gyrA Gene Sequences Research Articles

Characterization of Bacillus velezensis DCJ 2 isolated from the rhizosphere soil of aglaonema ‘Maria’ and investigation of its plant growth-promoting effects

Aglaonema ‘Maria’ is one of the key foliage plants that has gained high demand in foreign destinations in the last few years. This study was conducted to characterize Bacillus velezensis DCJ 2, assess its plant growth promotion effect and investigate the effectiveness of different delivery methods of the bacterial inoculum for the growth promotion of Aglaonema ‘Maria’. In this context, DCJ 2, isolated from the rhizosphere soil of A. Maria was screened to investigate its potential as a plant growth-promoting bacterium. The pure culture of DCJ 2 maintained on nutrient agar was utilized for morphological, biochemical and physiological examinations. The molecular characterization of the strain was performed with different primer pairs targeting 16SrRNA, gyrA, pgsB and sugar kinase genes of DCJ 2. A pot experiment was carried out to investigate the plant growth promotion effect. It was conducted in the greenhouse as per the complete randomized block design with eight treatments and five replicates. . Healthy plants of A. ‘Maria’ established in field soil and solarized soil were treated with 100 ml of the suspension of the DCJ 2 at the rate of 10 8 CFU/ml either as a soil drench or foliar spray. The partial 16SrRNA, gyrA, pgsB and sugar kinase gene sequences of DCJ 2, BLAST searched in the NCBI database revealed that it has a high similarity to Bacillus velezensis. The pot experiment showed that the application of strain DCJ 2 as a soil drench to solarized soil (Treatment 6) yielded a significant increase in growth parameters at p=0.05. Based on molecular analysis, morphological, biochemical, and physiological reactions, DCJ 2 was identified as Bacillus velezensis. This study revealed that B. velezensis DCJ 2 has the potential to boost the growth of A. Maria and hence serve as a base for developing a biofertilizer.

Selection and Characterization of Antagonistic Bacteria as a High Potential Control Agent for Xanthomonas oryzae pv. oryzae Causing Bacterial Leaf Blight Disease in Purple Rice

Bacterial leaf blight disease is one of the most serious diseases in rice caused by Xanthomonas oryzae pv. oryzae. Purple rice is one of the rice varieties that has been infected by this pathogen. Thus, screening and selecting highly efficient antagonistic bacteria for controlling the pathogen is necessary for disease control to reduce epidemics and crop loss. Forty-eight isolates of antagonistic bacteria were isolated from rice rhizosphere soil. The highly effective antagonistic bacteria were selected by dual culture method. The results showed bacteria strains KY16 and KY17 were the most effective antagonists that were able to inhibit X. oryzae pv. oryzae. The inhibition growth zone of antagonists were approximately 12.70 mm and 11.95 mm, respectively. The morphological and some biochemical characteristics of antagonistic bacteria belong to Bacillus sp. Additionally, the antagonists KY16 and KY17 were identified as Bacillus siamensis based on 16S rRNA, gyrA, and rpoB gene sequences. Antimicrobial biosynthesis genes of the antagonistic bacteria were also examined. The results revealed that iturin A and surfactin biosynthesis genes were detectable. From this study, the selected antagonistic bacteria can be used to control the bacterial leaf blight disease of purple rice in rice paddy fields.

The Bacillus velezensis CYS06 Strain Exhibits Promising Applications in Fighting Grass Carp Bacterial Diseases

