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Four novel Gram-negative, mesophilic, aerobic, motile, and cocci-shaped strains were isolated from tick samples (strains 546T and 573) and respiratory tracts of marmots (strains 1318T and 1311). The 16S rRNA gene sequencing revealed that strains 546T and 573 were 97.8% identical to Roseomonas wenyumeiae Z23T, whereas strains 1311 and 1318T were 98.3% identical to Roseomonas ludipueritiae DSM 14915T. In addition, a 98.0% identity was observed between strains 546T and 1318T. Phylogenetic and phylogenomic analyses revealed that strains 546T and 573 clustered with R. wenyumeiae Z23T, whereas strains 1311 and 1318T grouped with R. ludipueritiae DSM 14915T. The average nucleotide identity between our isolates and members of the genus Roseomonas was below 95%. The genomic G+C content of strains 546T and 1318T was 70.9% and 69.3%, respectively. Diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE) were the major polar lipids, with Q-10 as the predominant respiratory quinone. According to all genotypic, phenotypic, phylogenetic, and phylogenomic analyses, the four strains represent two novel species of the genus Roseomonas, for which the names Roseomonas haemaphysalidis sp. nov. and Roseomonas marmotae sp. nov. are proposed, with 546T (= GDMCC 1.1780T = JCM 34187T) and 1318T (= GDMCC 1.1781T = JCM 34188T) as type strains, respectively. Electronic Supplementary Material Supplementary material is available for this article at 10.1007/s12275-022-1428-1 and is accessible for authorized users.

Two unknown Gram-stain-positive, catalase- and oxidasenegative, non-motile, and coccus-shaped bacteria, designated MN-17T and MN-09, were isolated from yaks faeces (Bos grunniens) in the Qinghai-Tibet Plateau of China. 16S rRNA gene sequence-based comparative analyses revealed that the two strains were grouped within the genus Vagococcus, displaying the highest similarity with Vagococcus xieshaowenii CGMCC 1.16436T (98.6%) and Vagococcus elongatus CCUG 51432T (96.4%). Both strains grew optimally at 37°C and pH 7.0 in the presence of 0.5% (w/v) NaCl. The complete genome of MN-17T comprises 2,085 putative genes with a total of 2,190,262 bp and an average G + C content of 36.7 mol%. The major fatty acids were C16:0 (31.2%), C14:0 (28.5%), and C18:1ω9c (13.0%); the predominant respiratory quinone was MK-7 (68.8%); the peptidoglycan type was A4α(L-Lys-D-Asp); and the major polar lipid was diphosphatidylglycerol. Together, these supported the affiliation of strain MN-17T to the genus Vagococcus. In silico DNA-DNA hybridization and the average nucleotide identity values between MN-17T and all recognized species in the genus were 21.6-26.1% and 70.7-83.0%, respectively. MN-17T produced acid from D-cellobiose, D-fructose, glycerol, D-glucose, N-acetyl-glucosamine, gentiobiose, D-mannose, D-maltose, D-ribose, D-saccharose, salicin, D-trehalose, and D-xylose. These results distinguished MN-17T and MN-09 from closely related species in Vagococcus. Thus, we propose that strains MN-17T and MN-09 represent a novel species in the genus Vagococcus, with the name Vagococcus zengguangii sp. The type strain is MN-17T (= CGMCC 1.16726T = GDMCC 1.1589T = JCM 33478T).

Four unknown strains belonging to the genus Arthrobacter were isolated from plateau wildlife on the Qinghai–Tibet Plateau of PR China. Phylogenetic analysis based on 16S rRNA gene sequences showed that the four isolates were separated into two clusters. Cluster I (strains 785T and 208) had the greatest 16S rRNA gene sequence similarity to Arthrobacter citreus (98.6 and 98.7 %, respectively), Arthrobacter luteolus (98.0 and 98.1%, respectively), Arthrobacter gandavensis (97.9 and 98.0 %, respectively) and Arthrobacter koreensis (97.6 and 97.7 %, respectively). Likewise, cluster II (strains J391T and J915) had the highest sequence similarity to Arthrobacter ruber (98.6 and 98.3 %, respectively) and Arthrobacter agilis (98.1 and 97.9 %, respectively). Average nucleotide identity and the digital DNA–DNA hybridization values illustrated that the two type strains, 785T and J391T, represented two separate novel species that are distinct from all currently recognized species in the genus Arthrobacter . These strains had DNA G+C contents of 66.0–66.1 mol% (cluster I) and 68.0 mol% (cluster II). The chemotaxonomic properties of strains 785T and J391T were in line with those of the genus Arthrobacter : anteiso-C15:0 (79.3 and 40.8 %, respectively) as the major cellular fatty acid, MK-8(H2) (65.8 %) or MK-9(H2) (75.6 %) as the predominant respiratory quinone, a polar lipid profile comprising diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, glycolipids and phospholipid, and A3α or A4α as the cell wall peptidoglycan type. On the basis of our results, two novel species in the genus Arthrobacter are proposed, namely Arthrobacter yangruifuii sp. nov. (type strain, 785T=CGMCC 1.16725T=GDMCC 1.1592T=JCM 33491T) and Arthrobacter zhaoguopingii sp. nov. (type strain, J391T=CGMCC 1.17382T=GDMCC 1.1667T=JCM 33841T).

Four novel strains (592T, S592, MF47T and SMF47) were isolated from Tibetan antelopes (Pantholops hodgsonii) and plateau pikas (Ochotona curzoniae), respectively. The cells were aerobic, non-motile, Gram-stain- and catalase-positive, rod-shaped bacteria. The 16S rRNA gene sequences of the four strains showed highest similarities to Aeromicrobium fastidiosum DSM 10552T (98.1, 98.6, 98.7 and 98.7 %, respectively), and the phylogenetic analyses based on 16S rRNA gene and genomic sequences indicated that strains 592T and MF47T represent two novel species. The four isolates produced acid from l-rhamnose, d-xylose and cellobiose, but were unable to reduce nitrate. The DNA G+C contents of strains 592T and MF47T were 70.3 and 69.8 mol%, respectively. The digital DNA–DNA hybridization value between strains 592T and MF47T was 32.6 %, lower than the threshold of 70 %, indicating they belong to different species. The four strains’ genomes displayed less than 24.6 % DNA–DNA relatedness with all available genomes of the genus Aeromicrobium in the NCBI database, including Aeromicrobium fastidiosum NBRC 14897T and Aeromicrobium ginsengisoli JCM 14732T. The major fatty acids of the four strains were C18 : 1 ω9c and C18 : 0 10-methyl, and the main polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylinositol. The predominant respiratory quinones were MK-9(H4) and MK-8(H4). The cell-wall peptidoglycan contained ll-diaminopimelic acid. Based on these genotypic, phenotypic and biochemical analyses, it is proposed that the four unidentified bacteria be classified as two novel species, Aeromicrobium chenweiae sp. nov. and Aeromicrobium yanjiei sp. nov. The type strains are 592T (=CGMCC1.16526T=DSM 106289T) and MF47T (=CGMCC 1.17444T=JCM 33790T), respectively.

Eight Gram-stain-positive, rod-shaped bacterial strains were isolated from faeces of Tibetan antelopes on the Tibet-Qinghai Plateau of China. Genomic sequence analysis showed that the strains belong to the genera Actinomyces (strains 299T and 340), Corynebacterium (strains 2184T, 2185, 2183T and 2189) and Oceanobacillus (strains 160T and 143), respectively, with a percentage of similarity for the 16S rRNA gene under the species threshold of 98.7 % except for strains 160T and 143 with Oceanobacillus arenosus CAU 1183T (98.8 %). The genome sizes (and genomic G+C contents) were 3.1 Mb (49.4 %), 2.5 Mb (64.9 %), 2.4 Mb (66.1 %) and 4.1 Mb (37.1 %) for the type strains 299T, 2183T, 2184T and 160T, respectively. Two sets of the overall genome relatedness index values between our isolates and their corresponding closely related species were under species thresholds (95 % for average nucleotide identity, and 70 % for digital DNA–DNA hybridization). These results, together with deeper genotypic, genomic, phenotypic and biochemical analyses, indicate that these eight isolates should be classified as representing four novel species. Strain 299T (=CGMCC 1.16320T=JCM 33611T) is proposed as representing Actinomyces wuliandei sp. nov.; strain 2184T (=CGMCC 1.16417T=DSM 106203T) is proposed as representing Corynebacterium liangguodongii sp. nov.; strain 2183T (=CGMCC 1.16416T=DSM 106264T) is proposed as representing Corynebacterium yudongzhengii sp. nov.; and strain 160T (=CGMCC 1.16367T=DSM 106186T) is proposed as representing Oceanobacillus zhaokaii sp. nov.

A Gram-stain-positive, coccus-shaped, non-motile bacterium, designated CF-49T, was isolated from the cloacal content of a snow finch, which was incidentally captured in a plateau pika burrow on the Qinghai–Tibet Plateau, PR China. Analysis of the 16S rRNA gene sequence showed that strain CF-49T was closely related to Vagococcus elongatus CCUG 51432T (96.5 % similarity), Vagococcus fluvialis NCFB 2497T (96.0 %) and Vagococcus lutrae CCUG 39187T (95.9 %), whereas the similarity to another isolate (CF-210) was 99.9 %. Strains CF-49T and CF-210 grew optimally at 37 °C and pH 7.0 and in the presence of 0.5 % (w/v) NaCl. Acid was produced from N-acetylglucosamine, cellobiose, d-fructose, d-glucose, d-mannose, d-mannitol, maltose, d-ribose and salicin. The cell-wall peptidoglycan type was A4α (l-Lys–d-Asp). The major cellular fatty acids (>10 %) were C16 : 0 (35.6 %), C14 : 0 (17.3 %), C18 : 1 ω9c (16.2 %) and C16 : 1 ω9c (10.6 %). The predominant respiratory quinone was menaquinone MK-7 (68.8 %). The G+C content of the genomic DNA was 35.9 mol%. Digital DNA–DNA hybridization of strain CF-49T with V. fluvialis DSM 5731T, V. elongatus CCUG 51432Tand V. lutrae CCUG 39187T resulted in relatedness values of 21.4, 23.3 and 24.6 %, respectively. Based on results from polyphasic analyses, our two isolates are proposed to represent a novel species in the genus Vagococcus , with the name Vagococcus xieshaowenii. The type strain is CF-49T (=CGMCC 1.6436T=GDMCC 1.1588T=JCM 33477T).

