spiked into the cell substrate samples. The final achieved sensitivities of 2 GE/mL for cell culture supernatant and 20 GE/mL for cell lysate make this approach more sensitive than virus-specific PCR and qPCR assays. The study results allowed us to propose that TFF might be useful and valuable method for simple and rapid concentration of potential AAs in cell substrate samples prior to AAs detection by conventional in vivo, in vitro, or molecular methods.

Gene network analysis during early infection of human coronary artery smooth muscle cells by Trypanosoma cruzi and Its gp83 ligand.

Trypanosoma cruzi, the causative agent of Chagas' disease, infects heart and muscle cells leading to cardiac arrest, followed by death. The genetic architectures in the early T. cruzi infection process of human cells are unknown. To understand the genetic architectures of the early invasion process of T. cruzi, we conducted gene transcription microarray analysis, followed by gene network construction of the host cell response in primary human coronary artery smooth muscle (HCASM) cells infected with T. cruzi or exposed to T. cruzi gp83, a ligand used by the trypanosome to bind host cells. Using seven RT-PCR verified up-regulated genes (FOSB, ATF5, INPP1, CCND2, THBS1, LAMC1, and APLP2) as the seed for network construction, we built an interaction network of the early T. cruzi infection process containing 165 genes, connected by 598 biological interactions. This interactome network is centered on the BCL6 gene as a hub. Silencing the expression of two seed genes (THBS1 and LAMC1) by RNAi reduced T. cruzi infection. Overall, our results elucidate the significant and complex process involved in T. cruzi infection of HCASM cells at the transcriptome level. This is the first elucidation into the interactome network in human cells caused by T. cruzi and its gp83 ligand.

Human galectin-3 promotes Trypanosoma cruzi adhesion to human coronary artery smooth muscle cells.

Human galectin-3 binds to the surface of Trypanosoma cruzi trypomastigotes and human coronary artery smooth muscle (CASM) cells. CASM cells express galectin-3 on their surface and secrete it. Exogenous galectin-3 increased the binding of T. cruzi to CASM cells. Trypanosome binding to CASM cells was enhanced when either T. cruzi or CASM cells were preincubated with galectin-3. Cells stably transfected with galectin-3 antisense show a dramatic decrease in galectin-3 expression and very little T. cruzi adhesion to cells. The addition of galectin-3 to these cells restores their initial capacity to bind to trypanosomes. Thus, host galectin-3 expression is required for T. cruzi adhesion to human cells and exogenous galectin-3 enhances this process, leading to parasite entry.