Aeromonas septicemia and columnaris disease are major bacterial diseases in grass carp; however, the drugs currently used to control these diseases pose environmental and health risks. This study aimed to screen for a probiotic Bacillus strain with antagonistic activity to prevent and control bacterial diseases in grass carp and to evaluate the antimicrobial activities, biosafety, and biocontrol effects of this strain. A Bacillus strain with antagonistic activity against Aeromonas hydrophila, obtained from grass carp intestines, was screened, and the isolate CYS06 was identified by analyzing the 16S rRNA and gyrA gene sequences. The antimicrobial spectrum of the strain CYS06 was determined, and the activities of amylase, cellulase, protease, and lipase of the strain CYS06 were determined. The whole genome of the strain CYS06 was sequenced using the nanopore sequencing technology platform, followed by the analysis of the antagonistic substance synthesis gene clusters and CAZy enzyme gene families. The biosafety of the strain CYS06 was evaluated via intraperitoneal injection into healthy grass carp. After the strain CYS06 was fed to the grass carp, its biological control effect on this fish was evaluated through artificial infection experiments. The strain CYS06 was identified as Bacillus velezensis, based on molecular identification, which shows broad antimicrobial activity against various fish pathogens. The strain CYS06 secretes amylase, cellulase, protease, and lipase. The genome size of the strain CYS06 is 3,914,159 bp, and it contains eight antagonistic substance synthesis gene clusters and many CAZy enzymes. The strain CYS06 exhibits high biological safety for grass carp, based on the challenge test. Feeding grass carp with the strain CYS06 for 4 weeks significantly enhanced the resistance of the fish to A. hydrophila. Strain CYS06 could inhibit the growth of Flavobacterium columnare under co-culture and reduce the amount of F. columnare adherence on the gills of grass carp, indicating that CYS06 has good potential for the prevention and control of columnaris disease. In conclusion, we isolated an antagonistic probiotic strain, CYS06, which exhibits a biological control effect on septicemia and columnaris disease caused by Aeromonas spp. and F. columnare in grass carp, respectively. This strain contains many antagonistic substance synthesis-related gene clusters and holds the potential to degrade various types of carbohydrates. As a biological control agent, the strain CYS06 exhibits significant potential for the prevention and control of bacterial diseases in grass carp.

High prevalence of ciprofloxacin resistance in Escherichia coli isolated from chickens, humans and the environment: An emerging one health issue.

The emergence of antimicrobial resistance in commensal bacteria poses a serious public health burden worldwide. Commensals can disseminate the resistance genes to pathogenic bacteria causing life-threatening infections. This cross-sectional study was designed to investigate the antimicrobial resistance pattern and molecular mechanism(s) of ciprofloxacin resistance in commensal E. coli from three major one health components (humans, animals and the environment) in Bangladesh. Samples were randomly collected from broiler chickens, broiler farm environments and hospitalized human patients from the same geographical area. Isolation and identification of E. coli were performed following standard bacteriological techniques. Antimicrobial susceptibility testing (AST) was performed by disk diffusion and broth microdilution methods. Mutation at the quinolone-resistance determining region (QRDR) was analyzed by sequencing. Of 450 samples, a total of 287 (63.8%; 95% CI 59.2-68.1%) E. coli strains was isolated, where 240 (83.6%; 95% CI 78.9-87.5%) strains were phenotypically resistant to ciprofloxacin. The prevalence of ciprofloxacin-resistant E. coli in broiler chicken, broiler farm environments and hospitalized human patients are 77.6%, 88.8% and 89% respectively. In AST against nine antimicrobials, all the isolates were found to be multidrug-resistant (MDR). The minimum inhibitory concentration (MIC) of ciprofloxacin was ranged from 4 to >128mg/L. Point mutations were detected in several sites of QRDR, specifically at 83 and 87 amino acid positions in gyrA gene, and 56, 57, 78, 80 and 84 amino acid positions in parC gene. Mutations resulted in amino acid substitutions. Phylogenetic analysis of gyrA and parC gene sequences showed a close relationship between the strains isolated from different sources. This study demonstrates a high prevalence of ciprofloxacin resistance in commensal E. coli in humans, animals and environment interface and their genealogically similarity poses an alarming public health consequence.

Prevalence of Aminoglycoside Resistance Genes in Clinical Isolates of Pseudomonas aeruginosa from Taif, Saudi Arabia-An Emergence Indicative Study.

Hospital-acquired infections caused by P. aeruginosa contribute to global distress because of the elevated rates of microbial antibiotic resistance. Aminoglycosides are antipseudomonal agents that are effectively and frequently utilized to eradicate this infection. This current study is a retrospective study investigating plasmid-mediated aminoglycoside resistance by focusing on the prevalence of the genes encoding aminoglycoside-modifying enzymes (AMEs) and 16S rRNA methylase among P. aeruginosa clinical isolates from Taif, Saudi Arabia. A hundred clinical isolates of P. aeruginosa were collected. The isolates were identified from February 2021 to February 2022. Antibiotic susceptibility testing and MICs were determined using (DD) and (BM-MIC) testing, respectively. AMEs and 16S rRNA methylase variants in bacterial isolates were amplified via PCR for genetic detection. A relatively high multiple antibiotic resistance rate corresponding to 10-32% was reported. Eighteen percent of P. aeruginosa isolates were gentamicin-amikacin-tobramycin resistant according to the MIC levels. The aminoglycoside-resistant strains were additionally identified via GyrA gene sequencing. The phylogenic relatedness dendrogram of the sequenced GyrA genes was performed using a neighbor-joining method via MEGAX software version 10.2.6. The most prevalent AME encoding gene was aac(6')-Ib, observed in 94.4% of resistant isolates, while a resistance gene cocktail of [aac(6')-Ib and ant(3″)-I] was a highly frequent combination (27.8%). This study updated the knowledge about aminoglycoside resistance mechanisms in P. aeruginosa, which constitutes an urgent need, especially after the COVID-19 crisis, which was associated with increased antimicrobial use and resistance rates.