Two Gram-stain-positive, catalase-positive and oxidase-negative, aerobic, non-motile, cellobiose-utilizing, short-rod-shaped strains (Z28T and Z29) were isolated from faeces of Tibetan antelope (Pantholops hodgsonii) collected on the Qinghai–Tibet Plateau. Strain Z28T shared 98.1, 98.0, 97.8 and 97.4 % 16S rRNA gene similarity, 24.1, 22.8, 23.2 and 26.3 % digital DNA–DNA hybridization relatedness and 80.8, 80.0, 80.7 and 80.9 % average nucleotide identity values with Cellulomonas oligotrophica DSM 24482T, Cellulomonas flavigena DSM 20109T, Cellulomonas iranensis DSM 14785T and Cellulomonas terrae JCM 14899T, respectively. Results from further phylogenetic analyses based on the 16S rRNA gene and 148 core genes indicated that strains Z28T and Z29 were closest to C. oligotrophica DSM 24482T and C. flavigena DSM 20109T, but clearly separated from the currently recognized species of the genus Cellulomonas . The genomic DNA G+C content of strain Z28T was 75.3 mol%. The major cellular fatty acids were anteiso-C15 : 0, anteiso-C15 : 1 A, C16 : 0 and anteiso-C17 : 0. Ribose and mannose were detected as the whole-cell sugars. The major respiratory quinone was MK-9(H4) and ornithine was the diamino acid of the cell wall. The polar lipids present in strain Z28T were phosphatidylethanolamine, five phospholipids, two aminophospholipids, aminolipid and three unidentified lipids. Comparison of phenotypic and phylogenetic features between the two strains and the related organisms revealed that Z28T and Z29 represent a novel species of the genus Cellulomonas , for which the name Cellulomonas shaoxiangyii sp. nov. is proposed. The type strain is Z28T (=CGMCC 1.16477T=DSM 106200T).

Two Gram-staining-positive, catalase-positive, oxidase-negative, aerobic, non-motile, irregular rod-shaped bacterial strains (Z350T and Z527) were isolated from intestinal contents of plateau pika (Ochotona curzoniae) from the Qinghai–Tibet Plateau, PR China. Results of phylogenetic analyses based on 16S rRNA gene sequences indicated that strain Z350T belongs to the genus Mumia (family Nocardioidaceae ) but clearly differs from the currently recognized species Mumia xiangluensis DSM 101040T (98.4 % similarity) and Mumia flava DSM 27763T (97.4 %). Strain Z350T had a DNA G+C content of 70.7 mol% and shared 80.4 and 76.7 % average nucleotide identity values and 23.4 and 20.6 % in silico DNA–DNA hybridization relatedness with M. xiangluensis DSM 101040T and M. flava DSM 27763T, respectively. Further phylogenetic analyses based on 497 core genes indicated that our isolates were members of the genus Mumia but separated from all existing genera within the family Nocardioidaceae . The major cellular fatty acids were C18 : 1 ω9c and 10-methyl C18 : 0. The cell wall contained ll-diaminopimelic acid as the diamino acid, and rhamnose, ribose and glucose as whole cell-wall sugars. MK-9(H4) was detected as the major menaquinone. Polar lipids present were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and one unidentified phospholipid. Based on distinct differences in the genotypic and phenotypic data from the two Mumia species, a novel species, Mumia zhuanghuii sp. nov., is proposed. The type strain is Z350T (=CGMCC 4.7464T=DSM 106288T).

Four novel bacterial strains, designated Z294T, Z311, Z443T and Z446, were isolated from the intestinal contents of plateau pika (Ochotona curzoniae) on the Qinghai–Tibet Plateau of China. Cells were Gram-stain-positive, catalase-positive, oxidase-negative, aerobic, non-motile and short-rod shaped. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the four isolates belong to the genus Georgenia , but clearly separate from the currently recognized species. Both type strains (Z294T and Z443T) shared low 16S rRNA gene sequence similarity, digital DNA–DNA hybridization relatedness and average nucleotide identity values with Georginia satyanarayanai NBRC 107612T, G. subflava JCM 19765T, G. ruanii JCM 15130T and G. thermotolerans DSM 21501T and against each other. The genomic DNA G+C contents of strains Z294T and Z443T were 73.3 and 70 %, respectively. The major cellular fatty acids of strain Z294T were anteiso-C15 : 0, anteiso-C15 : 1 A and C16 : 0, in contrast to anteiso-C15 : 0 and anteiso-C15 : 1 A for strain Z443T. Both type strains (Z294T and Z443T) shared the following common features: glucose, rhamnose and ribose as cell-wall sugars; MK-8(H4) as major menaquinone; alanine, glutamic acid and lysine as cell-wall amino acids; and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannoside and one unidentified phosphoglycolipid as polar lipids. Comparing the phenotypic and phylogenetic features among the four strains and their related organisms, strains Z294T and Z443T represent two novel species within the genus Georgenia , for which the names Georgenia wutianyii sp. nov. (type strain Z294T=CGMCC 1.16428T=DSM 106344T) and Georgenia yuyongxinii sp. nov. (type strain Z443T=CGMCC 1.16435T=DSM 106174T) are proposed.

Two aerobic, Gram-stain-positive, catalase-positive, non-motile and rod-shaped bacterial strains, designated MF30-AT and MF845, were isolated from the intestinal contents of plateau pika collected from the Qinghai–Tibet Plateau. Optimal growth of these two strains was observed under aerobic conditions at pH 7.0 and 28 °C. The 16S rRNA gene sequences of the isolates had highest similarities of 98.5 and 98.4 % to Agromyces fucosus , respectively. In the 16S rRNA gene and polygenetic trees, strains MF30-AT and MF845 were clearly distinct from other species. The two strains could not produce acid from arbutin, d-fructose, D-sucrose, glycogen, salicin or starch. Production of β-glucosidase by these strains was negative. The major fatty acids of these strains were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. Strain MF30-AT contained galactose, rhamnose and ribose as cell wall sugars and MK-12 and MK-11 as predominant menaquinones. The major polar lipids in strain MF30-AT were diphosphatidylglycerol, phosphatidylglycerol and a glycolipid, while the peptidoglycan contained alanine, glutamic acid, glycine and 2,4-diaminobutyric acid. The G+C contents of the DNA of strains MF30-AT and MF845 were 69.8 mol% and 69.7 mol%, respectively. The average nucleotide identity and digital DNA–DNA relatedness values of the two strains with all available genomes of the genus Agromyces were far below the respective thresholds of 95 and 70 %, respectively. All genotypic and phenotypic data indicated that strains MF30-AT and MF845 should be classified as novel members of the genus Agromyces , for which the name Agromyces badenianii sp. nov. is proposed. The type strain is MF30-AT (=CGMCC 1.16469T=DSM 106183T).

Two strains of Gram-stain-positive, aerobic, non-spore-forming, non-motile, rod-shaped bacteria (designated dk512T and dk508) were isolated from the faeces of Tibetan gazelle (Procapra picticaudata) collected from the Qinghai-Tibet Plateau, PR China. The 16S rRNA gene sequences of the strains showed the highest identity to Microbacterium saccharophilum K-1T (98.0 and 97.9 % similarity, respectively). The phylogenetic analysis based on 16S rRNA gene sequences revealed that dk512T and dk508 were members of the genus Microbacterium , and most closely related to strains Microbacterium mitrae M4-8T and Microbacterium hatanonis FCC-01T. The strains grew optimally on brain-heart infusion (BHI) agar with 5.0 % (v/v) sheep blood at 30 °C, pH 7.0 and with 1.0 % (w/v) NaCl. The genome of type strain dk512T was 3.8 Mb with a G+C content of 70.6 mol%. The average nucleotide identity and digital DNA–DNA hybridization values between strain dk512T and previously characterized Microbacterium species were T, the detected primary cellular fatty acids were anteiso-C15 : 0 and anteiso-C17 : 0, the main respiratory quinones were MK-9 (37.9 %) and MK-10 (35.7 %), and the polar lipids included diphosphatidylglycerol, phosphatidylglycerol and three unidentified glycolipids. The major cell-wall sugars were rhamnose, ribose and galactose. Alanine, glutamic acid, glycine and ornithine were in the cell-wall peptidoglycan. Based on phenotypic data and phylogenetic inference, these two strains represent a novel species of the genus Microbacterium , named here as Microbacterium wangchenii sp. nov, where dk512T is designated the type strain (=CGMCC 1.16590T=JCM 33494T=KCTC 49313T).

