Species-level Microbiota of Biting Midges and Ticks from Poyang Lake.

Objective: The purpose of this study was to investigate the bacterial communities of biting midges and ticks collected from three sites in the Poyang Lake area, namely, Qunlu Practice Base, Peach Blossom Garden, and Huangtong Animal Husbandry, and whether vectors carry any bacterial pathogens that may cause diseases to humans, to provide scientific basis for prospective pathogen discovery and disease prevention and control. Methods: Using a metataxonomics approach in concert with full-length 16S rRNA gene sequencing and operational phylogenetic unit (OPU) analysis, we characterized the species-level microbial community structure of two important vector species, biting midges and ticks, including 33 arthropod samples comprising 3,885 individuals, collected around Poyang Lake. Results: A total of 662 OPUs were classified in biting midges, including 195 known species and 373 potentially new species, and 618 OPUs were classified in ticks, including 217 known species and 326 potentially new species. Surprisingly, OPUs with potentially pathogenicity were detected in both arthropod vectors, with 66 known species of biting midges reported to carry potential pathogens, including Asaia lannensis and Rickettsia bellii, compared to 50 in ticks, such as Acinetobacter lwoffii and Staphylococcus sciuri. We found that Proteobacteria was the most dominant group in both midges and ticks. Furthermore, the outcomes demonstrated that the microbiota of midges and ticks tend to be governed by a few highly abundant bacteria. Pantoea sp7 was predominant in biting midges, while Coxiella sp1 was enriched in ticks. Meanwhile, Coxiella spp., which may be essential for the survival of Haemaphysalis longicornis Neumann, were detected in all tick samples. The identification of dominant species and pathogens of biting midges and ticks in this study serves to broaden our knowledge associated to microbes of arthropod vectors. Conclusion: Biting midges and ticks carry large numbers of known and potentially novel bacteria, and carry a wide range of potentially pathogenic bacteria, which may pose a risk of infection to humans and animals. The microbial communities of midges and ticks tend to be dominated by a few highly abundant bacteria.

Unveiling the Mutations and Conservation of InlA in Listeria monocytogenes.

Listeria monocytogenes (L. monocytogenes) is a pathogen that is transmitted through contaminated food and causes the illness known as listeriosis. The virulence factor InlA plays a crucial role in the invasion of L. monocytogenes into the human intestinal epithelium. In addition, InlA enhances the pathogenicity of host strains, and different strains of L. monocytogenes contain varying variations of InlA. Our study analyzed a total of 4393 published L. monocytogenes genomes from 511 sequence types (STs) of diverse origins. We identified 300 unique InlA protein sequence types (PSTs) and revealed 45 highly mutated amino acid sites. The leucine-rich repeat (LRR) region was found to be the most conserved among the InlA, while the protein A (PA) region experienced the highest mutation rate. Two new types of mutations were identified in the B-repeat region of InlA. Correspondence analysis (CA) was used to analyze correlations between the lineages or 10 most common sequence types (STs) and amino acid (aa) sites. ST8 was strongly correlated with site 192_F, 454_T. ST7 exhibited a strong correlation with site 51_A, 573_E, 648_S, and 664_A, and it was also associated with ST6 and site 544_N, 671_A, 738_B, 739_B, 740_B, and 774_Y. Additionally, a strong correlation between ST1 and site 142_S, 738_N, ST2 and site 2_K, 142_S, 738_N, as well as ST87 and site2_K, 738_N was demonstrated. Our findings contribute significantly to the understanding of the distribution, composition, and conservation of InlA in L. monocytogenes. These findings also suggest a potential role of InlA in supporting molecular epidemiological tracing efforts.

Pararhizobium qamdonense sp. nov., Isolated from an Alpine Soil in Tibet and the Reclassification of Rhizobium gei Shi et al. 2016 as Pararhizobium gei comb. nov.

A novel Gram-stain-negative, aerobic, rod-shaped bacterium named T808T was isolated from an alpine soil in Qamdo, Tibet, PR China. Strain T808T grew at 5-30℃, pH 5.0-9.0 (optimum, 25℃ and pH 7.0-8.0) with 0-2% (w/v) NaCl (optimum, 0%). The 16S rRNA gene sequences of strain T808T showed the highest similarity with Pararhizobium herbae CCBAU83011T (98.8%), followed by Pararhizobium polonicum F5.1T (98.7%), Pararhizobium giardinii H152T (98.5%), Rhizobium gei ZFJT-2 T (98.4%), and Pararhizobium antarcticum NAQVI59T (97.5%). The highest digital DNA-DNA hybridization (dDDH), core-proteome average amino acid identity (cpAAI) and average nucleotide identity (ANI) values between strain T808T and related strains were estimated as 28.0%, 92.1% and 84.4%, respectively. Phylogenetic analysis based on 16S rRNA, core-proteome and whole-genome indicated that strain T808T belonged to the genus Pararhizobium. The genome size was 6.24 Mbp with genomic DNA G + C content of 60.1%. The major cellular fatty acids were Summed feature 8 (C18:1 ω7c or C18:1 ω6c), C16:0 and C19:0 cyclo ω8c. The polar lipids were diphosphatidyl glycerol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidyl choline and unidentified aminophospholipid. The isoprenoid quinone were ubiquinone-10 and ubiquinone-9. Based on phenotypic, phylogenetic, and genotypic data, strain T808T is considered to represent a novel species of the genus Pararhizobium, for which the name Pararhizobium qamdonense sp. nov. is proposed. The type strain is T808T (= JCM 36247 T = CICC 25216 T). According to phylogenetic coherence based on 16S rRNA, core-proteome and whole-genome, it is also proposed that the type strain Rhizobium gei Shi et al. 2016 should be reclassified as Pararhizobium gei comb. nov., the type strain is ZFJT-2 T (= CCTCC AB 2013015 T = KCTC 32301 T = LMG 27603 T).

