Pseudonocardia lacus sp. nov., An Actinomycete Isolated from a Lake Sediment Sample.

A novel actinomycete strain, designated H11425T, was isolated from a sediment sample collected from Baihua Lake, Guizhou Province, PR China, and a polyphasic approach was employed to determine its taxonomic position. 16S rRNA gene sequence comparisons showed that strain H11425T is most closely related to Pseudonocardia sulfidoxydans JCM 10411T (97.9%) and Pseudonocardia kunmingensis JCM 32122T (97.8%). Both of phylogenetic analysis based on 16S rRNA gene sequence and phylogenomic analysis based on whole-genome sequence showed that strain H11425T formed a separate clade within the genus Pseudonocardia. The draft genome had a length of 8,059,576 bp with a G + C content of 74.5%. The average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values between strain H11425T and its closely related Pseudonocardia species were 76.8-79.0%, 64.8-69.9% and 21.7-23.3%, respectively, which were significantly lower than the widely accepted species-defined threshold. Strain H11425T contained meso-diaminopimelic acid, arabinose, galactose, glucose and ribose in its whole-cell hydrolysates. Mycolic acids were absent. The menaquinone was identifed as MK-8(H4). The phospholipid profile consisted of diphosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylcholine, an unknown phospholipid and four unidentified aminophospholipids. The major fatty acids were iso-C16:0, iso-C14:0, iso H-C16:1 and iso-C16:0 2OH. On the basis of the taxonomic evidence, strain H11425T represents a novel species of the genus Pseudonocardia, for which the name Pseudonocardia lacus sp. nov. is proposed. The type strain is H11425T (= JCM 34851T = CICC 25118T).

Nonomuraea sediminis sp. nov., a novel actinobacterium with antimicrobial activity, isolated from sediment of Dianchi Lake.

A novel actinobacterium with antimicrobial activity, designated strain H16431T, was isolated from a sediment sample collected from Dianchi Lake, Yunnan Province, PR China. Phylogenetic analysis based on 16S rRNA gene sequence indicated that strain H16431T was most closely related to Nonomuraea rhizosphaerae CGMCC 4.7431T and Nonomuraea guangzhouensis CGMCC 4.7101T (98.1% similarity), but formed a monophyletic clade with Nonomuraea ceibae KCTC 39826T (98.0% similarity). Phylogenomic analysis based on whole-genome sequence showed that strain H16431T formed a separate clade within the genus Nonomuraea. The average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values between strain H16431T and its closely related Nonomuraea species were 80.0-81.5%, 71.2-74.6%, and 23.2-25.0%, respectively, which were significantly lower than the widely accepted species-defined threshold. The DNA G + C content was 70.2% based on the whole-genome sequence. The menaquinones were identified as MK-9(H4), MK-9(H6), and MK-9(H2). The major fatty acids were iso-C16:0, 10 methyl-C17:0, and iso-C16:0 2OH. The phospholipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, and phosphatidylinositol. These chemotaxonomic characteristics were corresponded to those of the genus Nonomuraea. On the basis of the taxonomic evidence, strain H16431T represents a novel species of the genus Nonomuraea, for which the name Nonomuraea sediminis sp. nov. is proposed. The type strain is H16431T (=JCM 34852T=CICC 25119T).

Sphaerisporangium fuscum sp. nov., Isolated from Sediment of Anmucuo Lake in Tibet Autonomous Region of China.

