Colorimetric platform based on synergistic effect between bacteriophage and AuPt nanozyme for determination of Yersinia pseudotuberculosis.

The development of a novel colorimetric method is reported, using vB_YepM_ZN18 phages along with AuPt nanozyme for the sensitive detection of Y. pseudotuberculosis. The phage used in this work has been extracted from hospital sewer water and is highly specific toward Y. pseudotuberculosis. The synthesized AuPt NPs possess peroxidase-like activity, which is suitable in the development of nanozyme based detection system. Furthermore, phages@MB and AuPt@phages are added into the bacterial samples for co-incubation, forming an intercalated complex. The magnetic separation and absorbance analysis of enzymatic reaction are carried out for the detection of targeted bacteria. The proposed method has a limit of detection of 14 CFU/mL, a wide linear range from 2.50 × 101 ~ 2.50 × 107 CFU/mL and the assay completion time is 40 min. Benefitting from the outperformance of this sensor, we have successfully employed the developed sensing platform for the detection of Y. pseudotuberculosis in food industry and hospital specimens.

Poly(indole-5-carboxylic acid)/reduced graphene oxide/gold nanoparticles/phage-based electrochemical biosensor for highly specific detection of Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis is an enteric bacterium causing yersiniosis in humans. The existing Yersinia pseudotuberculosis detection methods are time-consuming, requiring a sample pretreatment step, and are unable to discriminate live/dead cells. The current work reports a phage-based electrochemical biosensor for rapid and specific detection of Yersinia pseudotuberculosis. The conductive poly(indole-5-carboxylic acid), reduced graphene oxide, and gold nanoparticles are applied for surface modification of the electrode. They possess ultra-high redox stability and retain 97.7% of current response after performing 50 consecutive cycles of cyclic voltammetry.The specific bacteriophages vB_YepM_ZN18 we isolated from hospital sewage water were immobilized on modified electrodes by Au-NH2 bond between gold nanoparticles and phages. The biosensor fabricated with nanomaterials and phages were utilized to detect Yersinia pseudotuberculosis successfully with detection range of 5.30 × 102 to 1.05 × 107 CFU mL-1, detection limit of 3 CFU mL-1, and assay time of 35 min. Moreover, the biosensor can specifically detect live Yersinia pseudotuberculosis without responding to phage-non-host bacteria and dead Yersinia pseudotuberculosis cells. These results suggest that the proposed biosensor is a promising tool for the rapid and selective detection of Yersinia pseudotuberculosis in food, water, and clinical samples.

Complete genome sequence of the novel phage vB_BthS-HD29phi infecting Bacillus thuringiensis.

The phage vB_BthS-HD29phi infecting Bacillus thuringiensis strain HD29 was isolated and purified. The morphology of the phage showed that it belongs to the family Siphoviridae. The phage genome was 32,181 bp in length, comprised linear double-stranded DNA with an average G + C content of 34.9%, and exhibited low similarity to known phage genomes. Genomic and phylogenetic analysis revealed that vB_BthS-HD29phi is a novel phage. In total, 50 putative ORFs were predicted in the phage genome, and only 18 ORFs encoded proteins with known functions. This article reports the genome sequence of a new tailed phage and increases the known genetic diversity of tailed phages.

Pseudorhodobacter collinsensis sp. nov., isolated from a till sample of an icecap front.

A Gram-stain-negative, rod-shaped, aerobic and non-motile strain, designated 4-T-34T, was isolated from a till sample of Collins icecap front, Antarctica, and its taxonomic position was investigated by genotypic, phenotypic and chemotaxonomic analysis. The isolate grew at 4-30 °C (optimum 20-25 °C), at pH 6.0-10.0 and with 0-1.0 % (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 4-T-34T belonged to the genus Pseudorhodobacter, with the closest relatives being Pseudorhodobacter wandonensis WT-MW11T (96.9 % 16S rRNA gene sequence similarity), Pseudorhodobacter antarcticus ZS3-33T (96.8 %), Pseudorhodobacter ferrugineus IAM 12616T (96.5 %) and Pseudorhodobacter aquimaris HDW-19T (95.4 %). Strain 4-T-34T contained Q-10 as the only ubiquinone and C18 : 1ω7c as the major fatty acid. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, two unidentified aminophospholipids and two unidentified phospholipids. The DNA G+C content of strain 4-T-34T was 61 mol%. On the basis of phylogenetic, physiological and chemotaxonomic data, strain 4-T-34T is considered to represent a novel species of the genus Pseudorhodobacter, for which the name Pseudorhodobacter collinsensis sp. nov. is proposed. The type strain is 4-T-34T ( = CCTCC AB 2014005T = LMG 28256T).