Two strains, designated 2251T and 3058, that were aerobic, Gram-stain-negative, non-motile, coccoid or short rod-shaped bacilli, have recently been isolated from Tibetan antelopes on the Qinghai-Tibet Plateau. The results of phylogenetic analyses of 16S rRNA gene sequences indicated that strains 2251T and 3058 represent a new species within the genus Paracoccus and are most similar to ‘ Paracoccus gahaiensis ’ CUG00006T (98.9 and 99.3 %), Paracoccus nototheniae I-41R45T (98.3 and 98.7 %) and Paracoccus hibiscisoli THG-T2.31T (97.6 and 97.8 %). Results of genomic sequence-based phylogenomic analysis agreed with those from 16S rRNA gene sequence analysis. Optimal growth was achieved at pH 7.0–7.5 and 28 °C with marine medium. Cells contained C18 : 1 ω7c as the major cellular fatty acid and ubiquinone-10 as the predominant menaquinone. The polar lipids comprised phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phospholipid, glycolipid and an unidentified lipid. The cell-wall peptidoglycan amino acids were meso-2,6-diaminopimelic acid, alanine and glutamic acid; the major cell-wall sugar was galactose. The G+C content of strain 2251T was 66.5 mol%. Both strains (2251T and 3058) had DNA–DNA relatedness values less than 50 % with all available genomes of the genus Paracoccus in the ncbi database. Differential genotypic inferences, together with phenotypic and biochemical characteristics, demonstrated that strains 2251T and 3058 should be classified as a novel species of the genus Paracoccus , for which the name Paracoccus liaowanqingii sp. nov. is suggested. The type strain is 2251T (=CGMCC 1.16490T=DSM 106269T).

Two strains (S-1072T and 1626) of Gram-stain-negative, oxidase- and catalase-positive, aerobic, rod-shaped, motile bacteria with a single polar flagellum, were isolated from the leaves of Dandelion (Taraxacum officinale) on the Qinghai–Tibet Plateau of China. The cells grew optimally at 28 °C, pH 7.0 and with 0.5 % (w/v) NaCl on brain–heart infusion agar. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains S-1072T and 1626 belong to the genus Luteimonas , sharing the highest similarity with Luteimonas arsenica CCTCC AB 2014326T (97.0 %), Luteimonas terricola CGMCC 1.8985T (96.9 %) and Luteimonas aestuarii KCTC 22048T (96.6 %). The phylogenomic tree indicated that strains S-1072T and 1626 were most closely related to Luteimonas abyssi CGMCC 1.12611T. The biochemical characteristics revealed that strains S-1072T and 1626 could neither produce trypsin nor produce acid from d-glucose, N-acetylglucosamine and maltose, distinguishing them from four closest relatives. The DNA G+C contents of strains S-1072T and 1626 were 69.2 and 69.3 mol% respectively. The digital DNA–DNA hybridization values of our isolates with their four closely related species were below the 70 % threshold. The predominant menaquinone was Q-8 (98.7 %) and the major polar lipids included diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major fatty acids (>10 %) were iso-C15 : 0, iso-C16 : 0 and summed feature 9 (10-methyl C16 : 0 and/or iso-C17 : 1 ω9c). Based on the data obtained, strains S-1072T and 1626 should be classified as a novel species of the genus Luteimonas , for which the name Luteimonas yindakuii sp. nov. is proposed. The type strain is S-1072T (=CGMCC 1.13927T=JCM 33487T).

Two novel strains (HT111-2T and HT170-2) of the genus Lactobacillus were isolated from Marmota himalayana faecal samples collected on the Qinghai-Tibet Plateau, PR China. The isolates were Gram-stain-positive, rod-shaped, non-spore-forming bacteria with irregular circular colonies. Phylogenetic analysis and comparison of the 16S rRNA gene sequences demonstrated that the two strains form a subcluster and are closest to Lactobacillus hamsteri JCM 6256T (97.3 %) and Lactobacillus amylolyticus DSM 11664T (97.2 %). Phylogenetic analysis of two housekeeping genes (rpoA and pheS) found that strains HT111-2T and HT170-2 had the same closest relatives as the 16S rRNA gene sequence analysis did. The G+C content of strains HT111-2T and HT170-2 were 38.8 mol%. The values of in silico DNA–DNA hybridization with known Lactobacillus species were lower than the threshold (70%). Average nucleotide identity values of strain HT111-2T with L. hamsteri JCM 6256T and L. amylolyticus DSM 11664T were 77.84 % and 76.85 %, respectively. The major fatty acids of strains HT111-2T and HT170-2 were C16 : 0, C18 : 1ω9c and C18 : 0. Results of phenotypic, chemotaxonomic and phylogenetic analyses suggest strains HT111-2T and HT170-2 represent a novel species of the genus Lactobacillus , for which the name Lactobacillus xujianguonis sp. nov. is proposed with HT111-2T (=CGMCC 1.13855T=KCTC 15803T) as the type strain.

Strains HY041T and HY039 were oxidase- and Gram-stain-negative, catalase-positive, rod-shaped, non-motile, and facultatively anaerobic bacteria. They were isolated from the feces of bats of the Hipposideros and Taphozous spp. collected from Chongqing City and Guangxi province (PR China), respectively. Phylogenetic analysis based on the 16S rRNA gene and 463 core genes indicated that HY041T and HY039 represent members of the genus Apibacter , forming a clade with Apibacter adventoris wkB301T (95.2 % 16S rRNA gene sequence similarity) and Apibacter mensalis R-53146T (94.0 %). In silico DNA–DNA hybridization ( is DDH) and average nucleotide identity (ANI) values of our isolates with the most closely related species were lower than the 70 % and 95–96 % threshold, respectively, in contrast to values above these two thresholds (isDDH value: 89.1 %; ANI value: 98.5 %) between strains HY041T and HY039. The novel isolates could grow on nutrient and MacConkey agar. HY041T and HY039 could produce β-galactosidase and N-acetyl-β-glucosaminidase, and utilize d-adonitol, d-mannose, gentiobiose, glucose and salicin. The major fatty acids (>10.0 %) of HY041T were iso-C17 : 0 3OH, iso-C15 : 0, C16 : 0, summed feature 9 (C16 : 0 10-methyl and/or iso-C17 : 1ω9c) and C16 : 0 3OH. Polar lipids included phosphatidylethanolamine, glycolipid, two unidentified aminolipids and four unidentified lipids. Menaquinone 6 (MK-6) was the sole respiratory quinone. On the basis of all analyses so far, strains HY041T and HY039 represent a novel species of the genus Apibacter , for which the name Apibacter raozihei sp. nov. is proposed. The type strain is HY041T (=CGMCC 1.16567T=JCM 33423T) with a genomic DNA G+C content of 32.2 mol%.

Six novel facultatively anaerobic, Gram-stain-positive, rod-shaped, non-haemolytic bacteria (zg-320T/zg-336, zg-917T/zg-910 and zg-913T/zg-915) isolated from animal tissues and human faeces were found to belong to the genus Corynebacterium based on the phylogenetic analyses of 16S rRNA gene and 262 core genes set. Based on the greatest degree of 16S rRNA similarity, zg-320T/zg-336 had the highest 16S rRNA gene similarity to Corynebacterium falsenii DSM 44353T (97.51 %), zg-917T/zg-910 to Corynebacterium coyleae DSM 44184T (98.68 %), and zg-913T/zg-915 to Corynebacterium afermentans subsp. lipophilum CIP 103500T (98.79 %). The three novel type strains had a relatively high DNA G+C content (61.2–64.4 mol%), low DNA relatedness and ANI values with their respective neighbours: 23.5/72.7 %, 25.0/72.3%and 22.6/73.1 % (zg-320T vs. Corynebacterium auriscanis CIP 106629T, Corynebacterium resistens DSM 45100T and Corynebacterium suicordis DSM 45110T); 24.4/82.3% and 23.7/81.3 % (zg-917T vs. C. coyleae DSM 44184T and Corynebacterium jeddahense JCBT); 26.8/83.7% and 27.7/84.4 % (zg-913T vs. Corynebacterium mucifaciens ATCC 700355T and C. afermentans subsp. lipophilum CCUG 32105T). The three novel species had C16 : 0, C18 : 0, C18 : 1 ω9c and C18 : 0 ante/C18 : 2 ω6,9c as the major cellular fatty acids; MK-8(H2) in strain zg-917T and MK-9(H2) in strains zg-320T and zg-913T were found to be the major respiratory quinones. For the three novel species, the detected major polar lipids included diphosphatidylglycerol, phosphatidyl inositol mannoside, phosphatidylglycerol and phosphatidylinositol, the cell-wall peptidoglycan was based on meso-DAP, and the whole-cell sugars mainly included ribose, arabinose and galactose. The three novel species grew optimally at 35–37 °C, 0.5 % (w/v) NaCl and pH 7.0–8.0; notably, they were tolerant of 10.5 % (w/v) NaCl. Based on the results of these comprehensive analyses, three novel species in the genus Corynebacterium are proposed, aptly named Corynebacterium zhongnanshanii sp. nov. (zg-320T = GDMCC 1.1719T = JCM 34106T), Corynebacterium lujinxingii sp. nov. (zg-917T = GDMCC 1.1707T = JCM 34094T) and Corynebacterium wankanglinii sp. nov. (zg-913T = GDMCC 1.1706T = JCM 34398T).

Two pairs of aerobic, Gram-stain-positive, rod-shaped strains (HY164T/HY044, HY168T/HY211) were isolated from bat faecal samples. Strains HY164T and HY044 were motile with a polar flagellum, and had 16S rRNA gene similarity of 95.1–98.6 % to Haloactinobacterium album YIM 93306T and Haloactinobacterium glacieicola T3246-1T; strains HY168T and HY211 were most similar to Ruania albidiflava DSM 18029T (96.6 %). Phylogenetic trees based on 16S rRNA gene and whole genome sequences revealed affiliation of strains HY164T and HY168T to the family Ruaniaceae , representing novel lineages in the genera Haloactinobacterium and Ruania , respectively, which was also supported by the results for average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH). For all isolates, the principal cellular fatty acids were anteiso-C15 : 0 and iso-C14 : 0. HY164T and HY168T had MK-8(H4) as the predominant isoprenoid quinone, diphosphatidylglycerol, phosphatidylglycerol, several unidentified phospholipids and glycolipids as common polar lipids while the latter strain additionally contained one unidentified aminophospholipid and one unidentified phosphoglycolipid. Besides sharing alanine, glutamic acid and lysine with HY164T, HY168T additionally contained 2,4-diaminobutyric acid in the cell-wall peptidoglycan. The whole-cell sugars of HY164T were ribose and rhamnose, while HY168T only included the latter. The DNA G+C contents of HY164T and HY168T were 71.0 and 69.1 mol%, respectively. Combining the polyphasic taxonomic data, HY164T (=CGMCC 4.7606T=JCM 33464T) is classified as representing a novel species of the genus Haloactinobacterium with the proposed name Haloactinobacterium kanbiaonis sp. nov., and HY168T (=CGMCC 1.16970T=JCM 33465T) is proposed to represent a novel species of the genus Ruania with the name Ruania zhangjianzhongii sp. nov.