Taxonomic descriptions of Aeromicrobium duanguangcaii sp. nov., Aeromicrobium wangtongii sp. nov. and Aeromicrobium senzhongii sp. nov.

Six Gram-stain-positive, facultative anaerobic, nonmotile and rod-shaped strains, designated zg-Y50T, zg-Y1362, zg-Y1379T, zg-Y869, zg-629T and zg-Y636, were isolated from the intestinal contents of Marmota himalayana in Qinghai Province, PR China. Strains zg-Y50T, zg-Y1379T and zg-629T exhibited the highest 16S rRNA gene sequence similarities of 99.2, 98.9 and 98.8 % to Aeromicrobium choanae 9 H-4T, Aeromicrobium ginsengisoli JCM 14732T and Aeromicrobium flavum TYLN1T, respectively. Phylogenetic and phylogenomic analyses based on the 16S rRNA gene and genomic sequences, respectively, revealed that the six strains formed three distinct clades within the genus Aeromicrobium. The genome sizes of strains zg-Y50T, zg-Y1379T and zg-629T were 3.1-3.7 Mb, with DNA G+C contents of 69.6-70.4 mol%. Average nucleotide identity and digital DNA-DNA hybridization values between each novel strain and available members of the genus Aeromicrobium were all below species thresholds. All novel strains contained MK-9 (H4) as the major menaquinone and diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol as the polar lipids. The predominant fatty acid of the six isolates was C18 : 1 ω9c. The cell-wall peptidoglycan contained ʟʟ-diaminopimelic acid as the diagnostic diamino acid. Based on the results from this polyphasic taxonomic study, three novel species in the genus Aeromicrobium are proposed, namely, Aeromicrobium duanguangcaii sp. nov. (zg-Y50T=GDMCC 1.2981T=KCTC 49764T), Aeromicrobium wangtongii sp. nov. (zg-Y1379T=GDMCC 1.2982T=KCTC 49765T) and Aeromicrobium senzhongii sp. nov. (zg-629T=CGMCC 1.17414T=JCM 33888T).

Marnyiella aurantia, gen. nov., sp. nov., a novel bacterial species of the family Weeksellaceae that could produce flexirubin type pigments.

Two Gram-stain-negative, non-spore-forming, rod-shaped, and obligately aerobic bacteria, designated strains CX-624T and cx-311, were isolated from soil samples in Qinghai Province, China. The two strains grew best at 28 °C on the plate with Tryptone soya agar (TSA). Cells formed circular, convex, translucent, smooth, and orange colonies with approximately 1.0 mm diameter after 2 days of incubation on TSA at 28 °C. The strains were oxidase-negative and catalase-positive. The predominant cellular fatty acids were iso-C15 : 0 and anteiso-C15 : 0, and major polar lipids included phosphatidylethanolamine, an unidentified aminophospholipid, four unidentified lipids and an aminolipid. MK-6 was the sole menaquinone in strain CX-624T. Comparative analysis of the nearly full-length 16S rRNA gene sequences showed strains CX-624T and cx-311 were member of the family Weeksellaceae, with the highest similarity to Kaistella haifensis H38T (96.66 %), Epilithonimonas pallida DSM 18015T (96.59 %), and Chryseobacterium gambrini DSM 18014T (96.53 %). Both phylogenetic analysis of the 16S rRNA gene and 177 core genes revealed that strains CX-624T and cx-311 formed an independent clade. Average nucleotide identity values (< 72.64 %), average amino-acid identity values (<72.61 %) and digital DNA-DNA hybridization (< 21.10 %) indicated that the strains CX-624T and cx-311 should constitute a novel genus. The DNA G+C contents of strains CX-624T and cx-311 were 43.0 mol% and 42.7 mol%. According to the data obtained in this study, strain CX-624T represents a novel species belonging to a novel genus of the Weeksellaceae, for which the name Marnyiella aurantia gen. nov., sp. nov. is proposed. The type strain is CX-624T (=GDMCC 1.1714T = JCM 33925T).