Flavobacterium collinsense sp. nov., isolated from a till sample of an Antarctic glacier.

A novel rod-shaped, Gram-stain-negative, non-gliding and aerobic strain surrounded by a multilayer capsule, designated 4-T-2T, was isolated from a till sample of Collins glacier front, Antarctica. The bacterium formed yellow, circular, convex and smooth colonies. Growth occurred at 4-28 °C (optimum18-20 °C), at pH 7.0-10.0 (optimum pH 9.0) and with 0-1 % NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 4-T-2T belonged to the genus Flavobacterium. Strain 4-T-2T shared the highest 16S rRNA gene sequence similarity with the type strain of Flavobacterium algicola (96.7 %). The DNA G+C content of strain 4-T-2T was 36.2 mol%. The only menaquinone was MK-6. The major cellular fatty acids were iso-C15 : 0, iso-C15 : 1 G, iso-C16 : 0 and summed feature 9 (comprising iso-C17 : 1ω9c and/or 10-methyl C16 : 0). Polar lipid profile consisted phosphatidylethanolamine, one unidentified aminolipid, one unidentified aminophospholipid and one unidentified lipid. On the basis of phylogenetic, physiological and chemotaxonomic data, strain 4-T-2T is considered to represent a novel species of the genus Flavobacterium, for which the name Flavobacterium collinsense sp. nov. is proposed. The type strain is 4-T-2T ( = CCTCC AB 2014004T = LMG 28257T).

Roseomonas arctica sp. nov., isolated from arctic glacial foreland soil.

A novel psychrotolerant bacterium, designed strain M6-79T, was isolated from an arctic glacial foreland soil sample collected from Ny-Ålesund in the Svalbard Archipelago, Norway. Cells of strain M6-79T were Gram-stain-negative, rod-shaped and produced a red-pigment. Strain M6-79T was strictly aerobic, non-motile, non-endospore-forming, oxidase-negative and catalase-positive. Based on 16S rRNA gene sequence analysis, strain M6-79T was phylogenetically related to Roseomonas aquatica TR53T (95.2 % 16S rRNA gene sequence similarity), Roseomonas lacus TH-G33T (94.3 %), 'Roseomonas sediminicola' FW-3 (94.3 %), Roseomonas terrae DS-48T (94.1 %) and Roseomonas soli 5N26T (94.1 %). The unique isoprenoid quinone detected in strain M6-79T was Q-9. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, one unknown aminolipid and one unknown lipid. Strain M6-79T possessed C18 : 1ω7c, C16 : 0 and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) as the predominant fatty acids, and the DNA G+C content was 71.2 mol%. Combined data from phylogenetic, phenotypic and chemotaxonomic studies revealed that strain M6-79T represents a novel species of the genus Roseomonas, for which the name Roseomonas arctica sp. nov. is proposed. The type strain is strain M6-79T ( = CCTCC AB 2013101T = LMG 28251T).

Nocardioides antarcticus sp. nov., isolated from marine sediment.

