Spatial and Temporal Shifts of Endophytic Bacteria in Conifer Seedlings of Abies religiosa (Kunth) Schltdl. & Cham.

Endophytes play an important role in plant development, survival, and establishment, but their temporal dynamics in young conifer plants are still largely unknown. In this study, the bacterial community was determined by metabarcoding of the 16S rRNA gene in the rhizoplane, roots, and aerial parts of 1- and 5-month-old seedlings of natural populations of Abies religiosa (Kunth) Schltdl. & Cham. In 1-month-old seedlings, Pseudomonas dominated aerial parts (relative abundance 71.6%) and roots (37.9%). However, the roots exhibited significantly higher bacterial species richness than the aerial parts, with the dissimilarity between these plant sections mostly explained by the loss of bacterial amplification sequence variants. After 5 months, Mucilaginibacter dominated in the rhizoplane (9.0%), Streptomyces in the roots (12.2%), and Pseudomonas in the aerial parts (18.1%). The bacterial richness and community structure differed significantly between the plant sections, and these variations were explained mostly by 1-for-1 substitution. The relative abundance of putative metabolic pathways significantly differed between the plant sections at both 1 and 5 months. All the dominant bacterial genera (e.g., Pseudomonas and Burkholderia-Caballeronia-Paraburkholderia) have been reported to have plant growth-promoting capacities and/or antagonism against pathogens, but what defines their role for plant development has still to be determined. This investigation improves our understanding of the early plant-bacteria interactions essential for natural regeneration of A. religiosa forest.

Impact of captivity and natural habitats on gut microbiome in Epinephelus akaara across seasons.

BACKGROUND: The gut microbiota significantly influences the health and growth of red-spotted grouper (Epinephelus akaara), a well-known commercial marine fish from Fujian Province in southern China. However, variations in survival strategies and seasons can impact the stability of gut microbiota data, rendering it inaccurate in reflecting the state of gut microbiota. Which impedes the effective enhancement of aquaculture health through a nuanced understanding of gut microbiota. Inspired by this, we conducted a comprehensive analysis of the gut microbiota of wild and captive E. akaara in four seasons. RESULTS: Seventy-two E. akaara samples were collected from wild and captive populations in Dongshan city, during four different seasons. Four sections of the gut were collected to obtain comprehensive information on the gut microbial composition and sequenced using 16S rRNA next-generation Illumina MiSeq. We observed the highest gut microbial diversity in both captive and wild E. akaara during the winter season, and identified strong correlations with water temperature using Mantel analysis. Compared to wild E. akaara, we found a more complex microbial network in captive E. akaara, as evidenced by increased abundance of Bacillaceae, Moraxellaceae and Enterobacteriaceae. In contrast, Vibrionaceae, Clostridiaceae, Flavobacteriaceae and Rhodobacteraceae were found to be more active in wild E. akaara. However, some core microorganisms, such as Firmicutes and Photobacterium, showed similar distribution patterns in both wild and captive groups. Moreover, we found the common community composition and distribution characteristics of top 10 core microbes from foregut to hindgut in E. akaara. CONCLUSIONS: Collectively, the study provides relatively more comprehensive description of the gut microbiota in E. akaara, taking into account survival strategies and temporal dimensions, which yields valuable insights into the gut microbiota of E. akaara and provides a valuable reference to its aquaculture.

Tumour-associated and non-tumour-associated bacteria co-abundance groups in colorectal cancer.

BACKGROUND & AIMS: Gut microbiota is closely related to the occurrence and development of colorectal cancer (CRC). However, the differences in bacterial co-abundance groups (CAGs) between tumor tissue (TT) and normal tissue (NT), as well as their associations with clinical features, are needed to be clarified. METHODS: Bacterial 16 S rRNA sequencing was performed by using TT samples and NT samples of 251 patients with colorectal cancer. Microbial diversity, taxonomic characteristics, microbial composition, and functional pathways were compared between TT and NT. Hierarchical clustering was used to construct CAGs. RESULTS: Four CAGs were grouped in the hierarchical cluster analysis. CAG 2, which was mainly comprised of pathogenic bacteria, was significantly enriched in TT samples (2.27% in TT vs. 0.78% in NT, p < 0.0001). CAG 4, which was mainly comprised of non-pathogenic bacteria, was significantly enriched in NT samples (0.62% in TT vs. 0.79% in NT, p = 0.0004). In addition, CAG 2 was also significantly associated with tumor microsatellite instability (13.2% in unstable vs. 2.0% in stable, p = 0.016), and CAG 4 was positively correlated with the level of CA199 (r = 0.17, p = 0.009). CONCLUSIONS: Our research will deepen our understanding of the interactions among multiple bacteria and offer insights into the potential mechanism of NT to TT transition.