Neisseria lisongii sp. nov. and Neisseria yangbaofengii sp. nov., isolated from the respiratory tracts of marmots.

Four Gram-stain-negative, oxidase-positive, non-motile, cocci-shaped bacteria strains (ZJ106T, ZJ104, ZJ785T and ZJ930) were isolated from marmot respiratory tracts. Phylogenetic analyses based on 16S rRNA genes, 53 ribosomal protein sequences and 441 core genes supported that all four strains belonged to the genus Neisseria with close relatives Neisseria weixii 10022T and Neisseria iguanae ATCC 51483T. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values were below the species-level thresholds (95-96 % for ANI, and 70 % for dDDH). The major fatty acids of all four strains were C16 : 1 ω7c /C16 : 1 ω6c, C16 : 0 and C18 : 1 ω9c. Major polar lipids were composed of diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. MK-8 was the major menaquinone. Based on Virulence Factor Database analysis, the four strains were found to contain NspA and PorB H-factor binding proteins that promote evasion of host immunity. Strains ZJ106T and ZJ104 contained structures similar to the capsule synthesis manipulator of Neisseria meningitidis. Based on phenotypic and phylogenetic evidence, we propose that strains ZJ106T and ZJ785T represent two novel species of the genus Neisseria, respectively, with the names Neisseria lisongii sp. nov. and Neisseria yangbaofengii sp. nov. The type strains are ZJ106T (=GDMCC 1.3111T=JCM 35323T) and ZJ785T (=GDMCC 1.1998T=KCTC 82336T).

Species-level microbiota of ticks and fleas from Marmota himalayana in the Qinghai-Tibet Plateau.

Introduction: Ticks and fleas, as blood-sucking arthropods, carry and transmit various zoonotic diseases. In the natural plague foci of China, monitoring of Yersinia pestis has been continuously conducted in Marmota himalayana and other host animals, whereas other pathogens carried by vectors are rarely concerned in the Qinghai-Tibet Plateau. Methods: In this study, we investigated the microbiota of ticks and fleas sampling from M. himalayana in the Qinghai-Tibet Plateau, China by metataxonomics combined with metagenomic methods. Results: By metataxonomic approach based on full-length 16S rDNA amplicon sequencing and operational phylogenetic unit (OPU) analyses, we described the microbiota community of ticks and fleas at the species level, annotated 1,250 OPUs in ticks, including 556 known species and 492 potentially new species, accounting for 48.50% and 41.71% of the total reads in ticks, respectively. A total of 689 OPUs were detected in fleas, consisting of 277 known species (40.62% of the total reads in fleas) and 294 potentially new species (56.88%). At the dominant species categories, we detected the Anaplasma phagocytophilum (OPU 421) and potentially pathogenic new species of Wolbachia, Ehrlichia, Rickettsia, and Bartonella. Using shotgun sequencing, we obtained 10 metagenomic assembled genomes (MAGs) from vector samples, including a known species (Providencia heimbachae DFT2), and six new species affliated to four known genera, i.e., Wolbachia, Mumia, Bartonella, and Anaplasma. By the phylogenetic analyses based on full-length 16S rRNA genes and core genes, we identified that ticks harbored pathogenic A. phagocytophilum. Moreover, these potentially pathogenic novel species were more closely related to Ehrlichia muris, Ehrlichia muris subsp. eauclairensis, Bartonella rochalimae, and Rickettsia limoniae, respectively. The OPU 422 Ehrlichia sp1 was most related to Ehrlichia muris and Ehrlichia muris subsp. eauclairensis. The OPU 230 Bartonella sp1 and Bartonella spp. (DTF8 and DTF9) was clustered with Bartonella rochalimae. The OPU 427 Rickettsia sp1 was clustered with Rickettsia limoniae. Discussion: The findings of the study have advanced our understanding of the potential pathogen groups of vectors in marmot (Marmota himalayana) in the Qinghai-Tibet Plateau.

Identification of Nocardioides marmotae sp. nov. and Nocardioides faecalis sp. nov., two new members of the genus Nocardioides.