Four obligatory anaerobic, Gram-stain-positive, non-motile and rod-shaped organisms (HF-1365T, HF-1362, HF-1101T and HF-4214) were isolated from faecal samples of healthy Chinese subjects. Results of 16S rRNA gene sequence analyses showed that these isolates belong to the genera Enorma (strains HF-1365T and HF-1362) and Eggerthella (strains HF-1101T and HF-4214), closest to Enorma massiliensis (both 98.6 %) and Eggerthella sinensis (98.0 and 97.8 %), respectively. The whole genome sequences of strains HF-1365T and HF-1101T were 2.3 and 4.2 Mb in size with 61.7 and 66.2 mol% DNA G+C content, respectively. The average nucleotide identity and digital DNA–DNA hybridization values indicated that strains HF-1365T and HF-1101T represent novel species in the genera Enorma and Eggerthella . Major fatty acid constituents (>10 %) of strains HF-1365T and HF-1362 were C12 : 0 (24.7 and 23.9 %), C14 : 0 (21.9 and 20.6 %) and summed feature 1 (C15 : 1iso H/C13 : 0 3OH; 12.8 and 10.8 %); those of strains HF-1101T and HF-4214 were C18 : 1 ω9c (32.4 and 33.1 %) and C16 : 0 (13.9 and 14.0 %). Strain HF-1365T had phospholipid, glycolipid, lipid and phosphoglycolipid without any known quinones, while strain HF-1101T had diphosphatidylglycerol as the major polar lipid and MK-7 (80.7 %) as the predominant quinone. On the basis of their phylogenetic and phenotypic characteristics, strains HF-1365T and HF-1101T represent two distinct species, respectively, in the genera Enorma and Eggerthella , for which the names Enorma shizhengliae sp. nov. (type strain HF-1365T=CGMCC 1.17435T=GDMCC 1.1705T=JCM 33601T) and Eggerthella guodeyinii sp. nov. (type strain HF-1101T=CGMCC 1.17436T=GDMCC 1.1668T=JCM 33773T) are proposed.

Six novel strains (ZJ34T, ZJ561, ZJ750T, ZJ1629, zg-993T and zg-987) isolated from faeces and respiratory tracts of Marmota himalayana from the Qinghai–Tibet Plateau of PR China were characterized comprehensively. The results of analyses of the 16S rRNA gene and genome sequences indicated that the six strains represent three novel species of the genus Actinomyces , and are closely related to Actinomyces urogenitalis DSM 15434T (16S rRNA gene sequences similarities, 94.9–98.7 %), Actinomyces weissii CCUG 61299T (95.6–96.6 %), Actinomyces bovis CCTCC AB2010168T (95.7 %) and Actinomyces bowdenii DSM 15435T (95.2–96.4 %), with values of digital DNA–DNA hybridization less than 30.1 % when compared with their closest relatives but higher than 70 % within each pair of novel strains (ZJ34T/ZJ561, ZJ750T/ZJ1629 and zg-993T/zg-987). All the novel strains had C18 : 1 ω9c and C16 : 0 as the two most abundant major fatty acids. MK-9(H4) or MK-8(H4) was the sole or predominant respiratory quinone of strains ZJ34T, ZJ750T and zg-993T and their polar lipid profiles differed, but all had diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, and phosphatidyl inositol mannoside as major components. ZJ750T shared identical peptidoglycan amino acid profile with ZJ34T (alanine, glutamic acid, lysine and ornithine) and the same whole-cell sugar composition with zg-993T (glucose, rhamnose and ribose). Strain zg-993T contained alanine, aspartic acid, glutamic acid, glycine and lysine in the peptidoglycan, and the only sugar in ZJ34T was ribose. The DNA G+C contents of the novel strains were within the range of 65.8–70.1 mol%. On the basis of the results from the aforementioned analyses, the six novel strains were classified as representing three novel species of genus Actinomyces , for which the names Actinomyces faecalis sp. nov. [type strain ZJ34T (=GDMCC 1.1952T=JCM 34355T)], Actinomyces respiraculi sp. nov. [type strain ZJ750T (=GDMCC 1.1950T=JCM 34356T)] and Actinomyces trachealis sp. nov. [type strain zg-993T (=GDMCC 1.1956T=JCM 34357T)] were proposed, respectively.

Four aerobic, Gram-stain-positive, rod-shaped bacteria (HY60T, HY54, HY82T and HY89) were isolated from bat faeces of Hipposideros and Rousettus species collected in PR China. Phylogenetic analyses based on 16S rRNA gene sequences indicated that the four novel strains formed two separate but adjacent subclades close to Microbacterium agarici CGMCC 1.12260T (97.6–97.7 % similarity), Microbacterium humi JCM 18706T (97.3–97.5 %) and Microbacterium lindanitolerans JCM 30493T (97.3–97.4 %). The 16S rRNA gene sequence similarity was 98.3 % between strains HY60T and HY82T, and identical within strain pairs HY60T/HY54 and HY82T/HY89. The DNA G+C contents of strains HY60T and HY82T were 61.9 and 63.3 mol%, respectively. The digital DNA–DNA hybridization and average nucleotide identity values between each novel strain and their closest relatives were all below the 70 % and 95–96 % thresholds for species delimitation, respectively. All four novel strains contained anteiso-C15 : 0, anteiso-C17 : 0, iso-C16 : 0 and iso-C15 : 0 as the main fatty acids, MK-11 and MK-12 as the major respiratory quinones, and diphosphatidylglycerol, phosphatidylglycerol and one unidentified glycolipid as the predominant polar lipids. The cell-wall peptidoglycan was of B type and contained alanine, glutamate, glycine and ornithine. The acyl type of the muramic acid was glycolyl. The whole-cell sugars were rhamnose and ribose. Based on the foregoing polyphasic analyses, it was concluded that the four uncharacterized strains represented two novel species of the genus Microbacterium , for which the names Microbacterium chengjingii sp. nov. [type strain HY60T (=CGMCC 1.17468T=GDMCC 1.1951T=KACC 22102T)] and Microbacterium fandaimingii sp. nov. [type strain HY82T (=CGMCC 1.17469T=GDMCC 1.1949T=KACC 22101T)] are proposed, respectively.

Two novel, Gram-stain-positive, non-motile, facultatively anaerobic, rod-shaped bacteria (strains 2129T and 2119) were isolated from the faeces of Tibetan antelopes (Pantholops hodgsonii) on the Qinghai-Tibet Plateau, PR China. The 16S rRNA gene sequences of the strains showed highest similarity values to Actinomyces timonensis DSM 23838T (92.9 and 92.8 %, respectively), and phylogenetic analysis based on 16S rRNA gene and genomic sequences indicated that strains 2129T and 2119 represent a new lineage. Strains 2129T and 2119 could ferment d-adonitol and d-xylose, but were unable to utilize d-mannose and d-melibiose nor produce esterase (C4) and proline arylamidase. The G+C contents of the two strains were both 69.0 mol%. Their genomes exhibited less than 40.4 % relatedness in DNA-DNA hybridization tests (below 70 % as the recommended threshold for new species) with all available genomes of the genus Actinomyces in the NCBI database. The major fatty acids of the two strains were C18 : 1ω9c and C16 : 0, and the major polar lipids were diphosphatidylglycerol, glycolipid, phosphatidylinositol, phosphatidyl inositol mannoside and phosphoglycolipid. Based on the results of genotypic, phenotypic and biochemical analyses, it is proposed that the two unidentified bacteria be classified as representing a novel species, Actinomyces lilanjuaniae sp. nov. The type strain is 2129T (=CGMCC 4.7483T=DSM 106426T).

Two Gram-stain-positive, irregular rod-shaped (0.2–0.5×1.3–2.5 um) strains, HY056T and HY057, were isolated from the faeces of Equus kiang (the largest of the wild asses) collected at different regions from the Qinghai-Tibetan Plateau of PR China. Phylogenetic analyses indicated that strains HY056T and HY057 belong to the genus Nocardioides by sharing a similarity ranging from 96.3 to 97.0 % in the 16S rRNA gene sequence and forming a distinct cluster with Nocardioides daphniae JCM 16608T, Nocardioides houyundeii 78T, Nocardioides solisilvae Ka25T and Nocardioides gilvus XZ17T. The digital DNA–DNA hybridization value of strain HY056T was 96.9 % with strain HY057, but less than 30.0 % with the above four closest relatives. MK-8(H4) was the predominant (91.6 %) respiratory quinone. The cell wall contained ll-2,6-diaminopimelic acid as the diamino acid of the peptidoglycan. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol and phospholipid. The predominant fatty acids (>10.0 %) were C18 : 1ω9c and iso-C16:0. The DNA G+C contents of strains HY056T and HY057 were 68.9 and 69.1 mol%, respectively. β-Glucosidase expression was positive, and acid was produced from d-fructose. Strain HY056T (=CGMCC 4.7563T=JCM 33399T) is assigned as the type strain of a novel species within the genus Nocardioides , for which the name Nocardioides yefusunii sp. nov is proposed.