Valid publication of names for bacterial species from the chicken gut.

This article is a follow-up to Gilroy R, Ravi A, Getino M, Pursley I, Horton DL, et al. PeerJ 2021;9:e10941, detailing accession numbers from culture collections to ensure that names for 33 new species conform to the Rules of the International Code of Nomenclature of Prokaryotes required for valid publication of names for cultured species. The following species names are now proposed to be recognized as validly published: Acinetobacter pecorum sp. nov., Arthrobacter gallicola sp. nov., Arthrobacter pullicola sp. nov., Bacillus norwichensis sp. nov., Brevibacterium gallinarum sp. nov., Brevundimonas guildfordensis sp. nov., Cellulomonas avistercoris sp. nov., Clostridium gallinarum sp. nov., Comamonas avium sp. nov., Corynebacterium gallinarum sp. nov., Cytobacillus stercorigallinarum sp. nov., Escherichia whittamii sp. nov., Kaistella pullorum sp. nov., Luteimonas colneyensis sp. nov., Microbacterium commune sp. nov., Microbacterium gallinarum sp. nov., Microbacterium pullorum sp. nov., Oceanitalea stevensii sp. nov., Ochrobactrum gallinarum sp. nov., Oerskovia douganii sp. nov., Oerskovia gallyi sp. nov., Oerskovia merdavium sp. nov., Oerskovia rustica sp. nov., Paenibacillus gallinarum sp. nov., Phocaeicola gallinarum sp. nov., Planococcus wigleyi sp. nov., Psychrobacter communis sp. nov., Serpens gallinarum sp. nov., Solibacillus faecavium sp. nov., Sporosarcina gallistercoris sp. nov., Sporosarcina quadrami sp. nov., Stenotrophomonas pennii sp. nov. and Ureibacillus galli sp. nov.

Saccharopolyspora mangrovi sp. nov., a novel mangrove soil actinobacterium with distinct metabolic potential revealed by comparative genomic analysis.

A novel mangrove soil-derived actinomycete, strain S2-29T, was found to be most closely related to Saccharopolyspora karakumensis 5K548T based on 16 S rRNA sequence (99.24% similarity) and genomic phylogenetic analyses. However, significant divergence in digital DNA-DNA hybridization, average nucleotide identity, and unique biosynthetic gene cluster possession distinguished S2-29T as a distinct Saccharopolyspora species. Pan genome evaluation revealed exceptional genomic flexibility in genus Saccharopolyspora, with > 95% accessory genome content. Strain S2-29T harbored 718 unique genes, largely implicated in energetic metabolisms, indicating different metabolic capacities from its close relatives. Several uncharacterized biosynthetic gene clusters in strain S2-29T highlighted the strain's untapped capacity to produce novel functional compounds with potential biotechnological applications. Designation as novel species Saccharopolyspora mangrovi sp. nov. (type strain S2-29T = JCM 34,548T = CGMCC 4.7716T) was warranted, expanding the known Saccharopolyspora diversity and ecology. The discovery of this mangrove-adapted strain advances understanding of the genus while highlighting an untapped source of chemical diversity.

Comparison of Yamuna (India) and Mississippi River (United States of America) bacterial communities reveals greater diversity below the Yamunotri Glacier.