A polyphasic taxonomic characterization of two novel strain pairs (designated zg-579T/zg-578 and zg-536T/zg-ZUI104) isolated from the faeces of Marmota himalayana was conducted based on phylogenetic analysis of the nearly full-length 16S rRNA gene and genome, digital DNA-DNA hybridization, ortho-average nucleotide identity (Ortho-ANI), and phenotypic and chemotaxonomic traits. Comparative analysis of the nearly full-length 16S rRNA gene sequences showed that strain zg-579T was most closely related to Nocardioides dokdonensis FR1436T (97.57 %) and Nocardioides deserti SC8A-24T (97.36 %), whereas strain zg-536T had the highest similarity to Nocardioides caeni MN8T (98.33 %), Nocardioides convexus W2-2-3T (98.26 %) and Nocardioides daeguensis 2C1-5T (98.19 %). Low levels of DNA-DNA relatedness and Ortho-ANI values (19.8-31.0 %/78.6-88.2 %, zg-579T; 19.9-31.3 %/78.8-86.2 %, zg-536T) between the two new type strains and previously known species within the genus Nocardioides support the hypothesis that the four newly characterized strains could be considered to represent two novel species within this genus. The dominant cellular fatty acids found in strain pair zg-536T/zg-ZUI104 were iso-C16 : 0 and C18 : 1 ω9c, whereas C17 : 1 ω8c was major component in zg-579T/zg-578. Galactose and ribose were the main cell-wall sugars in these two new strain pairs. Diphosphatidylglycerol (DPG), phosphatidylcholine, phosphatidylglycerol (PG) and phosphatidylinositol (PI) were the major polar lipids in zg-579T, whereas DPG, PG and PI predominated in zg-536T. Both strain pairs had MK8(H4) as the major respiratory quinone and ll-diaminopimelic acid as the major cell-wall peptidoglycan. The optimal growth conditions for the two novel strain pairs were 30 °C, pH 7.0 and 0.5 % NaCl (w/v). Based on these polyphasic characterizations, two novel species within the genus Nocardioides are proposed, i.e. Nocardioides marmotae sp. nov. and Nocardioides faecalis sp. nov., with zg-579T (=CGMCC 4.7663T=JCM 33892T) and zg-536T (=CGMCC 4.7662T=JCM 33891T) as the type strains.

Cellulomonas xiejunii sp. nov., Cellulomonas chengniuliangii sp. nov. and Cellulomonas wangsupingiae sp. nov., three cellulolytic bacteria isolated from intestinal contents of Marmota himalayana.

Six facultative anaerobic, Gram-stain-positive, oxidase-negative, rod-shaped bacteria (strains zg-B89T, zg-B12, zg-Y338T, zg-Y138, zg-Y908T and zg-Y766), were isolated from the intestinal contents of Marmota himalayana in Qinghai Province, PR China. The 16S rRNA gene sequence analysis showed that zg-B89T showed highest similarity to Cellulomonas iranensis NBRC 101100T (99.5 %), zg-Y338T to Cellulomonas cellasea DSM 20118T (98.7 %), and zg-Y908T to Cellulomonas flavigena DSM 20109T (99.0 %). Phylogenetic and phylogenomic analysis based on 16S rRNA gene and 881 core genes revealed that these six strains formed three separate clades in the genus Cellulomonas. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between these three novel species and all members of the genus Cellulomonas were below species thresholds (95-96 % for ANI and 70 % for dDDH). The DNA G+C contents of zg-B89T, zg-Y338T and zg-Y908T were 73.6, 72.9 and 74.5 %, respectively. Strains zg-B89T and zg-Y908T had anteiso-C15 : 0, C16 : 0 and anteiso-C15 : 1 A, and zg-Y338T had anteiso-C15 : 0, C16 : 0 and iso-C16 : 0 as the main fatty acids. All novel type strains had MK-9 (H4) as the predominant respiratory quinone, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannoside as the major polar lipids, and rhamnose, ribose and glucose as the cell-wall sugars. The peptidoglycan amino acids of zg-B89T, zg-Y338T and zg-Y908T contained ornithine, alanine, glutamic acid and aspartic acid (except for zg-Y338T). Based on genotypic, phenotypic, phylogenetic and biochemical properties, the six uncharacterized strains represent three novel species in the genus Cellulomonas, for which the names Cellulomonas xiejunii sp. nov. (type strain zg-B89T=GDMCC 1.2821T=KCTC 49756T), Cellulomonas chengniuliangii sp. nov. (type strain zg-Y338T=GDMCC 1.2829T=KCTC 49754T) and Cellulomonas wangsupingiae sp. nov. (type strain zg-Y908T=GDMCC 1.2820T=KCTC 49755T) are proposed, respectively.

Species-Level Taxonomic Characterization of Uncultured Core Gut Microbiota of Plateau Pika.