A novel actinomycete strain, designated H8589T, was isolated from a lake sediment sample, and a polyphasic approach was employed to determine its taxonomic position. Phylogenetic analysis based on 16S rRNA gene indicated that strain H8589T formed a monophyletic clade within the genus Sphaerisporangium and was most closely related to Sphaerisporangium siamense DSM 45784 T (97.9% similarity) and Sphaerisporangium rufum DSM 46862 T (97.7% similarity). The draft genome had a length of 10,134,050 bp with a G + C content of 71.2%. The average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization values between strain H8589T and its closely related Sphaerisporangium species were 80.6 ~ 83.2%, 73.9 ~ 78.4% and 24.5 ~ 29.0%, respectively, which were significantly lower than the widely accepted species-defined threshold. The diagnostic diamino acid of the peptidoglycan was meso-diaminopimelic acid. Whole-cell sugars were glucose, ribose and madurose. The menaquinones were MK-9(H4), MK-9(H2), MK-9(H6) and MK-9. The phospholipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, unidentified phospholipids and unidentified aminophospholipids. The major fatty acids were identified as iso-C16:0, 10-methyl-C17:0 and C17:0. The results of phenotypic properties, genotypic distinctiveness and chemotaxonomic features indicated that strain H8589T should represent a novel species within the genus Sphaerisporangium, Sphaerisporangium fuscum sp.nov. The type strain is H8589T (= JCM 34848 T = CICC 25115 T).

Acrocarpospora catenulata sp. nov., a novel actinobacterium isolated from lake sediment.

A novel actinobacterium, designated strain H8750T, was isolated from sediment sampled at Lugu Lake, southwest PR China and its polyphasic taxonomy was studied. Strain H8750T produced well-developed substrate mycelium, and formed club-shaped and hooked structures borne on the tip of the aerial mycelia. It contained meso-diaminopimelic, glucose, ribose and madurose in whole-cell hydrolysates. The predominant menaquinones were MK-9(H4), MK-9(H2) and MK-9(H6). The phospholipid profile contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, hydroxy-phosphatidylethanolamine, unidentified phospholipids and unidentified aminophospholipids. The major fatty acid (>10 %) were cis 9 C17 : 1, iso-C16 : 0 and C15 : 0. The DNA G+C content was 69.7 mol% based on the whole genome sequence. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences indicated that strain H8750T was closely related to Acrocarpospora macrocephala JCM 10982T (98.0 %), Acrocarpospora pleiomorpha JCM 10983T (97.9 %) and Acrocarpospora phusangensis DSM 45867 T (97.8 %) and formed a monophyletic clade within the genus Acrocarpospora in the phylogenetic trees. The average nucleotide identity and digital DNA-DNA hybridization values between strain H8750T and its closely related Acrocarpospora species were 79.8~87.2 % and 25.9~28.0 %, respectively, which showed that it belonged to a distinct species. Furthermore, the morphological and phenotypic characteristics allowed the isolate to be differentiated from its closely related species. Therefore, it is concluded that strain H8750T can be classified as representing a novel species of the genus Acrocarpospora, for which the name Acrocarpospora catenulata sp. nov. is proposed. The type strain is H8750T (=JCM 34849T=CICC 25116T). Moreover, based on the gene prediction results, strain H8750T may have the genetic potential to synthesize many new secondary metabolites, which further increases its bioactive value.

A novel antibiotic combination of linezolid and polymyxin B octapeptide PBOP against clinical Pseudomonas aeruginosa strains.

BACKGROUND: Antibiotic-resistant Gram-negative bacteria are becoming a major public health threat such as the important opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa). The present study investigated enhancement of the linezolid spectrum, which is normally used to treat Gram-positive bacteria, at inhibiting P. aeruginosa growth. METHODS: The checkerboard test or time-kill assay were carried out to determine the antibacterial effects of linezolid in cooperation with polymyxin B octapeptide PBOP (LP) against P. aeruginosa based on in vitro model. The protective effect of LP against P. aeruginosa infection was assessed based on a Caenorhabditis elegans (C. elegans) model. RESULTS: The synergistic activity and antibacterial effects were significantly increased against P. aeruginosa by LP treatment, while linezolid and PBOP as monotherapies exhibited no remarkably bactericidal activity against the clinical strains. Additionally, LP treatment modified biofilm production, morphology, swimming motility of P. aeruginosa, and protected C. elegans from P. aeruginosa infection. CONCLUSIONS: This research demonstrates that LP combination has significant synergistic activity against P. aeruginosa, and PBOP is potential to be an activity enhancer. Notably, this strategy improved the antibacterial activity spectrum of linezolid and other anti-Gram-positive agents and represents an effective choice to surmount the antibiotic resistance of bacteria in the long term.