The Yamuna River in India and the Mississippi River in the United States hold significant commercial, cultural, and ecological importance. This preliminary survey compares the bacterial communities sampled in surface waters at 11 sites (Yamuna headwaters, Mississippi headwaters, Yamuna River Yamunotri Town, Mississippi River at Winona, Tons River, Yamuna River at Paonta Sahib, Yamuna River Delhi-1, Yamuna River Delhi-2, Yamuna River before Sangam, Sangam, Ganga River before Sangam). Bacterial 16S rDNA analyses demonstrate dominance of Proteobacteria and Bacteroidetes phyla. Actinobacteria were also dominant at sites near Sangam in India and sites in Minnesota. A dominance of Epsilonbacteraeota were found in Delhi, India. Principal component analysis (PCA) using unique operational taxonomic units (OTUs) resulted in the identification of 3 groups that included the Yamuna River locations in Delhi (Delhi locations), Yamuna headwaters and Yamuna River at Yamunotri (Yamuna River locations below the Glacier) and Mississippi, Ganga, Tons, and other Yamuna River locations. Diversity indices were significantly higher at the Yamuna River locations below the Glacier (Simpson D = 0.986 and Shannon H = 5.06) as compared (p value <0.001) to the Delhi locations (D = 0.951 and H = 4.23) and as compared (p value < 0.001) to Mississippi, Ganga, Tons, and other Yamuna River locations (D = 0.943 and H = 3.96). To our knowledge, this is the first survey to compare Mississippi and Yamuna River bacterial communities. We demonstrate higher diversity in the bacterial communities below the Yamunotri glacier in India.

Nocardioides bizhenqiangii sp. nov. and Nocardioides renjunii sp. nov., isolated from soil and faeces of Tibetan antelope (Pantholops hodgsonii) on the Qinghai-Tibet Plateau.

Two novel strain pairs (HM61T/HM23 and S-34T/S-58) were isolated from soil and the faeces of Tibetan antelope (Pantholops hodgsonii) collected at the Qinghai-Tibet Plateau of PR China. All four new isolates were aerobic, non-motile, Gram-stain-positive, catalase-positive, oxidase-negative, and short rod-shaped bacteria. The results of phylogenetic analysis based on the full-length 16S rRNA genes and 283 core genomic genes indicated that the four strains were separated into two independent branches belonging to the genus Nocardioides. Strains HM61T and HM23 were most closely related to Nocardioides pelophilus THG T63T (98.58 and 98.65 % 16S rRNA gene sequence similarity). Strains S-34T and S-58 were most closely related to Nocardioides okcheonensis MMS20-HV4-12T (98.89 and 98.89 % 16S rRNA gene sequence similarity). The G+C contents of the genomic DNA of strains HM61T and S-34T were 70.6 and 72.5 mol%, respectively. Strains HM61T, S-34T and the type strains of closely related species in the analysis had average nucleotide identity values of 75.4-90.5 % as well as digital DNA-DNA hybridization values between 20.1 and 40.8 %, which clearly indicated that the four isolates represent two novel species within the genus Nocardioides. The chemotaxonomic characteristics of strains HM61T and S-34T were consistent with the genus Nocardioides. The major fatty acids of all four strains were iso-C16 : 0, C17 : 1 ω8c or C18 : 1 ω9c. For strains HM61T and S-34T, MK-8(H4) was the predominant respiratory quinone, ll-2,6-diaminopimelic acid was the diagnostic diamino acid in the cell-wall peptidoglycan, and the polar lipids profiles were composed of diphosphatidylglycerol and phosphatidylglycerol. Based on phylogenetic, phenotypic, and chemotaxonomic data, we propose that strains HM61T and S-34T represent two novel species of the genus Nocardioides, respectively, with the names Nocardioides bizhenqiangii sp. nov. and Nocardioides renjunii sp. nov. The type strains are HM61T (=GDMCC 4.343T=JCM 36399T) and S-34T (=CGMCC 4.7664T=JCM 33792T).

Crenobacter oryzisoli sp. nov., a novel phosphate-solubilizing bacterium isolated from the paddy soil.

Two Gram-staining-negative, facultative anaerobic, rod-shaped and phosphate-solubilizing strains designated SG2303T and SG2305, were isolated from paddy soil in China. Phylogenetic analysis based on 16 S rRNA gene sequences indicated that SG2303T and SG2305 represented a member of the genus Crenobacter within the family Neisseriaceae of the phylum Pseudomonadota. Strain SG2303T displayed higher 16 S rRNA gene sequence similarities with members of the genus Crenobacter ranging from 93.5 to 94.0%. Strains C. luteus YIM 78141T and C. cavernae K1W11S-77T were closest related to the isolated strains and were considered as type strains. Growth of strain SG2303T occurred at 10-55 °C (optimum 37 °C), pH 5.0-9.0 (optimum pH 6.0-7.0) and 0-1% (w/v) NaCl (optimum 0%). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain SG2303T and its closely related taxa were 76.1-78.2% and 20.5-22.1%, respectively. The genomic DNA G + C content was 62.2%. The quinone of strain SG2303T was Q-8. The major fatty acids (> 10%) of strain SG2303T were C16:0 (30.6%), summed feature 3 (C16:1ω7c and/or C16:1ω6c) (26.0%) and C12:0 3OH (12.1%). The polar lipids were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phospholipids (PL), glycolipid (GL) and unidentified lipids (UL). Based on the results of the phylogenetic, physiological, biochemical, and morphological analysis, strain SG2303T is recognized as a novel species of the genus Crenobacter, for which the name Crenobacter oryzisoli sp. nov. is proposed. The type strain is SG2303T (= GDMCC 1.3970T = JCM 36468T). In addition, SG2303T was also able of phosphorus solubilization and promoting the growth of rice seeds. Strain SG2303T exhibited a relatively high dissolvable phosphorus content of 2.52 µg·mL- 1.