Rarely has the vast diversity of bacteria on Earth been profiled, particularly on inaccessible plateaus. These uncultured microbes, which are also known as "microbial dark matter," may play crucial roles in maintaining the ecosystem and are linked to human health, regarding pathogenicity and prebioticity. The plateau pika (Ochotona curzoniae) is a small burrowing steppe lagomorph that is endemic to the Qinghai-Tibetan Plateau and is a keystone species in the maintenance of ecological balance. We used a combination of full-length 16S rRNA amplicon sequencing, shotgun metagenomics, and metabolomics to elucidate the species-level community structure and the metabolic potential of the gut microbiota of the plateau pika. Using a full-length 16S rRNA metataxonomic approach, we clustered 618 (166 ± 35 per sample) operational phylogenetic units (OPUs) from 105 plateau pika samples and assigned them to 215 known species, 226 potentially new species, and 177 higher hierarchical taxa. Notably, 39 abundant OPUs (over 60% total relative abundance) are found in over 90% of the samples, thereby representing a "core microbiota." They are all classified as novel microbial lineages, from the class to the species level. Using metagenomic reads, we independently assembled and binned 109 high-quality, species-level genome bins (SGBs). Then, a precise taxonomic assignment was performed to clarify the phylogenetic consistency of the SGBs and the 16S rRNA amplicons. Thus, the majority of the core microbes possess their genomes. SGBs belonging to the genus Treponema, the families Muribaculaceae, Lachnospiraceae, and Oscillospiraceae, and the order Eubacteriales are abundant in the metagenomic samples. In addition, multiple CAZymes are detected in these SGBs, indicating their efficient utilization of plant biomass. As the most widely connected metabolite with the core microbiota, tryptophan may relate to host environmental adaptation. Our investigation allows for a greater comprehension of the composition and functional capacity of the gut microbiota of the plateau pika. IMPORTANCE The great majority of microbial species remain uncultured, severely limiting their taxonomic characterization and biological understanding. The plateau pika (Ochotona curzoniae) is a small burrowing steppe lagomorph that is endemic to the Qinghai-Tibetan Plateau and is considered to be the keystone species in the maintenance of ecological stability. We comprehensively investigated the gut microbiota of the plateau pika via a multiomics endeavor. Combining full-length 16S rRNA metataxonomics, shotgun metagenomics, and metabolomics, we elucidated the species-level taxonomic assignment of the core uncultured intestinal microbiota of the plateau pika and revealed their correlation to host nutritional metabolism and adaptation. Our findings provide insights into the microbial diversity and biological significance of alpine animals.

Whole-genome sequencing of Paracoccus species isolated from the healthy human eye and description of Paracoccus shanxieyensis sp. nov.

Currently, the genus Paracoccus comprises 76 recognized species. Members of Paracoccus are mostly isolated from environmental, animal, and plant sources. This report describes and proposes a novel species of Paracoccus isolated from clinical specimens of the human ocular surface. We isolated two aerobic, Gram-stain-negative, non-spore-forming, coccoid or short rod-shaped, and non-motile strains (designated DK398T and DK608) from conjunctival sac swabs of two healthy volunteers. The results showed that the strains grew best under the conditions of 28°C, pH 7.0, and 1.0 % (w/v) NaCl. Sequence analysis based on the 16S rRNA gene showed that strains DK398T and DK608 were members of Paracoccus, most similar to Paracoccus laeviglucosivorans 43PT (98.54 and 98.62 %), Paracoccus litorisediminis GHD-05T (98.34 and 98.41 %), and Paracoccus limmosus NB88T (98.21 and 98.29 %). Phenotypic analysis showed that DK398T and DK608 were positive for catalase and oxidase, negative for producing N-acetyl-ß-glucosaminic acid, arginine dihydrolase, and ß-glucuronidase but positive for leucine arylamidase. The predominant isoprenoid quinone was Q-10, and the major polar lipids included phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, and an unidentified glycolipid. The major fatty acids (>10%) were summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c) and C16 : 0. The meso-diaminopimelic acid was found in the cell wall peptidoglycan of DK398T. The major cell wall sugars were ribose and galactose. Based on the results of phylogenetic analyses, low (<83.22 %) average nucleotide identity, digital DNA-DNA hybridization (<26.0%), chemotaxonomic analysis, and physiological properties, strain DK398T represents a novel species of the genus Paracoccus, for which the name Paracoccus shanxieyensis sp. nov. is proposed. The type strain is DK398T (=CGMCC 1.17227T=JCM 33719T).

Morphological and genomic characteristics of two novel actinomycetes, Ornithinimicrobium sufpigmenti sp. nov. and Ornithinimicrobium faecis sp. nov. isolated from bat faeces (Rousettus leschenaultia and Taphozous perforates).

Four Gram-staining-positive, aerobic, non-motile, circle-shaped bacteria were isolated from the faeces of bats (Rousettus leschenaultia and Taphozous perforates) collected from Guangxi autonomous region (E106°49'20″, N22°20'54″) and Yunnan province (E102°04'39″, N25°09'10″) of South China. Strains HY006T and HY008 shared highly 16S rRNA gene sequence similarity to those of Ornithinimicrobium pratense W204T (99.3%) and O. flavum CPCC 203535T (97.3%), while the strains HY1745 and HY1793T were closest to the type strains O. ciconiae H23M54T (98.7%), O. cavernae CFH 30183T (98.3%), and O. murale 01-Gi-040T (98.1%). Furthermore, when compared to the other members of the genus Ornithinimicrobium, the digital DNA-DNA hybridization and average nucleotide identity values of the four novel strains were within the ranges of 19.6-33.7% and 70.6-87.4%, respectively, both of which were below the respective recommended cutoff values of 70.0% and 95-96%. Significantly, strain HY006T was resistant to chloramphenicol and linezolid whereas strain HY1793T was resistant to erythromycin, clindamycin (intermediately), and levofloxacin (intermediately). The main cellular fatty acids (>20.0%) of our isolates were iso-C15:0 and iso-C16:0. Strains HY006T and HY1793T contained ornithine as the diagnostic diamino acid, also along with the alanine, glycine and glutamic acid in their cell wall. Based on phylogenetic, chemotaxonomic and phenotypic analyses, these four strains could be classified as two novel species of the genus Ornithinimicrobium, for which the names Ornithinimicrobium sufpigmenti sp. nov. and Ornithinimicrobium faecis sp. nov. are proposed. The type strains are HY006T (=CGMCC 1.16565T =JCM 33397T) and HY1793T (=CGMCC 1.19143T =JCM 34881T), respectively.