Inhibition of Virulence Factors and Biofilm Formation by Wogonin Attenuates Pathogenicity of Pseudomonas aeruginosa PAO1 via Targeting pqs Quorum-Sensing System.

Pseudomonas aeruginosa, an important opportunistic pathogen, is capable of producing various virulence factors and forming biofilm that are regulated by quorum sensing (QS). It is known that targeting virulence factor production and biofilm formation instead of exerting selective pressure on growth such as conventional antibiotics can reduce multidrug resistance in bacteria. Therefore, many quorum-sensing inhibitors (QSIs) have been developed to prevent or treat this bacterial infection. In this study, wogonin, as an active ingredient from Agrimonia pilosa, was found to be able to inhibit QS system of P. aeruginosa PAO1. Wogonin downregulated the expression of QS-related genes and reduced the production of many virulence factors, such as elastase, pyocyanin, and proteolytic enzyme. In addition, wogonin decreased the extracellular polysaccharide synthesis and inhibited twitching, swimming, and swarming motilities and biofilm formation. The attenuation of pathogenicity in P. aeruginosa PAO1 by wogonin application was further validated in vivo by cabbage infection and fruit fly and nematode survival experiments. Further molecular docking analysis, pathogenicity examination of various QS-related mutants, and PQS signal molecule detection revealed that wogonin could interfere with PQS signal molecular synthesis by affecting pqsA and pqsR. Taken together, the results indicated that wogonin might be used as an anti-QS candidate drug to attenuate the infection caused by P. aeruginosa.

Genetic and Functional Diversity of Pseudomonas aeruginosa in Patients With Chronic Obstructive Pulmonary Disease.

Pseudomonas aeruginosa is the most relevant pathogen to the severe exacerbations of patients with chronic obstructive pulmonary disease (COPD). However, the genetic and functional characteristics of P. aeruginosa isolates from COPD airways still remain less understood. In this study, the genetic, phylogenetic, phenotypic, and transcriptional features of P. aeruginosa isolates from COPD sputa were comprehensively explored by susceptibility testing, comparative-genomic analysis, phylogenetic analysis, phenotypic profiling, and comparative-transcriptomic analysis. We found that P. aeruginosa was prevalent in elder COPD patients and highly resisted to many commonly used antibiotics. P. aeruginosa COPD isolates harbored a substantial number of variant sites that might influence the primary metabolism and substance transport system. These isolates were discretely distributed in the phylogenetic tree and clustered with internationally collected P. aeruginosa in two major groups, and could be classified into three groups according to their differences in virulence-related phenotypes. Furthermore, the transcriptional patterns of COPD isolates could be classified into PAO1-like group with reduced protein secretion and motility and PAO1-distinct group with decreased substance transport but enhanced primary metabolism. In conclusion, this study demonstrates that P. aeruginosa isolates from COPD patients have abundant genetic and phenotypic diversity, and provides an important reference for further exploring the survival strategy of P. aeruginosa in COPD airways and the development of anti-pseudomonal therapy.

Catellatospora sichuanensis sp. nov., a novel actinobacterium isolated from soil.

A novel actinobacterium, designated strain H14505T, was isolated from a soil sample collected in Hong Yuan, Sichuan, southwest PR China. The temperature, pH and NaCl ranges for growth were determined to be 15-35 °C (optimum, 28 °C), 6.0-8.0 (optimum, pH 7.0) and 0-2 % (w/v; optimum without NaCl), respectively. The polar lipdis detected for strain H14505T were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, glycolipid and four unidentified lipids. The predominant menaquinones of strain H14505T were MK-9(H4) and MK-9(H6), and the prevalent fatty acids (>10 %) were C18 : 1 ω9c, C17 : 1 ω8c, summed feature 5 (anteiso-C18 : 0/ C18 : 2 ω6,9c) and C16 : 0. Phylogenetic analysis based on 16S rRNA gene and whole-genome sequences indicated that strain H14505T showed high similarity to Catellatospora vulcania NEAU-JM1T (99.0 %) and Catellatospora paridis NEAU-CL2T (99.0 %), and formed a monophyletic clade within the the genus Catellatospora in the phylogenetic trees. However, the average nucleotide indentity and DNA-DNA hybridization values between strain H14505T and closely related Catellatospora species showed that it belonged to a distinct species. Furthermore, the results of morphological, physiological and biochemical tests allowed further phenotypic differentiation of strain H14505T from its closest relatives. Thus, it is proposed that strain H14505T represents a novel species of the genus Catellatospora, for which the name Catellatospora sichuanensis sp. nov. is proposed. The type strain of Catellatospora sichuanensis is H14505T (=JCM 32394T=CICC 11042T).