Mesobacterium hydrothermale sp. nov., isolated from shallow-sea hydrothermal systems off Kueishantao Island.

A Gram-negative, rod-shaped, non-motile, aerobic bacterium, designated as strain TK19101T, was isolated from the intermediate seawater of yellow vent in the shallow-sea hydrothermal system located near Kueishantao Island. The strain was found to grow at 10-40 °C (optimum, 35 °C), at pH 6.0-8.0 (optimum, 7.0), and in 0-5% (w/v) NaCl (optimum, 1%). Strain TK19101T was catalase-positive and oxidase-positive. The predominant fatty acids (> 10%) in strain TK19101T cells were C16:0, summed feature 8 (C18:1 ω6c and/or C18:1 ω7c), and C18:0. The predominant isoprenoid quinone of strain TK19101T was ubiquinone-10. The polar lipids of strain TK19101T comprised phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phospholipid, and unknown polar lipid. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain TK19101T belonged to the genus Mesobacterium. Strain TK19101T exhibited highest 16S rRNA gene sequence similarity value to Mesobacterium pallidum MCCC M24557T (97.48%). The estimated average nucleotide identity and digital DNA-DNA hybridization values between strain TK19101T and the closest related species Mesobacterium pallidum MCCC M24557T were 74.88% and 20.30%, respectively. The DNA G + C content was 63.49 mol%. On the basis of the analysis of 16S rRNA gene sequences, genotypic and phylogenetic data, strain TK19101T has a unique phylogenetic status and represents a novel species of genus Mesobacterium, for which the name Mesobacterium hydrothermale sp. nov. is proposed. The type strain is TK19101T (= MCCC 1K08936T = KCTC 8354T).

Lacticaseibacillus parahuelsenbergensis sp. nov., Lacticaseibacillus styriensis sp. nov. and Lacticaseibacillus zeae subsp. silagei subsp. nov., isolated from different grass and corn silage.

Four rod-shaped, non-motile, non-spore-forming, facultative anaerobic, Gram-stain-positive lactic acid bacteria, designated as EB0058T, SCR0080, LD0937T and SCR0063T, were isolated from different corn and grass silage samples. The isolated strains were characterized using a polyphasic approach and EB0058T and SCR0080 were identified as Lacticaseibacillus zeae by 16S rRNA gene sequence analysis. Based on whole-genome sequence-based characterization, EB0058T and SCR0080 were separated into a distinct clade from Lacticaseibacillus zeae DSM 20178T, together with CECT9104 and UD2202, whose genomic sequences are available from NCBI GenBank. The average nucleotide identity (ANI) values within the new subgroup are 99.9 % and the digital DNA-DNA hybridization (dDDH) values are 99.3-99.9 %, respectively. In contrast, comparison of the new subgroup with publicly available genomic sequences of L. zeae strains, including the type strain DSM 20178T, revealed dDDH values of 70.2-72.5 % and ANI values of 96.2-96.6 %. Based on their chemotaxonomic, phenotypic and phylogenetic characteristics, EB0058T and SCR0080 represent a new subspecies of L. zeae. The name Lacticaseibacillus zeae subsp. silagei subsp. nov. is proposed with the type strain EB0058T (=DSM 116376T=NCIMB 15474T). According to the results of 16S rRNA gene sequencing, LD0937T and SCR0063T are members of the Lacticaseibacillus group. The dDDH value between the isolates LD0937T and SCR0063T was 67.6 %, which is below the species threshold of 70 %, clearly showing that these two isolates belong to different species. For both strains, whole genome-sequencing revealed that the closest relatives within the Lacticaseibacillus group were Lacticaseibacillus huelsenbergensis DSM 115425 (dDDH 66.5 and 65.9 %) and Lacticaseibacillus casei DSM 20011T (dDDH 64.1 and 64.9 %). Based on the genomic, chemotaxonomic and morphological data obtained in this study, two novel species, Lacticaseibacillus parahuelsenbergensis sp. nov. and Lacticaseibacillus styriensis sp. nov. are proposed and the type strains are LD0937T (=DSM 116105T=NCIMB 15471T) and SCR0063T (=DSM 116297T=NCIMB 15473T), respectively.