Strain M-SA3-94T, an aerobic, Gram-stain-positive, ovoid- to rod-shaped, non-motile bacterium, was isolated from the marine sediment of Ardley cove, King George Island, Antarctica. Strain M-SA3-94T grew optimally at pH 5.0-6.0, 20 °C and in the presence of 1.0 % (w/v) NaCl. Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that strain M-SA3-94T belonged to the genus Nocardioides in the family Nocardioidaceae, clustering with Nocardioides plantarum NCIMB 12834T, Nocardioides ginsengagri BX5-10T, Nocardioides marinquilinus CL-GY44T and Nocardioides lianchengensis D94-1T (with 96.1 %, 95.9 %, 94.5 % and 94.7 % 16S rRNA gene sequence similarity, respectively). The chemotaxonomic properties of strain M-SA3-94T were similar to those of members of the genus Nocardioides with validly published names. The major fatty acid was iso-C16 : 0. The polar lipid pattern contained diphosphatidylglycerol, phosphatidylglycerol and three unknown phospholipids. The diagnostic diamino acid in the cell-wall peptidoglycan was ll-2, 6-diaminopimelic acid. MK-8(H4) was the predominant menaquinone and the DNA G+C content of this strain was 66.7 mol%. On the basis of these phenotypic, phylogenetic and chemotaxonomic data, strain M-SA3-94T represents a novel species of the genus Nocardioides, for which the name Nocardioides antarcticus sp. nov. is proposed. The type strain is M-SA3-94T ( = CCTCC AB2014053T = LMG 28254T).

Pedobacter ardleyensis sp. nov., isolated from soil in Antarctica.

A red-pigmented, non-motile, Gram-stain-negative, rod-shaped bacterium, strain R2-28T, was isolated from a soil sample collected from Ardley Island, South Shetland Islands, Antarctica, and was characterized taxonomically by using a polyphasic approach. The organism grew optimally at 18 °C in TSB. On the basis of phylogenetic analyses of the 16S rRNA gene sequence, strain R2-28T was most closely related to the genus Pedobacter of the family Sphingobacteriaceae. The highest levels of 16S rRNA gene sequence similarity were found with respect to Pedobacter alluvionis NWER-II11T (95.6 %) and Pedobacter terrae DS-57T (95.2 %). The DNA G+C content was 39.9 mol%, and MK-7 was the only respiratory quinone. The major polar lipids were phosphatidylethanolamine and a sphingolipid. The predominant cellular fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c). These chemotaxonomic and phylogenetic data supported the allocation of strain R2-28T to the genus Pedobacter. Additionally, the results of physiological and biochemical tests allowed phenotypic differentiation of strain R2-28T from species of the genus Pedobacter with validly published names. Therefore, strain R2-28T represents a novel species within the genus Pedobacter, for which the name Pedobacter ardleyensis sp. nov. is proposed. The type strain is R2-28T ( = CCTCC AB 2013365T = LMG 28255T).

Roseovarius antarcticus sp. nov., isolated from a decayed whale bone.

A pale yellow, ovoid- to rod-shaped and budding bacterium, designated strain M-S13-148(T), was isolated from a decayed bone of whale from the eastern coast of King George Island, South Shetlands, Antarctica. Strain M-S13-148(T) exhibited motility, aerobic growth and was Gram-stain-negative. Strain M-S13-148(T) was positive for catalase and oxidase. Growth was observed at pH 6.0-9.0, at 4-42 °C and with 0-14% (w/v) NaCl. The novel strain contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine and an unknown phospholipid as the major polar lipids. The dominant cellular fatty acids were summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c), (58.8%) and C16 : 0 (11.7%). The respiratory quinone was Q-10 and the DNA G + C content was 60.9 mol%. Neighbour-joining, maximum-likelihood and minimum-evolution phylogenetic trees, based on 16S rRNA gene sequences, indicated that strain M-S13-148(T) belonged to the genus Roseovarius and was most closely related to Roseovarius nanhaiticus CCTCC AB 208317(T) (93.72% 16S rRNA gene sequence similarity). The 16S rRNA gene sequence similarity with respect to members of the genus Roseovarius ranged from 91.81 to 93.94%. On the basis of phenotypic, molecular and chemotaxonomic characteristics, strain M-S13-148 is considered to represent a novel species of the genus Roseovarius, for which the name Roseovarius antarcticus sp. nov., is proposed. The type strain is M-S13-148(T) ( = CCTCC AB2014072(T) = LMG 28420(T)).