Comparative Evaluation of Standard RT-PCR Assays and Commercial Real-Time RT-PCR Kits for Detection of Lassa Virus.

Lassa virus (LASV) is a causative agent of hemorrhagic fever epidemic in West Africa. In recent years, it has been transmitted several times to North America, Europe, and Asia. Standard reverse transcription (RT)-PCR and real-time RT-PCR are extensively used for early detection of LASV. However, the high nucleotide diversity of LASV strains complicates the development of appropriate diagnostic assays. Here, we analyzed LASV diversity clustered with geographic location and evaluated the specificity and sensitivity of two standard RT-PCR methods (GPC RT-PCR/1994 and 2007) and four commercial real-time RT-PCR kits (namely, Da an, Mabsky, Bioperfectus, and ZJ) to detect six representative LASV lineages using in vitro synthesized RNA templates. The results showed that the GPC RT-PCR/2007 assay had better sensitivity compared to the GPC RT-PCR/1994 assay. The Mabsky and ZJ kits were able to detect all RNA templates of six LASV lineages. Contrastingly, the Bioperfectus and Da an kits failed to detect lineages IV and V/VI. The limit of detection for lineage I with the Da an, Bioperfectus, and ZJ kits were significantly higher than that of the Mabsky kit at an RNA concentration of 1 × 1010 to 1 × 1011 copies/mL. The Bioperfectus and Da an kits detected lineages II and III at an RNA concentration of 1 × 109 copies/mL, higher than that of the other kits. In conclusion, the GPC RT-PCR/2007 assay and the Mabsky kit were suitable assays for the detection of LASV strains based on good analytical sensitivity and specificity. IMPORTANCE Lassa virus (LASV) is a significant human pathogen causing hemorrhagic fever in West Africa. Increased traveling around the world raises the risk of imported cases to other countries. The high nucleotide diversity of LASV strains clustered with geographic location complicates the development of appropriate diagnostic assays. In this study, we showed that the GPC reverse transcription (RT)-PCR/2007 assay and the Mabsky kit are suitable for detecting most LASV strains. Future assays for molecular detection of LASV should be based on specific countries/regions along with new variants.

Description of Ornithinimicrobium cryptoxanthini sp. nov., a Novel Actinomycete Producing ß-cryptoxanthin Isolated from the Tongtian River Sediments.

Two novel Gram-stain-positive, aerobic, non-motile, and yellow-pigmented, irregular rod-shaped bacteria (JY.X269 and JY.X270T) were isolated from the near-surface sediments of river in Qinghai Province, P. R. China (32°37'13″N, 96°05'37″E) in July 2019. Both strains were shown to grow at 15-35 °C and pH 7.0-10.0, and in the presence of 0-6.0% (w/v) NaCl. The 16S rRNA gene sequence analysis showed that the isolates were closely related to Ornithinimicrobium cavernae CFH 30183 T (98.6-98.8% 16S rRNA gene sequence similarity), O. ciconiae H23M54T (98.5-98.6%) and O. murale 01-Gi-040T (98.3-98.5%). The phylogenetic and phylogenomic trees based on the 16S rRNA gene and 537 core gene sequences, respectively, revealed that the two strains formed a distinct cluster with the above three species. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between our two isolates (JY.X269 and JY.X270T) and other Ornithinimicrobium species were within the ranges of 19.0-23.9% and 70.8-80.4%, respectively, all below the respective recommended 70.0% and 95-96% cutoff point. Furthermore, the major cellular fatty acids (> 10.0%) of strains JY.X269 and JY.X270T were iso-C15:0, iso-C16:0, and summed feature 9. Strain JY.X270T contained MK-8(H4) and ornithine as the predominant menaquinone and diagnostic diamino acid component within the cell wall teichoic acids. ß-cryptoxanthin (C40H56O) can be extracted from strain JY.X270T, and its content is 6.3 µg/ml. Based on results from the phylogenetic, chemotaxonomic, and phenotypic analyses, the two strains could be classified as a novel species of the genus Ornithinimicrobium, for which the name Ornithinimicrobium cryptoxanthini sp. nov. is proposed (type strain JY.X270T = CGMCC 1.19147T = JCM 34882T).

Emergence of an ancient and pathogenic mammarenavirus.