Characterization and Genomic Analysis of ValSw3-3, a New Siphoviridae Bacteriophage Infecting Vibrio alginolyticus.

A novel lytic bacteriophage, ValSw3-3, which efficiently infects pathogenic strains of Vibrio alginolyticus, was isolated from sewage water and characterized by microbiological and in silico genomic analyses. Transmission electron microscopy indicated that ValSw3-3 has the morphology of siphoviruses. This phage can infect four species in the Vibrio genus and has a latent period of 15 min and a burst size of 95 ± 2 PFU/infected bacterium. Genome sequencing results show that ValSw3-3 has a 39,846-bp double-stranded DNA genome with a GC content of 43.1%. The similarity between the genome sequences of ValSw3-3 and those of other phages recorded in the GenBank database was below 50% (42%), suggesting that ValSw3-3 significantly differs from previously reported phages at the DNA level. Multiple genome comparisons and phylogenetic analysis based on the major capsid protein revealed that phage ValSw3-3 is grouped in a clade with five other phages, including Listonella phage phiHSIC (GenBank accession no. NC_006953.1), Vibrio phage P23 (MK097141.1), Vibrio phage pYD8-B (NC_021561.1), Vibrio phage 2E1 (KX507045.1), and Vibrio phage 12G5 (HQ632860.1), and is distinct from all known genera within the Siphoviridae family that have been ratified by the International Committee on Taxonomy of Viruses (ICTV). An in silico proteomic comparison of diverse phages from the Siphoviridae family supported this clustering result and suggested that ValSw3-3, phiHSIC, P23, pYD8-B, 2E1, and 12G5 should be classified as a novel genus cluster of Siphoviridae A subsequent analysis of core genes also revealed the common genes shared within this new cluster. Overall, these results provide a characterization of Vibrio phage ValSw3-3 and support our proposal of a new viral genus within the family SiphoviridaeIMPORTANCE Phage therapy has been considered a potential alternative to antibiotic therapy in treating bacterial infections. For controlling the vibriosis-causing pathogen Vibrio alginolyticus, well-documented phage candidates are still lacking. Here, we characterize a novel lytic Vibrio phage, ValSw3-3, based on its morphology, host range and infectivity, growth characteristics, stability under various conditions, and genomic features. Our results show that ValSw3-3 could be a potent candidate for phage therapy to treat V. alginolyticus infections due to its stronger infectivity and better pH and thermal stability than those of previously reported Vibrio phages. Moreover, genome sequence alignments, phylogenetic analysis, in silico proteomic comparison, and core gene analysis all support that this novel phage, ValSw3-3, and five unclassified phages form a clade distant from those of other known genera ratified by the ICTV. Thus, we propose a new viral genus within the Siphoviridae family to accommodate this clade, with ValSw3-3 as a representative member.

Two-step biological approach for treatment of rapeseed meal.