Risungbinella pyongyangensis gen. nov., sp. nov., a mesophilic member of the family Thermoactinomycetaceae isolated from an agricultural soil sample.

A mesophilic strain, designed MC 210T, was isolated from an agricultural soil sample from Pyongyang, Democratic People's Republic of Korea, and its taxonomic position was investigated by using a polyphasic approach. The novel strain grew well on PYI medium, and no diffusible pigments were produced. The optimum temperature for growth was 37 °C. The aerial mycelium was well developed, but not fragmented. The strain was Gram-reaction-positive and non-motile and formed endospores on the aerial mycelium. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain MC 210T belongs to the family Thermoactinomycetaceae. Strain MC 210T showed 16S rRNA gene sequence similarities of 92.90 and 92.54% to the type strains of Geothermomicrobium terrae and Shimazuella kribbensis, respectively. The cell wall of strain MC 210T contained meso-diaminopimelic acid, glutamic acid and alanine as the diagnostic amino acids, and whole-cell hydrolysates contained glucose, arabinose and galactose. Strain MC 210T contained anteiso-C13 : 0, iso-C14 : 0, C14 : 0, anteiso-C15 : 0, C16 : 0 and iso-C13 : 0 as the major cellular fatty acids. The main polar lipids were phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, two unknown aminophospholipids, an unknown aminolipid, three unknown phospholipids and five unknown polar lipids. The predominant menaquinone was MK-7.The G+C content of the genomic DNA was 42.1 mol%. On the basis of our phylogenetic, physiological and chemotaxonomic data, strain MC 210T is considered to represent a novel genus and species, for which we propose the name Risungbinella pyongyangensis gen. nov., sp. nov., in the family Thermoactinomycetaceae. The type strain of Risungbinella pyongyangensis is MC 210T (CCTCC AA 2013021T = NRRL B-59118T).

Genomic, Antimicrobial, and Aphicidal Traits of Bacillus velezensis ATR2, and Its Biocontrol Potential against Ginger Rhizome Rot Disease Caused by Bacillus pumilus.

Ginger rhizome rot disease, caused by the pathogen Bacilluspumilus GR8, could result in severe rot of ginger rhizomes and heavily threaten ginger production. In this study, we identified and characterized a new Bacillus velezensis strain, designated ATR2. Genome analysis revealed B. velezensis ATR2 harbored a series of genes closely related to promoting plant growth and triggering plant immunity. Meanwhile, ten gene clusters involved in the biosynthesis of various secondary metabolites (surfactin, bacillomycin, fengycin, bacillibactin, bacilysin, difficidin, macrolactin, bacillaene, plantazolicin, and amylocyclicin) and two clusters encoding a putative lipopeptide and a putative phosphonate which might be explored as novel bioactive compounds were also present in the ATR2 genome. Moreover, B. velezensis ATR2 showed excellent antagonistic activities against multiple plant pathogenic bacteria, plant pathogenic fungi, human pathogenic bacteria, and human pathogenic fungus. B. velezensis ATR2 was also efficacious in control of aphids. The antagonistic compound from B. velezensis ATR2 against B.pumilus GR8 was purified and identified as bacillomycin D. In addition, B. velezensis ATR2 exhibited excellent biocontrol efficacy against ginger rhizome rot disease on ginger slices. These findings showed the potential of further applications of B. velezensis ATR2 as a biocontrol agent in agricultural diseases and pests management.

Genomic Analysis of a Novel Phage Infecting the Turkey Pathogen Escherichia coli APEC O78 and Its Endolysin Activity.