Emerging zoonoses of wildlife origin caused by previously unknown agents are one of the most important challenges for human health. The Qinghai-Tibet Plateau represents a unique ecological niche with diverse wildlife that harbours several human pathogens and numerous previously uncharacterized pathogens. In this study, we identified and characterized a novel arenavirus (namely, plateau pika virus, PPV) from plateau pikas (Ochotona curzoniae) on the Qinghai-Tibet Plateau by virome analysis. Isolated PPV strains could replicate in several mammalian cells. We further investigated PPV pathogenesis using animal models. PPV administered via an intraventricular route caused trembling and sudden death in IFNαßR-/- mice, and pathological inflammatory lesions in brain tissue were observed. According to a retrospective serological survey in the geographical region where PPV was isolated, PPV-specific IgG antibodies were detected in 8 (2.4%) of 335 outpatients with available sera. Phylogenetic analyses revealed that this virus was clearly separated from previously reported New and Old World mammarenaviruses. Under the co-speciation framework, the estimated divergence time of PPV was 77-88 million years ago (MYA), earlier than that of OW and NW mammarenaviruses (26-34 MYA).

Linking Microbial Functional Gene Abundance and Daqu Extracellular Enzyme Activity: Implications for Carbon Metabolism during Fermentation.

Daqu is the starter of Baijiu, it provides the microbes and enzymes necessary for fermentation. Studies have already established carbohydrate metabolism as the primary functional module in Daqu fermentation. The present study investigated the changes in microbial functions and the relationship between carbohydrate metabolism-related functional genes and extracellular enzyme activity during the Daqu fermentation. Amplicon sequencing identified 38 bacterial and 10 fungal phyla in Daqu samples, while shotgun metagenomic sequencing classified and annotated 40.66% of the individual features, of which 40.48% were prokaryotes. KEGG annotation showed that the pathways related to metabolites were less in the early fermentation stage, but higher in the middle and late stages. The functional genes related to pyruvate metabolism, glyoxylate and dicarboxylate metabolism, and propanoate metabolism were relatively high in the early and late stages of fermentation, while that for start and cross metabolism was relatively low. The study also found that amino sugar and nucleoside sugar metabolism were dominant in the middle stage of fermentation. Finally, the correlation network analysis showed that amylase activity positively correlated with many carbon metabolism-related pathways, while liquefaction activity negatively correlated with these pathways. In conclusion, the present study provides a theoretical basis for improving and stabilizing the quality of Daqu.

Nocardia sputi sp. nov. isolated from the sputum of patients with pulmonary infection.

Two Gram-stain-positive, aerobic and rod-shaped actinomycetes (strains CY18T and CY8) were isolated from the sputum of two patients with pulmonary infections, and their taxonomic status was investigated. The 16S rRNA gene sequences and the results of phylogenetic analyses indicated that CY18T and CY8 were identical (100 %) and were most closely related to Nocardia beijingensis CGMCC 4.1521T (99.9 %) and Nocardia araoensis NBRC 100135T (99.5 %). The predominant cellular fatty acids of CY18T and CY8 were C16 : 0, C18 : 0, C18 : 1ω9c and summed feature 3 (comprising C16 : 1É·7c and/or C16 : 1É·6c), and the major menaquinone was MK-8(H4ω-cycl).The diagnostic diamino acid in the cell-wall peptidoglycan was meso-diaminopimelic acid. The whole-cell hydrolytic sugar pattern consisted of arabinose and glucose. The polar lipid profile contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannoside, two unidentified phospholipids, three unidentified glycolipids and two unidentified lipids.The DNA G+C contents of CY18T and CY8 were 67.9 and 68.0 % respectively. The digital DNA-DNA hybridization and average nucleotide identity values between the two novel strains and closely related species were well under the 70 % and 95-96 % thresholds, respectively, but these values between the two novel strains were 95.5 % and 99.5 %, respectively. On the basis of morphological and chemotaxonomic characteristics and the results of phylogenetic analyses, strains CY18T and CY8 represent a novel species of the genus Nocardia, for which the name Nocardia sputi sp. nov. is proposed. The type strain is CY18T (=GDMCC 1.3318T = JCM 33932T).

Nocardioides ochotonae sp. nov., Nocardioides campestrisoli sp. nov. and Nocardioides pantholopis sp. nov., isolated from the Qinghai-Tibet Plateau.