Investigation of potentially pathogenic Vibrionaceae in Saint-Louis city, Senegal.

Introduction: as cholera, due to toxigenic bacteria Vibrio cholera (serogroups O1 and O139), is a major public health threat in Africa, the aim of this work was to investigate potentially pathogenic Vibrionaceae bacteria firstly from human stool samples, and secondly from various environmental water points of Saint-Louis city in Senegal. Methods: a hospital-based study was conducted between 2013 and 2015. Stool samples were taken and cultured from daily incoming patients or hospitalized for acute diarrhea at Saint-Louis´ regional hospital. For environment, a monthly longitudinal sampling from January to October 2016 was carried out at 10 sites in the city. We used total DNA extracted from APW (alkaline peptone water) broth solutions and on suspect bacterial colonies to run PCR Multiplex targeting specific DNA fragments to detect Vibrio genus and specific species. In case of positivity, a simplex PCR was performed to test for cholera toxins Ctx, and V. parahaemolyticus TRH and TDH. Results: for 43 patients screened, bacterial culture was positive in 6% of cases but no strain of V. cholerae or other Vibrio sp. was isolated. PCR on 90 APW solutions were positive for Vibrio sp.(n = 43), V. cholera(n = 27), V. mimicus(n = 16), V. parahaemolyticus(8), V. alginolyticus(n = 4), and V. vulnificus(n = 2). Unlike for those on suspected colonies which were positive for a majority of V. parahaemolyticus (n = 40) and V. cholerae non-O1 / O139 (n = 35). Six strains of V. parahaemolyticus carried TRH gene, 3 of which expressed simultaneously virulence TRH and TDH genes. For physicochemical parameters, all temperatures varied similarly according to a unimodal seasonality, as well as salinity. Conclusion: despite the presence of natural populations of Vibrionaceae, even toxigenic ones, was noted in water environment, along with favorable habitat conditions that could play a role in transmission of Vibriosis in the Saint Louis population, we did not isolate any of them from patients screened at the hospital.

Elongatibacter sediminis gen. nov., sp. nov., isolated from intertidal sediment, and genomic comparison with all genera in the family Wenzhouxiangellaceae.

A novel slightly halophilic, aerobic, and Gram-stain-negative strain, designated as CH-27T, was isolated during a bacterial resource investigation of intertidal sediment collected from Xiaoshi Island in Weihai, PR China. Cells of strain CH-27T were rod-shaped with widths of 0.3-0.6 µm and lengths of 2.0-11.0 µm. Strain CH-27T grew optimally at 37 °C, pH 7.0 and with 2.0 % (w/v) NaCl. Catalase activity was weakly positive and oxidase activity was positive. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain CH-27T was most related to Marinihelvus fidelis KCTC 92639T (93.6 %), followed by Wenzhouxiangella marina MCCC 1K00261T (92.0 %). Based on genome comparisons between strain CH-27T and M. fidelis KCTC 92639T, the average amino acid identity was 63.6 % and the percentage of conserved proteins was 48.3 %. The major cellular fatty acid of strain CH-27T (≥10 %) was iso-C15 : 0 and the sole respiratory quinone was quinone-8. The polar lipids were phosphatidylglycerol, phosphatidylethanolamine, diphosphatidylglycerol, and aminophospholipid. The DNA G+C content was 62.7 mol%. Based on comprehensive analysis of its phylogenetic, physiological, biochemical, and chemotaxonomic characteristics, strain CH-27T represents a novel species in a novel genus, for which the name Elongatibacter sediminis gen. nov., sp.nov. is proposed. The type strain is CH-27T (=MCCC 1H00480T=KCTC 8011T).