Two Gram-stain-negative, catalase- and oxidase-positive, non-spore-forming, aerobic, motile, flagellated, and coccus-shaped strains (Z23T and Z24) were isolated from faeces of Tibetan antelopes (Pantholops hodgsonii) on the Qinghai–Tibet Plateau, PR China. Results of the morphological, biochemical, and phylogenetic studies indicated that they were similar to each other, but distinct from existing species of the genus Roseomonas . The proposed type strain, Z23T, had 97.8, 97.1 and 96.8 % 16S rRNA similarity to Roseomonas ludipueritiae DSM 14915T, Roseomonas aerofrigidensis JCM 31878T and Roseomonas aerophila KACC 16529T. Results from further phylogenetic analyses based on the 16S rRNA gene and 857 core genes indicated that the two strains were members of Roseomonas , but clearly separated from the currently recognized species. Strains Z23T had 43.8 %, 25.0 % DNA–DNA relatedness and 91.2, 81.3 % ANI values with R. ludipueritiae DSM 14915T and R. aerophila KACC 16529T. The genomic DNA G+C content of strain Z23T was 68.6 mol%. The major cellular fatty acids of strain Z23T were C18 : 1ω7c and/or C18 : 1ω6c and C19 : 0cyclo ω8c. The cell-wall sugars included glucose, rhamnose and ribose. Q-10 was the sole respiratory quinone, and spermidine was the major polyamine component. Polar lipids present in strain Z23T were phosphatidylcholine, diphosphatidylglycerol, phosphatidylethanolamine, aminophospholipid, phosphatidylglycerol, three aminolipids, two phospholipids and two unidentified lipids. Based on the distinct differences from other Roseomonas species judged from the genotypic and phenotypic data, a novel species represented by Z23T and Z24, Roseomonas wenyumeiae sp. nov., is proposed. The type strain is Z23T (=CGMCC 1.16540T=DSM 106207T).

Two rod-shaped, slightly halophilic and extremely halotolerant bacterial strains (X-1125T and X-1174), which were Gram-stain-positive, facultatively anaerobic and motile with peritrichous flagella, were isolated from the faeces of Tibetan antelopes. Their optimal temperature, NaCl concentration and pH for growth were 28 °C, 3 % (w/v) NaCl and pH 7.5, respectively. Based on the results of 16S rRNA gene sequences, and phylogenetic and phylogenomic analyses, their nearest phylogenetic neighbours were Paraliobacillussediminis KCTC 33762T (98.4 % similarity), Paraliobacillusquinghaiensis CGMCC 1.6333T (96.9 %) and Paraliobacillusryukyuensis NBRC 100001T (95.9 %) while the 16S rRNA genes of strains X-1125T and X-1174 were highly similar (99.7 %) to each other. The polar lipids comprised diphosphatidylglycerol, two unidentified phospholipids and four unidentified lipids. MK-7 was the sole menaquinone (100 %). The cell wall contained alanine, glycine, glutamic acid and meso-diaminopimelic acid. The major fatty acids (>9 %) were anteiso-C15 : 0, anteiso-C17 : 0 and C16 : 1ω11c. The in silico DNA–DNA hybridization value between strains X-1125T and X-1174 was 97.8 % (well above the species threshold), but their values were lower than the 70 % threshold with the three closely related type strains. Strains X-1125T and X-1174 had DNA G+C contents (mol%) of 35.2 and 35.1 %, respectively. Based on the presented data, strains X-1125T and X-1174 hereby represent a novel species of the genus Paraliobacillus , for which the name Paraliobacillus zengyii sp. nov. is proposed. The type strain is X-1125T (=DSM 107811T=CGMCC 1.16464T).

A novel, Gram-stain-positive, catalase-positive, non-spore-forming, short rod-shaped strain (VUL4_3T) was isolated from rectal swabs of Old World vultures (Aegypius monachus) from the Tibet–Qinghai Plateau, China. Based on the results of biochemical tests and 16S rRNA gene sequence comparison, strain VUL4_3T was determined to be a member of the genus Actinomyces that is closely related to the type strains of Actinomyces liubingyangii (97.7 % 16S rRNA gene sequence similarity) and Actinomyces marimammalium (96.5 %). Optimal growth occurred at 37 °C, pH 6–7 and with 1 % (w/v) NaCl. The typical major cellular fatty acids of strain VUL4_3T were C18 : 1ω9c, C16 : 0 and C18 : 0. The VUL4_3T genome contained 2 207 832 bp with an average G+C content of 51.9 mol%. DNA–DNA hybridization values between strain VUL4_3T and the above two species of the genus Actinomyces showed less than 32 % DNA–DNA relatedness, supporting a novel species status of strain VUL4_3T. Based on the phenotypic data and phylogenetic inference, the novel species Actinomyces tangfeifanii sp. nov. is proposed. The type strain is VUL4_3T (=CGMCC 4.7369T=DSM 103436T).

Four Gram-stain-positive, non-motile and asporous bacilli (strains ZJ-599T, ZJ-621, MC1420T and MC1482), isolated from animal tissue and environmental samples collected on the Qinghai-Tibet Plateau, PR China, were taxonomically characterized. Based on the results of 16S rRNA gene sequence analyses, the closest relatives of strains ZJ-599T and ZJ-621 were Corynebacterium endometrii LMM-1653T (97.5 %), Corynebacterium phocae M408/89/1T (96.5 %) and Corynebacterium flavescens OJ8T (96.3 %), whereas strains MC1420T and MC1482 were closest to Corynebacterium sanguinis CCUG 58655T (98.9 %), Corynebacterium mycetoides DSM 20632T (98.4 %) and Corynebacterium lipophiloflavum DSM 44291T (97.9 %). The results of rpoB gene sequence similarity analysis indicated that C. phocae M408/89/1T and C. sanguinis CCUG 58655T were closest to strains ZJ-599T/ZJ-621 (83.5 %) and MC1420T/MC1482 (91.8 %), respectively. The two novel type strains shared a similarity of 95.2 % in 16S rRNA and 81.3 % in rpoB gene sequences. The TAP-PCR DNA fingerprint and MALDI-TOF MS spectrum patterns clearly differentiated the novel isolates within and between each pair of strains. Strain ZJ-599T had 21.9–22.4 % digital DNA–DNA hybridization (dDDH) scores with C. endometrii LMM-1653T, C. phocae M408/89/1T and C. flavescens OJ8T, and 72.3–72.9 % of average nucleotide identity (ANI) with them. Similarly, strain MC1420T had 22.9–23.7 % dDDH values with C. sanguinis CCUG 58655T, C. mycetoides DSM 20632T and C. lipophiloflavum DSM 44291T, and 80.4–81.3 % ANI scores with them. Strain ZJ-599T had a 23.1 % dDDH value and 70.5 % ANI score with strain MC1420T, both below the corresponding thresholds for species delineation. Strains ZJ-599T and MC1420T both contain mycolic acids and have MK-8(H2) and MK-9(H2) as the predominant respiratory quinones, meso-diaminopimelic acid as the diagnostic diamino acid, and C18 : 1 ω9c as the main fatty acid. C17 : 1 ω8c and C15 : 1 ω8c were predominant in strain ZJ-599T in contrast to C17 : 1 ω7c being predominant in strain MC1420T. The main polar lipids in strain ZJ-599T were diphosphatidylglycerol, phosphatidylinositol and one unidentified glycolipid, while strain MC1420T had diphosphatidylglycerol, phosphatidylglycerol and one unidentified lipid as the major components. Since the two pairs of novel strains (ZJ-599T/ZJ-621, MC1420T/MC1482) distinctly differ from each other and from their nearest relatives, two novel species of the genus Corynebacterium are proposed, namely Corynebacterium lizhenjunii (type strain ZJ-599T=GDMCC 1.1779T=JCM 34341T) and Corynebacterium qintianiae (type strain MC1420T=GDMCC 1.1783T=JCM 34340T), respectively.

Four unknown strains, characterized as Gram-stain-negative, strictly anaerobic, non-motile and rod-shaped, were isolated from fresh faeces of healthy humans in PR China. Pairwise sequence comparisons of the 16S rRNA genes showed that these isolates were separated into two clusters. Cluster I (strains HF-5141T and HF-106) was most closely related to Bacteroides xylanisolvens XB1AT (98.0–98.3 % similarity) and Bacteroides ovatus ATCC 8483T (97.3–97.5 %), whereas cluster II (strains HF-5287T and HF-5300) exhibited a similarity range of 96.8–97.0 % to Bacteroides finegoldii JCM 13345T, 96.7–96.9 % to Bacteroides faecis MAJ27T and 96.4–96.5 % to Bacteroides xylanisolvens XB1AT. The DNA G+C contents of type strains HF-5141T and HF-5287T were 41.5 and 42.6 mol%, respectively. These strains had anteiso-C15 : 0 as the major cellular fatty acid, MK-9 and MK-11 as the predominant respiratory quinones, and phosphatidylethanolamine, aminophospholipids and phospholipids as major polar lipids, which is typical for members of the genus Bacteroides . However, the average nucleotide identity and digital DNA–DNA hybridization values, accompanied by different phenotypic and biochemical characteristics, distinguished them from their corresponding closest relatives as well as from other recognized members of the genus Bacteroides . Therefore, strains HF-5141T and HF-5287T represent two novel species in the genus Bacteroides , for which the names Bacteroides luhongzhouii sp. nov. and Bacteroides zhangwenhongii sp. nov. are proposed, with HF-5141T (=CGMCC 1.16787T=GDMCC 1.1591T=JCM 33480T) and HF-5287T (=CGMCC 1.16724T=GDMCC 1.1590T=JCM 33481T) as type strains.