Due to the increasing spread of multidrug-resistant (MDR) bacteria, phage therapy is considered one of the most promising methods for addressing MDR bacteria. Escherichia coli lives symbiotically in the intestines of humans and some animals, and most strains are beneficial in terms of maintaining a healthy digestive tract. However, some E. coli strains can cause serious zoonotic diseases, including diarrhea, pneumonia, urinary tract infections, and hemolytic uremic syndrome. In this study, we characterized a newly isolated Myoviridae phage, vB_EcoM_APEC. The phage vB_EcoM_APEC was able to infect E. coli APEC O78, which is the most common MDR E. coli serotype in turkeys. Additionally, the phage's host range included Klebsiella pneumoniae and other E. coli strains. The genome of phage vB_EcoM_APEC (GenBank accession number MT664721) was 35,832 bp in length, with 52 putative open reading frames (ORFs) and a GC content of 41.3%. The genome of vB_EcoM_APEC exhibited low similarity (79.1% identity and 4.0% coverage) to the genome of Acinetobacter phage vB_AbaM_IME284 (GenBank no. MH853787.1) according to the nucleotide Basic Local Alignment Search Tool (BLASTn). Phylogenetic analysis revealed that vB_EcoM_APEC was a novel phage, and its genome sequence showed low similarity to other available phage genomes. Gene annotation indicated that the protein encoded by orf11 was an endolysin designated as LysO78, which exhibited 64.7% identity (91.0% coverage) with the putative endolysin of Acinetobacter baumannii phage vB_AbaM_B9. The LysO78 protein belongs to glycoside hydrolase family 19, and was described as being a chitinase class I protein. LysO78 is a helical protein with 12 α-helices containing a large domain and a small domain in terms of the predicted three-dimensional structure. The results of site-directed mutagenesis indicated that LysO78 contained the catalytic residues E54 and E64. The purified endolysin exhibited broad-spectrum bacteriolytic activity against Gram-negative strains, including the genera Klebsiella, Salmonella, Shigella, Burkholderia, Yersinia, and Pseudomonas, as well as the species Chitinimonas arctica, E. coli, Ralstonia solanacearum, and A. baumannii. An enzymatic assay showed that LysO78 had highly lytic peptidoglycan hydrolases activity (64,620,000 units/mg) against E. coli APEC O78, and that LysO78 had lytic activity in the temperature range of 4-85 °C, with an optimal temperature of 28 °C and optimal pH of 8.0, and was active at pH 3.0-12.0. Overall, the results suggested that LysO78 might be a promising therapeutic agent for controlling MDR E. coli APEC O78 and nosocomial infections caused by multidrug-resistant bacteria.

Novel spore lytic enzyme from a Bacillus phage leading to spore killing.

Bacterial spores maintain metabolic dormancy and have high resistance to external pressure. Germination requires degradation of the spore cortex and the participation of germination-specific cortex-lytic enzymes (GSLEs). Previously reported GSLEs have been identified in bacteria and facilitate germination. In this study, we have characterized a novel spore lytic enzyme, Ply67, from Bacillus pumilus phage vB_BpuM_BpSp. Ply67 had a similar cortex-lytic activity to GSLEs but disrupted the inner membranes (IMs) of spores, leading to spore killing rather than germination. The amino acid sequence of the complete protein, Ply67FL, exhibited 40% homology to the GSLE SleB. Domain prediction showed that Ply67FL was composed of three domains: a signal peptide, N-terminal domain protein and C-terminal domain protein. Ply67FL rapidly caused E. coli cells lysis when it was expressed in E. coli. The protein containing the C-terminal domain protein, Ply67C, could kill B. pumilus spores. The protein containing the N-terminal domain protein, Ply67N, could combine with the decoated B. pumilus spores, indicating that N-terminal was the binding domain and C-terminal was the hydrolase domain. The protein lacking the signal peptide but containing the N-terminal and C-terminal domain proteins, Ply67, had activity against spores of various Bacillus species. The surface of spores treated with Ply67 shrank and the permeability barrier was disrupted, and the inner contents leaked out. Immunoelectron microscopic observation showed that Ply67 was mainly acted on the spore cortex. Overall, Ply67 is a novel spore lytic enzyme that differs from other GSLEs not only in amino acid sequence but also in activity against spores, and Ply67 might have the potential to kill spores of pathogenic Bacillus species, e.g., B. cereus and B. anthracis.