Streptococcus raffinosi sp. nov., isolated from human breast milk samples.

Novel Gram-positive, catalase-negative, α-haemolytic cocci were isolated from breast milk samples of healthy mothers living in Hanoi, Vietnam. The 16S rRNA gene sequences of these strains varied by 0-2 nucleotide polymorphisms. The 16S rRNA gene sequence of one strain, designated as BME SL 6.1T, showed the highest similarity to those of Streptococcus salivarius NCTC 8618T (99.4 %), Streptococcus vestibularis ATCC 49124T (99.4 %), and Streptococcus thermophilus ATCC 19258T (99.3 %) in the salivarius group. Whole genome sequencing was performed on three selected strains. Phylogeny based on 631 core genes clustered the three strains into the salivarius group, and the strains were clearly distinct from the other species in this group. The average nucleotide identity (ANI) value of strain BME SL 6.1T exhibited the highest identity with S. salivarius NCTC 8618T (88.4 %), followed by S. vestibularis ATCC 49124T (88.3 %) and S. thermophilus ATCC 19258T (87.4 %). The ANI and digital DNA-DNA hybridization values between strain BME SL 6.1T and other species were below the cut-off value (95 and 70 %, respectively), indicating that it represents a novel species of the genus Streptococcus. The strains were able to produce α-galactosidase and acid from raffinose and melibiose. Therefore, we propose to assign the strains to a new species of the genus Streptococcus as Streptococcus raffinosi sp. nov. The type strain is BME SL 6.1T (=VTCC 12812T=NBRC 116368T).

Using clotted, pelleted blood samples for direct molecular detection of Bartonella spp. in small mammal wildlife surveillance studies.

OBJECTIVE: Bartonella are emerging bacterial zoonotic pathogens. Utilization of clotted blood samples for surveillance of these bacteria in wildlife has begun to supersede the use of tissues; however, the efficacy of these samples has not been fully investigated. Our objective was to compare the efficacy of spleen and blood samples for DNA extraction and direct detection of Bartonella spp. via qPCR. In addition, we present a protocol for improved DNA extraction from clotted, pelleted (i.e., centrifuged) blood samples obtained from wild small mammals. RESULTS: DNA concentrations from kit-extracted blood clot samples were low and A260/A280 absorbance ratios indicated high impurity. Kit-based DNA extraction of spleen samples was efficient and produced ample DNA concentrations of good quality. We developed an in-house extraction method for the blood clots which resulted in apposite DNA quality when compared to spleen samples extracted via MagMAX DNA Ultra 2.0 kit. We detected Bartonella in 9/30 (30.0%) kit-extracted spleen DNA samples and 11/30 (36.7%) in-house-extracted blood clot samples using PCR. Our results suggest that kit-based methods may be less suitable for DNA extraction from blood clots, and that blood clot samples may be superior to tissues for Bartonella detection.

Chitinophaga pollutisoli sp. nov., isolated from contaminated sediment.

A Gram-stain-negative, yellow-pigmented, and facultatively aerobic bacterium, designated strain GPA1T, was isolated from plastic waste landfill soil in the Republic of Korea. The cells were non-motile short rods exhibiting oxidase-negative and catalase-positive activities. Growth was observed at 15-40 °C (optimum, 30 °C), at pH 6.0-9.0 (optimum, pH 7.0-8.0) and in the presence of 0-2.5 % (w/v) NaCl (optimum, 0 %). Menaquinone-7 was the sole respiratory quinone, and iso-C15 : 0, C16 : 1 ω5c, and iso-C17 : 0 3-OH were the major cellular fatty acids (>10 % of the total fatty acids). Phosphatidylethanolamine was identified as a major polar lipid. Phylogenetic analyses based on 16S rRNA gene sequences and 120 concatenated marker protein sequences revealed that strain GPA1T formed a distinct lineage within the genus Chitinophaga. The genome of strain GPA1T was 6078 kb in size with 53.8 mol% G+C content. Strain GPA1T exhibited the highest similarity to Chitinophaga rhizosphaerae T16R-86T, with a 98.6 % 16S rRNA gene sequence similarity, but their average nucleotide identity and digital DNA-DNA hybridization values were 82.5 and 25.9 %, respectively. Based on its phenotypic, chemotaxonomic, and phylogenetic characteristics, strain GPA1T represents a novel species of the genus Chitinophaga, for which the name Chitinophaga pollutisoli sp. nov. is proposed. The type strain is GPA1T (=KACC 23415T=JCM 36644T).