Four novel bacterial strains (ST-M6T, L-033, L-031T and Z-333) were isolated from the intestinal contents of plateau pikas (Ochotona curzoniae) collected on the Qinghai–Tibet Plateau, PR China. Cells were aerobic, non-motile, Gram-stain-positive, catalase-positive, oxidase-negative, capsuled and short-rod-shaped. Phylogenetic analyses based on the 16S rRNA gene sequences and 387 core genes indicated that the four isolates belong in the genus Microbacterium and clearly separate from recognized species. The two type strains (ST-M6T and L-031T) shared low 16S rRNA similarity, average nucleotide identity values and digital DNA–DNA hybridization relatedness with their phylogenetic neighbours ( Microbacterium ginsengisoli DSM 18659T, Microbacterium hatanonis DSM 19179T, Microbacterium rhizomatis JCM 30598T, Microbacterium radiodurans CCTCC M208212T, Microbacterium oleivorans DSM 16091T and Microbacterium arborescens DSM 20754T). The genomic DNA G+C contents of strains ST-M6T and L-031T were 70.4 and 70.7 mol%, respectively. The major cellular fatty acids of strain ST-M6T were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0, in contrast to anteiso-C17 : 0, anteiso-C15 : 0 and anteiso-C17 : 1 ω9c of strain L-031T. Both type strains (ST-M6T and L-031T) were glycolate test positive and shared the following common features: MK-11 and MK-12 as major menaquinones; rhamnose, ribose, mannose and galactose as major cell-wall sugars; diphosphatidylglycerol, phosphatidylglycerol and two glycolipids as polar lipids; and ornithine, alanine, glycine and glutamic acid as cell-wall amino acids. Comparing the phenotypic, phylogenetic and chemotaxonomic features of the four strains and their related taxa, strains ST-M6T and L-031T represent two novel species of the genus Microbacterium , for which the names Microbacterium caowuchunii sp. nov. (type strain ST-M6T=CGMCC 1.16364T=DSM 104058T) and Microbacterium lushaniae sp. nov. (type strain L-031T =CGMCC 1.16363T=DSM 106170T) are proposed.

Six aerobic, non-motile, non-haemolytic, Gram-stain-negative, oxidase-negative strains (185T, 187, 323-1T, 194, dk386T and dk771) were recovered from different faecal samples of Equus kiang on the Qinghai–Tibet Plateau. In the 16S rRNA gene sequences, one strain pair, 185T/187, shared highest similarity to Acinetobacter equi 114T (97.9 %), and the other two (323-1T/194 and dk771T/dk386) to Acinetobacter harbinensis CGMCC 1.12528T (98.6 and 97.0 %, respectively). Phylogenomic tree analysis showed that these six strains formed three separate clades in the genus Acinetobacter . Digital DNA–DNA hybridization values of each pair of the isolates with all members of the genus Acinetobacter were far below 70 %. The main cellular fatty acids of all six strains were C18 : 1 ω9c, C16 : 0 and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). Q-9 was the predominant respiratory quinone for strains 185T, 323-1T and dk386T. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Based on the genotypic, phenotypic and biochemical analyses, these six strains represent three novel species of the genus Acinetobacter , for which the names Acinetobacter lanii sp. nov., Acinetobacter shaoyimingii sp. nov. and Acinetobacter wanghuae sp. nov. are proposed. The type strains are 185T (=CGMCC 1.13636T=JCM 33607T), 323-1T (=CGMCC 1.13940T=JCM 33608T) and dk386T (=CGMCC 1.16589T=JCM 33592T), respectively.

Two previously undescribed, Gram-stain-positive, rod-shaped strains, 410T and 553, were isolated from faeces of the Tibetan antelope (Pantholops hodgsonii) from the Tibet-Qinghai Plateau, PR China. The optimum growth conditions of the two novel strains were 1 % (w/v) NaCl, 37 °C and pH 7. The end products from glucose fermentation included ethanol and lactic acid. Based on results of 16S rRNA gene sequence comparison and phylogenetic and phylogenomic analyses, strains 410T and 553 were classified into the genus Actinomyces , and were closely related to Actinomyces ruminicola (97.6 %), Actinomyces oricola (93.5 %) and Actinomyces dentalis (90.8 %). The genomic G+C content of strain 410T was 67.4 mol%. Digital DNA–DNA hybridization values between strain 410T and each of the closely related species were under 70 %. The respiratory quinones were MK-10 (68 %) and MK-9 (32 %). The main cellular fatty acids of the isolates were C16 : 0, followed by C18 : 1 ω9c. The major polar lipids were diphosphatidylglycerol and phosphatidylinositol-mannoside. The whole-cell sugars contained rhamnose, ribose and glucose. The diagnostic amino acids of cell-wall peptidoglycan included alanine, glutamic acid, lysine and ornithine. The results of biochemical, chemotaxonomic and genotypic analyses revealed that the two novel strains represent a novel species of genus Actinomyces , for which the name Actinomyce s qiguomingii sp. nov. is proposed. The type strain is 410T (=CGMCC 1.16361T= DSM 106201T).

Two strains (pika_113T and pika_114) of a previously undescribed Actinomyces -like bacterium were recovered from the intestinal contents of plateau pika (Ochotona curzoniae) on the Tibet-Qinghai Plateau, China. Results from biochemical characterization indicated that the two strains were phenotypically homogeneous and distinct from other previously described species of the genus Actinomyces . Based on the comparison of 16S rRNA gene sequences and genome analysis, the bacteria were determined to be a hitherto unknown subline within the genus Actinomyces , being most closely related to type strains of Actinomyces denticolens and Actinomyces timonensis with a respective 97.2 and 97.1 % similarity in their 16S rRNA gene sequences. Phylogenetic analyses confirmed that pika_113T was well separated from any other recognized species of the genus Actinomyces and within the cluster with A. denticolens and A. timonensis. The genome of strain pika_113T displayed less than 42 % relatedness in DNA–DNA hybridization with all the available genomes of existing species of the genus Actinomyces in the NCBI database. Collectively, based on the phenotypic characteristics and phylogenetic analyses results, we propose the novel isolates as representatives of Actinomyces gaoshouyii sp. nov. The type strain of Actinomyces gaoshouyii is pika_113T (=CGMCC 4.7372T=DSM 104049T), with a genomic DNA G+C content of 71 mol%.

Five strains of Gram-positive-staining, catalase-negative, coccus-shaped, chain-forming organisms isolated separately from the respiratory tracts of five Marmota himalayana animals in the Qinghai-Tibet Plateau of China were subjected to phenotypic and molecular taxonomic analyses. Comparative analysis of the 16S rRNA gene indicated that these singular organisms represent a new member of the genus Streptococcus , being phylogenetically closest to Streptococcus marmotae DSM 101995T (98.4 % similarity). The groEL, sodA and rpoB sequence analysis showed interspecies similarity values between HTS2T and Streptococcus . marmotae DSM 101995T, its closest phylogenetic relative based on 16S rRNA gene sequences, of 98.2, 78.8 and 93.7 %, respectively. A whole-genome phylogenetic tree built from 82 core genes of genomes from 16 species of the genus Streptococcus validated that HTS2T forms a distinct subline and exhibits specific phylogenetic affinity with S. marmotae . In silico DNA–DNA hybridization of HTS2T showed an estimated DNA reassociation value of 40.5 % with Streptococcus . marmotae DSM 101995T. On the basis of their phenotypic characteristics and phylogenetic findings, it is proposed that the five isolates be classified as representatives of a novel species of the genus Streptococcus , Streptococcus himalayensis sp. nov. The type strain is HTS2T (=DSM 101997T=CGMCC 1.15533T). The genome of Streptococcus himalayensis sp. nov. strain HTS2T contains 2195 genes with a size of 2 275 471 bp and a mean DNA G+C content of 41.3 mol%.

Four Gram-stain-positive, catalase-negative, non-spore-forming, rod-shaped bacterial strains (zg-325T, zg329, dk561T and dk752) were isolated from the respiratory tract of marmot (Marmota himalayana) and the faeces of Tibetan gazelle (Procapra picticaudata) from the Qinghai-Tibet Plateau of PR China. The results of 16S rRNA gene sequence-based phylogenetic analyses indicated that strains zg-325T and dk561T represent members of the genus Actinomyces , most similar to Actinomyces denticolens DSM 20671T and Actinomyces ruminicola B71T, respectively. The DNA G+C contents of strains zg-325T and dk561T were 71.6 and 69.3 mol%, respectively. The digital DNA–DNA hybridization values of strains zg-325T and dk561T with their most closely related species were below the 70 % threshold for species demarcation. The four strains grew best at 35 °C in air containing 5 % CO2 on brain heart infusion (BHI) agar with 5 % sheep blood. All four strains had C18:1ω9c and C16:0 as the major cellular fatty acids. MK-8 and MK-9 were the major menaquinones in zg-325T while MK-10 was predominant in dk561T. The major polar lipids included diphosphatidylglycerol and phosphatidylinositol. On the basis of several lines of evidence from phenotypic and phylogenetic analyses, zg-325T and dk561T represent novel species of the genus Actinomyces , for which the name Actinomyces marmotae sp. nov. and Actinomyces procaprae sp. nov. are proposed. The type strains are zg-325T (=GDMCC 1.1724T=JCM 34091T) and dk561T (=CGMCC 4.7566T=JCM 33484T). We also propose, on the basis of the phylogenetic results herein, the reclassification of Actinomyces liubingyangii and Actinomyces tangfeifanii as Boudabousia liubingyangii comb. nov. and Boudabousia tangfeifanii comb. nov., respectively.