Rapeseed meal (RSM) is an important source of protein, but its value is limited due to the poor digestibility and the presence of many antinutritional factors. In this study, a two-step biological method was developed for detoxifying RSM and increasing its protein value. In the first stage, various detoxifying enzymes and proteases were produced by Aspergillus niger during solid-state fermentation (SSF). In the second stage, coordinated enzymatic hydrolysis was employed to further degrade the antinutritional factors and macromolecular proteins in the fermented RSM. Following fermentation at 30 °C for 48 hr and enzymatic hydrolysis at 45 °C for 24 hr, the content of trichloroacetic acid soluble protein (TCA-SP) and glucosinolates (GLS) in RSM was increased by 81.70% and reduced by 30.06%, respectively, compared with that obtained using the SSF process alone. Moreover, to improve the efficiency of enzymatic hydrolysis, the yield of acid protease was increased by optimizing the composition of the medium so that the TCA-SP content was increased to 208.39 mg/g and accounted for 51.62% of the total RSM protein, which was 99.36% and 629.66% higher than that in the fermented RSM and control, respectively. Overall, these results demonstrate that the two-step process could be more effective for the degradation of the antinutritional factors and improvement of the protein quality of RSM compared to use of the SSF method alone, which may improve the utilization of RSM in food and animal feed. PRACTICAL APPLICATION: Rapeseed meal (RSM) is a protein source that provides high-quality nutrition and can be applied to the development of value-added products for humans and animal feed. To improve the utilization of RSM, a combined method of solid-state fermentation and enzymatic digestion was developed. Compared with the traditional solid-state fermentation method, the present method further improves the quality of RSM and demonstrates improved efficacy in increasing the small peptide content while reducing the levels of antinutritional factors.

Analysis of the bacterial community in aged and aging pit mud of Chinese Luzhou-flavour liquor by combined PCR-DGGE and quantitative PCR assay.

BACKGROUND: The community structure of bacteria in aged and aging pit mud, which was judged according to their sensory and physicochemical characteristics, was analysed using polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative real-time PCR (qPCR). RESULTS: The phyla Firmicutes, Actinobacteria, Proteobacteria, Synergistetes and Unclassified Bacteria were detected and the fermentative Firmicutes was predominant in both types of pit mud in the PCR-DGGE analysis. Among Firmicutes, Clostridiales was dominant in aged pit mud while Bacillales and Lactobacillales were dominant in aging pit mud. The diversity of bacterial communities in aged pit mud was higher than that in aging pit mud. In the qPCR analysis the abundance of Clostridium IV in aged pit mud was higher than that in aging pit mud and there were significant differences in the quantity of Clostridium IV between aged and aging pit mud of the same cellar (P < 0.05). CONCLUSION: There were some significant differences in the microbial community structure between aged and aging pit mud. The differences in the quantity of Clostridium IV might be involved in the distinction that the aged pit mud has a strong aroma while the aging pit mud does not.

Clostridium swellfunianum sp. nov., a novel anaerobic bacterium isolated from the pit mud of Chinese Luzhou-flavor liquor production.

A novel Gram-positive, strictly anaerobic, spore-forming, rod-shaped bacterium, designated strain S11-3-10(T), was isolated from the pit mud used for Chinese Luzhou-flavor liquor production. Phylogenetic analysis based on 16S rRNA gene sequencing revealed that the strain formed a monophyletic clade with the closely related type strains of Clostridium cluster I and was most closely related to Clostridium amylolyticum JCM 14823(T) (94.38%). The temperature, pH, and NaCl range for growth was determined to be 20-45 °C (optimum 37 °C), 4.0-10.0 (optimum pH 7.3), and 0-3.0% (w/v), respectively. The strain was able to tolerate up to 7.5 % (v/v) ethanol. Yeast extract or peptone was found to be required for growth. Acids were found to be produced from glucose, mannose and trehalose. The major end products from glucose fermentation were identified as ethanol, acetate and hydrogen. The polar lipids were found to consist of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and unidentified phospholipids and polar lipids. The major fatty acids (>5%) were identified as iso-C(15:0), C(16:0), C(16:0)dma, C(14:0), anteiso-C(15:0) and iso-C(13:0). No respiratory quinone was detected. The diamino acid in the cell wall peptidoglycan was identified as meso-diaminopimelic acid and the whole-cell sugars were found to include galactose and glucose as major components. The DNA G+C content was determined to be 36.4 mol%. Based on the phylogenetic, chemotaxonomic and phenotypic evidence, the isolate is considered to represent a novel species of the genus Clostridium for which the name Clostridium swellfunianum sp. nov. is proposed. The type strain is S11-3-10(T) (=DSM 27788(T) = JCM 19606(T) = CICC 10730(T)).