Spartinivicinus poritis sp. nov., a red pigment-producing bacterium isolated from a scleractinian coral Porites lutea.

A Gram-stain-negative, red pigment-producing, aerobic, and rod-shaped bacterial strain (A2-2T) was isolated from a bleached scleractinian coral (Porites lutea). Strain A2-2T grew with 1.0-7.0 % (w/v) NaCl (optimum, 3.0 %), at pH 6.0-11.0 (optimum, pH 8.0), and at 18-41 °C (optimum, 35 °C). Results of phylogenetic analysis based on 16S rRNA gene sequences suggested that strain A2-2T fell within the genus Spartinivicinus and was closely related to Spartinivicinus ruber S2-4-1HT (98.1 % sequence similarity) and Spartinivicinus marinus SM1973T (98.0 % sequence similarity). The predominant cellular fatty acids of strain A2-2T were C16 : 0 (31.0 %), summed feature 3 (29.0 %), summed feature 8 (11.7 %), C12 : 0 3-OH (6.4 %), and C10 : 0 3-OH (5.5 %), while the major respiratory quinone was Q-9. The polar lipids mainly comprised phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and an unidentified phospholipid. The genome size of strain A2-2T was 6.8 Mb, with a G+C content of 40.2 mol%. The DNA-DNA hybridization value was 24.2 % between A2-2T and S. ruber S2-4-1HT and 36.9 % between A2-2T and S. marinus SM1973T, while the average nucleotide identity values were 80.1 and 88.8 %, respectively. Based on these findings, strain A2-2T could be recognized to represent a novel species of the genus Spartinivicinus, for which the name Spartinivicinus poritis sp. nov. is proposed. The type strain is A2-2T (=MCCC 1K08228T=KCTC 8323T).

Thioclava litoralis sp. nov., a novel species of alphaproteobacterium, isolated from surface seawater.

A Gram-negative, aerobic, rod-shaped, non-motile bacterium, designated as FTW29T, was isolated from surface seawater sampled in Futian district, Shenzhen, China. Growth of strain FTW29T was observed at 15-42 ℃ (optimum, 28-30 ℃), pH 4.0-9.0 (optimum, pH 5.5-7.5) and in the presence of 0.5-10% NaCl (optimum, 3.0% NaCl). Strain FTW29T showed 95.0-96.8% 16 S rRNA gene sequence similarity to various type strains of the genera Thioclava, Sinirhodobacter, Rhodobacter, Haematobacter and Frigidibacter of the family Paracoccaceae, and its most closely related strains were Thioclava pacifica DSM 10,166T (96.8%) and Thioclava marina 11.10-0-13T (96.7%). The phylogenomic tree constructed on the bac120 gene set showed that strain FTW29T formed a clade with the genus Thioclava, with a bootstrap value of 100%. The evolutionary distance values between FTW29T and type strains of the genus Thioclava were 0.17-0.19, which are below the recommended standard (0.21-0.23) for defining a novel genus in the family Paracoccaceae. In strain FTW29T, the major fatty acids identified were summed feature 8 (C18:1ω7c) and C16:0, and the predominant respiratory quinones were ubiquinone-10 and ubiquinone-9. The composition of polar lipids in strain FTW29T included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, an unidentified aminolipid, two unidentified glycolipids and an unidentified lipid. The genome of strain FTW29T comprised one circle chromosome and six plasmids, with a G + C content of 61.4%. The average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values between strain FTW29T and seven type strains of the genus Thioclava were 76.6-78.4%, 53.2-56.4% and 19.3-20.4%, respectively. Altogether, the phenotypic, phylogenetic and chemotaxonomic evidence illustrated in this study suggested that strain FTW29T represents a novel species of the genus Thioclava, with the proposed name Thioclava litoralis sp. nov. The type strain is FTW29T (= KCTC 82,841T = MCCC 1K08523T).

Pleionea litopenaei sp. nov., isolated from the gastric tract of juvenile Pacific white shrimp Litopenaeus vannamei.