Three independent isolates (10022T, 10 009 and 10011) of a novel catalase-positive, Gram-stain-negative coccus in the genus Neisseria were obtained from the rectal contents of plateau pika on the Qinghai–Tibet Plateau, PR China. Based on 16S rRNA gene sequence analysis, our newly identified organisms were most closely related to Neisseria iguanae , Neisseria flavescens and Neisseria perflava with similarities ranging from 98.02 to 98.45 %, followed by seven other species in the genus Neisseria . Phylogenetic analysis based on 16S rRNA and rplF genes showed that our three novel isolates group with members of the genus Neisseria . Results of the average nucleotide identity (ANI) analysis confirmed that our isolates are of the same species, and the ANI values between type strain 10022T and other Neisseria species are 74.12–85.06 %, lower than the threshold range of 95–96 %. The major cellular fatty acids for our novel species are C16 : 0 and C16:1ω7c/C16:1ω6c, which along with their phenotypic characteristics can distinguish our isolates from other Neisseria species. On the basis of polyphasic analyses, our isolates are proposed to represent a novel species in genus Neisseria , with the name Neisseria weixii sp. nov. The type strain is 10022T (=DSM 103441T=CGMCC 1.15732T).

Strains 449T and 622 are both aerobic, Gram-stain-positive, short, rod-shaped bacilli that were recently isolated from the faeces of Tibetan antelopes on the Qinghai–Tibet Plateau in China. Their 16S rRNA gene sequences were most similar to those of Mycetocola zhadangensis ZD1-4T(97.9–98.0 %) and Mycetocola miduiensis CGMCC 1.11101T(97.3–97.4 %). Phylogenetic analysis of the 16S rRNA gene sequences further suggested that strains 449T and 622 represent a new lineage within the genus Mycetocola . The G+C content of strain 449T is 64.9 mol%. Optimal growth was achieved at pH 7.0 and 28 °C. Cells contained anteiso-C15 : 0 as the major cellular fatty acid, MK-10 and MK-11 as predominant menaquinones, diphosphatidylglycerol and phosphatidylglycerol as major polar lipids, and lysine as the diagnostic diamino acid. The DNA–DNA hybridization values of strains 449T and 622 were below the 70 % cut-off with respect to known strains of the genus Mycetocola . Based on these genotypic, phenotypic and biochemical characteristics, it seems rational to conclude that strains 449T and 622 belong to the genus Mycetocola and thus represent a novel species, for which the name Mycetocola zhujimingii sp. nov. is proposed. The type strain is 449T (=CGMCC 1.16372T=DSM 106173T).

Two novel aerobic, Gram-staining-positive and non-spore-forming bacterial strains, 194T and S1194, were isolated from faeces of Tibetan antelopes sampled at the Qinghai–Tibet Plateau of China. The strains were able to grow in medium up to 10 % NaCl, similar to the NaCl-resistant property of the genus Salinibacterium members. The 16S rRNA gene sequences of the strains showed the highest similarity to Salinibacterium xinjiangense(98.1–98.2 %), and phylogenetic analysis based on 16S rRNA gene sequences indicated that strains 194T and S1194 represent a new lineage. The DNA G+C contents of strain 194T and S1194 are 64.1 and 64.2 mol%. Their genomes exhibit less than 96 % average nucleotide identity and 70 % DNA–DNA relatedness to known species of Salinibacterium . Strains 194T and S1194 are unable to utilize d-mannose or produce naphthol-AS-BI-phosphohydrolase. The two strains had anteiso-C15 : 0 and anteiso-C17 : 0 as major fatty acids, and their cell walls contained lysine, alanine, glycine and glutamic acid. The predominant menaquinones identified were MK-11 and MK-10, with diphosphatidylglycerol and phosphatidylglycerol as major polar lipids. Overall, the major cellular content profiles of 194T agreed with those of Salinibacterium xinjiangense and Salinibacterium amurskyense, though the proportions were distinct. Based on genotypic, phenotypic and biochemical analyses, the novel species Salinibacterium hongtaonis sp. nov. is proposed. The type strain is 194T (=CGMCC 1.16371T=DSM 106171T).

Five strains of a Gram-stain-positive, catalase-negative, α-haemolytic, coccus-shaped chain-forming organism were isolated separately from the lower respiratory tracts of five animals of Marmota himalayana in the endemic area of plague, the Qinghai-Tibet Plateau, China. Based on their morphological characteristics, biochemical features and molecular phylogenetic studies, the strains were placed as representing a new member of the genus Streptococcus . Comparative 16S rRNA gene sequence studies indicated that strain HTS5T shared 96.5, 96.2 and 96.0 % similarity with Streptococcus gallinaceus CCUG 42692T, Streptococcus parasanguinis ATCC 15912T and Streptococcus suis ATCC 43765T, respectively. Sequence analysis of its rpoB and sodA genes showed that strain HTS5T was most closely related to Streptococcus cuniculi CCUG 65085T with 9.2 and 10.9 % interspecies divergence, respectively. The whole genome phylogenetic tree based on 339 core genes of 65 Streptococcus genomes confirmed that HTS5T belongs to a distinct lineage that is well separated from recognized species of the genus Streptococcus . In silico DNA–DNA hybridization using 65 available genomes from GenBank showed that HTS5T displayed less than 70 % DNA–DNA relatedness with the other 65 species of the genus Streptococcus deposited in the GenBank database. The genome of strain HTS5T (2 322 791 bp) contained 2377 genes and had a G+C content of 41.6 mol%. Therefore, the five strains are considered to represent a novel species of the genus Streptococcus for which the name Streptococcus marmotae sp. nov. is proposed. The type strain is HTS5T (=DSM 101995T=CGMCC 1.15534T).

Francisella tularensisis the causative pathogen of tularemia and a Tier 1 bioterror agent on the CDC list. Considering the fact that some subpopulation of theF. tularensisstrains is more virulent, more significantly associated with mortality, and therefore poses more threat to humans, rapid identification and characterization of this subpopulation strains is of invaluable importance. This review summarizes the up-to-date developments of assays for mainly detecting and characterizingF. tularensisand a touch of caveats of some of the assays.

A total of 48 endophytic bacteria were isolated from surface-sterilized tissues of the medicinal plant Lonicera japonica, which is grown in eastern China; six strains were selected for further study based on their potential ability to promote plant growth in vitro (siderophore and indoleacetic acid production). The bacteria were characterized by phylogenetically analyzing their 16S rRNA gene similarity, by examining their effect on the mycelial development of pathogenic fungi, by testing their potential plant growth-promoting characteristics, and by measuring wheat growth parameters after inoculation. Results showed that the number of endophytic bacteria in L. japonica varied among different tissues, but it remained relatively stable in the same tissues from four different plantation locations. Among the three endophytic strains, strains 122 and 124 both had high siderophore production, with the latter showing the highest phosphate solubilization activity (45.6 mg/L) and aminocyclopropane-1-carboxylic acid deaminase activity (47.3 nmol/mg/h). Strain 170 had the highest indoleacetic acid (IAA) production (49.2 mg/L) and cellulase and pectinase activities. After inoculation, most of the six selected isolates showed a strong capacity to promote wheat growth. Compared with the controls, the increase in the shoot length, root length, fresh weight, dry weight, and chlorophyll content was most remarkable in wheat seedlings inoculated with strain 130. The positive correlation between enzyme (cellulose and pectinase) activity and inhibition rate on Fusarium oxysporum, the IAA production, and the root length of wheat seedlings inoculated with each tested endophytic strain was significant in regression analysis. Deformity of pathogenic fungal mycelia was observed under a microscope after the interaction with the endophytic isolates. Such deformity may be directly related to the production of hydrolytic bacterial enzymes (cellulose and pectinase). The six endophytic bacterial strains were identified to be Paenibacillus and Bacillus strains based on the results of 16S rRNA gene sequencing analysis and their physiological and biochemical characteristics. Results indicate the promising application of endophytic bacteria to the biological control of pathogenic fungi and the improvement of wheat crop growth.

Two Gram-stain negative, catalase positive, coccus shaped bacteria, designated 10023T and 10010, were isolated from the rectal contents of a plateau pika (Ochotona curzoniae) in Qinghai–Tibet Plateau, China. Based on 16S rRNA gene sequence analysis, phylogenetic trees showed that these two isolates (10023T, 10010) group with members of the genus Neisseria. Additionally, these two isolates exhibited high 16S rRNA gene sequence similarity with Neisseria zalophi CSL 7565T (96.98%), Neisseria wadsworthii WC 05-9715T (96.92%) and Neisseria canis ATCC 14687T (96.79%). Further phylogenetic analysis based on the rplF gene showed that these two novel strains can be easily discriminated from phylogenetically closely related species. Optimal growth was found to occur on BHI agar with 5% defibrinated sheep blood at 37 °C and growth was also observed on nutrient agar, Columbia blood agar and chocolate agar plates; however, growth was not observed on MacConkey agar after 7 days. The major cellular fatty acids of these strains were identified as C16:0 and C16:1ω7c/C16:1ω6c. The complete genome size of the type strain 10023T is 2,496,444 bp, with DNA G+C content of 54.0 mol %. The average nucleotide identity values were 73.5–79.3% between isolate 10023T and reference Neisseria spp. Based on polyphasic analysis, these isolates (10023T and 10010) are considered to represent a novel species in the genus Neisseria, for which the name Neisseria chenwenguii sp. nov. is proposed. The type strain is 10023T (= DSM 103440T = CGMCC 1.15736T). Electronic supplementary material The online version of this article (10.1007/s10482-019-01234-2) contains supplementary material, which is available to authorized users.