Six Gram-stain-positive, aerobic and irregular-rod-shaped actinobacteria (ZJ1313T, ZJ1307, MC1495T, Y192, 603T and X2025) were isolated from the Qinghai-Tibet Plateau of China and were characterized using a polyphasic taxonomic method. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the six new strains formed three distinct clusters within the genus Nocardioides, and strains ZJ1313T and ZJ1307 were most closely related to N. solisilvae JCM 31492T (16S rRNA gene sequence similarity, 98.0 %), MC1495T and Y192 to N. houyundeii 78T (98.5 %), and 603T and X2025 to N. dokdonensis JCM 14815T (97.6 %). The digital DNA-DNA hybridization values of strains ZJ1313T, MC1495T and 603T among each other and with type strains of their closest relatives were all below the 70 % cut-off point, but values within each pair of new strains were all higher than the threshold. The major fatty acids of these strains were iso-C16 : 0, C17 : 1 ω8c or C18 : 1 ω9c. MK-8(H4) was the predominant respiratory menaquinone and ʟʟ-2,6-diaminopimelic acid was the diagnostic diamino acid. All the strains shared diphosphatidylglycerol (predominant), phosphatidylglycerol, phosphatidylcholine and phosphatidylinositol as the common polar lipids, with minor difference in the types of unidentified phospholipids, glycolipids and lipids. The G+C contents based on genomic DNA of strains ZJ1313T, MC1495T and 603T were 72.5, 72.1 and 73.2 mol%, respectively. The above results suggested that strain pairs ZJ1313T/ZJ1307, MC1495T/Y192 and 603T/X2025 represent three new species of genus Nocardioides, for which the names Nocardioides ochotonae sp. nov. (ZJ1313T=GDMCC 4.177T=KCTC 49537T=JCM 34185T), Nocardioides campestrisoli sp. nov. (MC1495T=GDMCC 4.176T=KCTC 49536T=JCM 34307T) and Nocardioides pantholopis sp. nov. (603T=CGMCC 4.7510T=DSM 106494T) are proposed accordingly.

Gordonia zhenghanii sp. nov. and Gordonia liuliyuniae sp. nov., isolated from bat faeces.

Four mesophilic actinobacteria (HY002T, HY442, HY366T and HY285) isolated from the faeces of bats collected in southern China were found to be strictly aerobic, non-motile, rod-shaped, oxidase-negative, Gram-stain-positive and catalase-positive. Strains HY002T and HY366T contained meso-diaminopimelic acid as the diagnostic diamino acid and MK-9(H2) the sole respiratory quinone. Arabinose, galactose and ribose were detected in the whole-cell hydrolysates of both type strains. The main cellular fatty acids (> 10.0%) of all strains were C16 : 0, C18 : 1 ω9c, 10-methyl-C18 : 0 and summed feature 3. Strains HY002T and HY366T contained diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidyl inositol mannosides as the major polar lipids. The phylogenetic/phylogenomic analyses based on 16S rRNA gene and genomic sequence comparison revealed that the four strains belong to the genus Gordonia, most closely related to G. neofelifaecis NRRL B-59395T(98.2-98.3% sequence similarity) on the EzBioCloud database. The G+C contents of strains HY002T and HY366T based on genomic DNA were 66.5 and 66.9%, respectively. The DNA-DNA relatedness values between the two types strains and members of the genus Gordonia were far below 70 % (18.6-23.1 %). All genotypic and phenotypic data indicated that the four strains are representatives of two novel separate species, for which the names Gordonia zhenghanii sp. nov. and Gordonia liuliyuniae sp. nov. are proposed, with HY002T (=CGMCC 4 7757T=JCM 34 878T) and HY366T (=CGMCC 1 19146T=JCM 34 879T) as the respective type strains.

The Threat of Potentially Pathogenic Bacteria in the Feces of Bats.

Bats have attracted global attention because of their zoonotic association with severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Previous and ongoing studies have predominantly focused on bat-borne viruses; however, the prevalence or abundance of bat-borne pathogenic bacteria and their potential public health significance have largely been neglected. For the first time, this study used both metataxonomics (16S rRNA marker gene sequencing) and culturomics (traditional culture methods) to systematically evaluate the potential public health significance of bat fecal pathogenic bacteria. To this end, fecal samples were obtained from five bat species across different locations in China, and their microbiota composition was analyzed. The results revealed that the bat microbiome was most commonly dominated by Proteobacteria, while the strictly anaerobic phylum Bacteroidetes occupied 35.3% of the relative abundance in Rousettus spp. and 36.3% in Hipposideros spp., but less than 2.7% in the other three bat species (Taphozous spp., Rhinolophus spp., and Myotis spp.). We detected 480 species-level phylotypes (SLPs) with PacBio sequencing, including 89 known species, 330 potentially new species, and 61 potentially higher taxa. In addition, a total of 325 species were identified by culturomics, and these were classified into 242 named species and 83 potentially novel species. Of note, 32 of the 89 (36.0%) known species revealed by PacBio sequencing were found to be pathogenic bacteria, and 69 of the 242 (28.5%) known species isolated by culturomics were harmful to people, animals, or plants. Additionally, nearly 40 potential novel species which may be potential bacterial pathogens were identified. IMPORTANCE Bats are one of the most diverse and widely distributed groups of mammals living in close proximity to humans. In recent years, bat-borne viruses and the viral zoonotic diseases associated with bats have been studied in great detail. However, the prevalence and abundance of pathogenic bacteria in bats have been largely ignored. This study used high-throughput sequencing techniques (metataxonomics) in combination with traditional culture methods (culturomics) to analyze the bacterial flora in bat feces from different species of bats in China, revealing that bats are natural hosts of pathogenic bacteria and carry many unknown bacteria. The results of this study can be used as guidance for future investigations of bacterial pathogens in bats.