A Gram-stain-negative, aerobic, rod-shaped and motile strain HL-JVS1T, was isolated from the gastric tract of a juvenile Pacific white shrimp. Molecular phylogenetic analysis based on 16S rRNA gene sequences of strain HL-JVS1T revealed its affiliation with the genus Pleionea, with close relatives including Pleionea mediterranea MOLA115T (97.5%) and Pleionea sediminis S1-5-21T (96.2%). The complete genome of strain HL-JVS1T consisted of a circular 4.4 Mb chromosome and two circular plasmids (6.6 and 35.0 kb) with a G + C content of 43.1%. The average nucleotide identity and digital DNA-DNA hybridization values between strain HL-JVS1T and the type strains of described Pleionea species were 69.7-70.4% and 18.3-18.6%, respectively. Strain HL-JVS1T grew at 10-40 °C (optimum, 30 °C) in the presence of 0.5 - 9.0% (w/v) sea salts (optimum, 2.0 - 2.5%), and at pH range of 5.5 - 10.0 (optimum, pH 6.5). The major fatty acids (> 10%) were summed feature 9 (iso-C17:1 ω9c and/or C16:0 10-methyl) (23.3%), iso-C16:0 (14.5%), iso-C11:0 3-OH (13.8%) and iso-C15:0 (11.0%). The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, an unidentified aminophospholipid, two unidentified aminolipids, and two unidentified lipids. The respiratory quinone was ubiquinone-8. The comprehensive phylogenetic, phylogenomic, phenotypic and chemotaxonomic results showed that strain HL-JVS1T is distinct from other Pleionea species. Hence, we propose strain HL-JVS1T as a novel species belonging to the genus Pleionea, for which the name Pleionea litopenaei sp. nov. is proposed with HL-JVS1T (= KCCM 90514T = JCM 36490T) as the type strain.

Polymerase Chain Reaction and Dot-Blot Hybridization for Leptospira Detection in Water Samples.

The dot-blot is a simple, fast, sensitive, and versatile technique that enables the identification of minimal quantities of DNA specifically targeted by probe hybridization in the presence of carrier DNA. It is based on the transfer of a known amount of DNA onto an inert solid support, such as a nylon membrane, utilizing the dot-blot apparatus and without electrophoretic separation. Nylon membranes have the advantage of high nucleic acid binding capacity (400 µg/cm2), high strength, and are positively or neutrally charged. The probe used is a highly specific ssDNA fragment of 18 to 20 bases long labeled with digoxigenin (DIG). The probe will conjugate with the Leptospira DNA. Once the probe has hybridized with the target DNA, it is detected by an anti-digoxigenin antibody, allowing its easy detection through its emissions revealed in an X-ray film. The dots with an emission will correspond to the DNA fragments of interest. This method employs the non-isotopic labeling of the probe, which may have a very long half-life. The drawback of this standard immuno-label is a lower sensitivity than isotopic probes. Nevertheless, it is mitigated by coupling polymerase chain reaction (PCR) and dot-blot assays. This approach enables the enrichment of the target sequence and its detection. Additionally, it may be used as a quantitative application when compared against a serial dilution of a well-known standard. A dot-blot application to detect Leptospira from the three main clades in water samples is presented here. This methodology can be applied to large amounts of water once they have been concentrated by centrifugation to provide evidence of the presence of Leptospiral DNA. This is a valuable and satisfactory tool for general screening purposes, and may be used for other non-culturable bacteria that may be present in water, enhancing the comprehension of the ecosystem.

Improved Diagnosis and Treatment Monitoring of Tuberculosis Using Stool and the Tuberculosis Bacterial Load Assay (TB-MBLA).

The diagnosis and monitoring of tuberculosis treatment is difficult as many patients are unable to produce sputum. This means that many patients are treated on the basis of clinical findings and consequently some will be exposed to anti-tuberculosis drugs unnecessarily. Moreover, for those appropriately on treatment and unable to produce a sputum sample, it will be impossible to monitor the response to treatment. We have shown that stool is a potential alternative sample type for diagnosis of tuberculosis. Currently, available protocols like the Xpert MTB/RIF use DNA as a target to detect Mycobacterium tuberculosis in stool but DNA survives long after the organism is dead so it is not certain whether a positive test is from an old or a partially treated infection. The TB MBLA only detects live organisms and thus, can be used to follow the response to treatment. In this chapter, we describe a protocol for TB-MBLA, an RNA-based assay, and apply it to quantify TB bacteria in stool.