Two bacterial strains were individually isolated from Marmota himalayana respiratory tracts; the animals were from the Tibet–Qinghai Plateau, PR China. The isolates were Gram-stain-positive, catalase-negative, coccus-shaped, chain-forming organisms. Analysis of 16S rRNA gene sequences indicated that the type strain HTS25T shared 98.0, 97.4, 97.2 and 97.1 % similarity with Streptococcus cuniculi , Streptococcus acidominimus , Streptococcus marmotae and Streptococcus himalayensis respectively. Sequence analysis of the sodA and rpoB genes indicated that HTS25T was closely related to S. marmotae (similarities of 94.7 and 91.4 % respectively). Analysis of groEL sequences showed interspecies similarity of 84.8 % between HTS25T and S. himalayensis . A whole-genome phylogenetic tree reconstructed from 81 core genes from the genomes of 17 members of the genus Streptococcus was used to validate that HTS25T forms a distinct subline from other recognized species of the genus Streptococcus . DNA–DNA hybridization of HTS25T showed a maximum estimated DNA reassociation value of 32.1 % to Streptococcus cuniculi CCUG 65085T. On the basis of the results of phenotypic and phylogenetic analyses, we propose that the two isolates be classified as representing a novel species of the genus Streptococcus , named Streptococcus respiraculi sp. nov. The type strain is HTS25T (=DSM 101998T=CGMCC 1.15531T). The genome of Streptococcus respiraculi sp. nov. strain HTS25T (2 067 971 bp) contains 2001 genes with an average DNA G+C content of 42.7 mol%.

The taxonomic position of four phenotypically closely related strains isolated from faecal samples of yaks (Bos grunniens) collected from the Qinghai-Tibetan plateau, China, was determined by a polyphasic approach. The strains were non-spore-forming, non-motile Gram-stain-positive, ovoid cocci, occurring predominantly in pairs and short chains or in irregular clusters. The 16S rRNA gene of strain MN05T was related phylogenetically to those of Enterococcus haemoperoxidus , Enterococcus rotai , Enterococcus quebecensis , Enterococcus plantarum , Enterococcus crotali , Enterococcus moraviensis , Enterococcus silesiacus , Enterococcus caccae , Enterococcus termitis , Enterococcus ureasiticus and Enterococcus ureilyticus , all belonging to the Enterococcus faecalis species group. The sequence similarities of three selected genes of MN05T to those of the type strains of phylogenetically related species were measured, with values within the range of 99.2–99.5 % (16S rRNA gene), 90.0–97.3 % (rpoA) and 80.0–85.3 % (pheS), respectively. The genome of MN05T (3 842 361 bp) contained 4299 genes with a DNA G+C content of 37.5 mol%. A whole-genome phylogenetic tree based on 808 core genes confirmed that MN05T belongs to a distinct lineage, well separated from all recognized species of the Enterococcus faecalis species group. DNA–DNA hybridization in silico showed that MN05T displayed less than 70 % DNA–DNA relatedness with the other 13 species of the Enterococcus faecalis species group. Moreover, their phenotypic features distinguished the four strains from the other species of the Enterococcus faecalis species group. Based upon these data obtained from the polyphasic characterization performed in the present study, a novel species of the genus Enterococcus , Enterococcus wangshanyuanii sp. nov., is proposed, with the type strain MN05T (=DSM 104047T=CGMCC 1.15942T).

Two strains (VUL4_1T and VUL4_2) of Gram-staining-positive, catalase-negative, non-spore-forming short rods were isolated from rectal swabs of Old World vultures (Gypaetus barbatus) in the Tibet-Qinghai Plateau, China. Analysis of morphological characteristics and biochemical tests indicated that the two strains closely resembled each other but were distinct from other species of the genus Actinomyces previously described. Based on the results of 16S rRNA gene sequence comparison and genome analysis, strains were determined to be members of the genus Actinomyces , closely related to the type strains of Actinomyces marimammalium (96.4 % 16S rRNA gene sequence similarity), Actinomyces hongkongensis (92.4 %), Actinomyces hordeovulneris (92.3 %) and Actinomyces nasicola (92.2 %), respectively. Optimal growth conditions were 37 °C, pH 6–7, with 1 % (w/v) NaCl. Strain VUL4_1T contained C18 : 1ω9c and C16 : 0 as the major cellular fatty acids and diphosphatidylglycerol as the major component of the polar lipids. The genomic DNA G+C content of VUL4_1T was 54.9 mol%. Strain VUL4_1T showed less than 70 % DNA–DNA relatedness with other species of the genus Actinomyces , further supporting strain VUL4_1T as a representative of a novel species. Based on the phenotypic data and phylogenetic inference, a novel species, Actinomyces liubingyangii sp. nov., is proposed with VUL4_1T (=CGMCC 4.7370T=DSM 104050T) as the type strain.

Two strains of Gram-stain-positive, facultatively anaerobic, non-spore-forming short rods (VUL7T and VUL8) were isolated from rectal swabs of Old World vultures, namely Gyps himalayensis, in Tibet-Qinghai Plateau, China. Optimal growth occurred at 37 °C, pH 6–7, with 1 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences classified the two strains to the genus Actinomyces , with highest 16S rRNA gene sequence similarity (95 %) to type strains of Actinomyces haliotis , Actinomyces radicidentis and Actinomyces urogenitalis . The major cellular fatty acids were C18 : 1ω9c and C16 : 0. MK-10(H4) was the major respiratory quinone. The genomic DNA G+C content of the isolate was 54.4 mol%. DNA–DNA hybridization values with the most closely related species of the genus Actinomyces was 24.6 %. The two strains can be differentiated from the most closely related species such as A. haliotis , A. radicidentis, A. graevenitzii and A. urogenitalis by a list of carbohydrate fermentations and enzyme activities. On the basis of physiological, biochemical and phylogenetic analysis, strains VUL7T and VUL8 represent novel species of the genus Actinomyces , for which the name Actinomyces vulturis sp. nov. is proposed. The type strain is VUL7T (=CGMCC 4.7366T=DSM 103437T).

Macrophage-pathogen interaction is a complex process and the outcome of this tag-of-war for both sides is to live or die. Without attempting to be comprehensive, this review will discuss the complexity and significance of the interaction outcomes between macrophages and some facultative intracellular bacterial pathogens as exemplified by Francisella, Salmonella, Shigella and Yersinia. Upon bacterial infection, macrophages can die by a variety of ways, such as apoptosis, autophagic cell death, necrosis, necroptosis, oncosis, pyronecrosis, pyroptosis etc, which is the focus of this review.

Historically, Francisella strains have been described as nonhemolytic. In this study, we show by use of solid and liquid hemolysis assays that some Francisella strains have hemolytic properties. The Francisella novicida type strain U112 is hemolytic to horse erythrocytes and Francisella philomiragia type strain FSC144 is hemolytic towards both human and horse erythrocytes. The F. novicida strain U112 released a protein (novilysin A) into the culture supernatant which cross-reacted with antiserum against Escherichia coli HlyA whereas there was no similar protein detectable with this cross-reactive property from the supernatant of the F. philomiragia strain.

The murine macrophage-like cell line J774.A1 ingests and allows intracellular growth ofFrancisella tularensis. We demonstrate that, after 24 h of infection, a pronounced cytopathogenicity resulted and the J774 cells were undergoing apoptosis. Despite this host cell apoptosis, no decrease in bacterial numbers was observed. When internalization of bacteria was prevented or intracellularly locatedF. tularensisbacteria were eradicated within 12 h, the progression of host cell cytopathogenicity and apoptosis was prevented.

The standard Escherichia coli collection of reference (ECOR) strains was examined for ability to exert cytotoxicity towards mammalian cells. A group of strains with functional haemolysin expression caused strong cytotoxicity and detachment in J774 macrophage cells as measured by lactate dehydrogenase release and as observed under a microscope. The expression of haemolysin was monitored by using antisera recognizing the E. coli α-haemolysin, the HlyA protein, and by quantitative haemolysis assays. The presence of the hlyA gene, which may be part of a pathogenicity island, was also confirmed. These analyses revealed that different ECOR strains express quantitatively different levels of haemolysin. One putative enteroaggregative E. coli (EAEC) strain was also found in the ECOR collection. The EAEC strain was characterized by the clump formation assay, PCR amplification of the EAEC DNA probe sequence and confirmative sequence analysis of the amplified fragment. The EAEC heat-stable enterotoxin 1 gene, astA, was found in 14% (10/72) of the ECOR strains and a consensus sequence for astA was proposed by comparing these sequences with those from pathogens. The astA gene appeared to be plasmid-located. Based on evidence from the work of other laboratories and from the present findings, it is concluded that the ECOR collection contains strains that may represent pathogenic E. coli. It is noted that caution is necessary when handling or disposing of those potentially pathogenic ECOR strains.

One hundred and seventy-two strains of Escherichia coli isolated from diarrheal patients in Beijing, P. R. China, were analyzed for plasmid DNA profile, HEp-2 cell adherence ability and reactivity to 10 previously described DNA probes. They had not been recognized as pathogenic E. coli in China. Of the 110 strains tested, 76 (69%) contained one or multiple large plasmids. Of the 71 strains with the large plasmids 64 could adhere to HEp-2 cells. Of the 172 strains, 102 (59.3%) were hybridized with at least one of the 10 probes. Of those, seven strains hybridized with enteroaggregative E. coli (EAggEC) probe. Their serotypes were O128 (two strains), O6 (one strain), and O111 (one strain). Three strains were untypable. Six and three strains were hybridized with enteropathogenic E. coli (EPEC) attaching and effacing genes (eae) or EPEC adherence factor (EAF) probe, respectively. Two non-O157: H7 strains hybridized with enterohemorrhagic E. coli (EHEC) probe. Seventy-two strains (41.9%) hybridized with shiga-like toxin 2 or 1 (SLT2 or SLT1) probes. Among the SLT1 or SLT2 probe-positive strains, 54 hybridized with invasive (INV) plasmid probe developed for identification of enteroinvasive E. coli (EIEC) and Shigella species. The INV and SLT probe-positive strains might represent a new variety of verotoxin-producing E. coli (VTEC).