Isolation and characterization of Bacillus velezensis strain B19 for biocontrol of Panax notoginseng root rot

Medicine plants such as Panax notoginseng (Sanqi) frequently experience replant failure owing to the accumulation of soilborne pathogens and autotoxins in continuous cropping soil. Bacillus sp. is a commonly used biocontrol agent to combat soilborne pathogens, but further research is needed to understand its impact on controlling Sanqi root rot and the underlying mechanisms. Here, we isolated and screened a Bacillus strain, B19, from Sanqi rhizosphere soil that exhibited potential for inhibiting Sanqi root rot pathogens. We then tested the strain's effect on the incidence of Sanqi root rot in continuous cropping soil, along with its motility/chemotaxis and biofilm formation ability (i.e., potential for root colonization). We also analyzed the strain's genomic characteristics, including potential secondary metabolite synthesis gene clusters. Our results showed that the strain B19 exhibited high inhibitory activity against fungal pathogens (F. solani and F. oxysporum) and could metabolize saponins as a sole carbon source. The application of B19 to continuous cropping soil significantly reduced the incidence of root rot in Sanqi seedlings. B19 was identified as B. velezensis through morphological characteristics, 16S rRNA and gyrA gene sequence analysis, and genome-level annotation. Notably, B19′s genome encodes many genes associated with functions such as motility/chemotaxis and biofilm formation, as well as 13 biosynthetic gene clusters that could encode secondary metabolites, including surfactin, fengycin, and siderophore. Additionally, B19 demonstrated the capability to produce volatile organic compounds that inhibit pathogenic fungi. The motility/chemotaxis and biofilm-forming ability of B19 were enhanced by saponins. Collectively, these properties could enhance B19′s biocontrol ability, thereby reducing the incidence of root rot.

Occurrence, Identification and Preliminary Biological Control of Bulb Rot of Onion (Allium cepa)

Onion bulb rot was observed in low-temperature warehouses in Mae Wang District, Chiang Mai, Thailand in 2022. The pathogens were isolated and identifi ed as Pantoea agglomerans and Burkholderia gladioli based on morphological and biochemical characterization, pathogenicity tests, and 16S rRNA gene sequences. Biological control is regarded as a safer and more sustainable strategy than chemical control and has elicited great attention for disease control. Bacillus sp. isolate BB22 was isolated from rhizosphere soil and was evaluated for its preliminary potential to inhibit the growth of onion bulb rot bacterial pathogens by the dual culture technique. The results showed inhibition zones of P. agglomerans of 30.1 mm and B. gladioli of 13.0 mm. Molecular identifi cation of Bacillus sp. isolate BB22 was performed by using 16S rRNA and gyrA gene sequences and confi rmed as Bacillus velezensis. PCR amplifi cation of the bmyB, fenD, ituA, and srfAA genes coding for bacillomycin, fengycin D, iturin A, and surfactin, respectively, showed the potential for the production of these antibiotics. This study revealed the potential application of B. velezensis as a biocontrol agent against bulb rot-causing bacterial pathogens of onions.

Identification, Biocontrol Activity, and Field Application Effect of Bacillus velezensis Yb-1

Colletotrichum capsici is one of the most important pathogens on chili peppers. Unreasonable application of chemical fungicides will lead to threats to human and animal health, environmental damage, and increased fungicide resistance to pathogens. As an alternative strategy, biological control has been paid more and more attention by academics. In this study, the Yb-1 strain was isolated from healthy cucumber leaves, which could significantly restrict the mycelium growth of C. capsici and Botrytis cinerea, with inhibition rates of 93.49% and 74.03%, respectively. Strain Yb-1 was identified as Bacillus velezensis by morphological features and 16S rRNA gene, gyrA gene, and gyrB gene sequence analysis. When pepper seeds were treated with different concentrations of bacterial suspension, we found that a medium-concentration treatment (104 CFU/mL and 105 CFU/mL) could promote seed germination and growth, while high-concentration treatments (108 CFU/mL) limited seed germination and growth. In addition, the root-irrigation method, acupuncture-leaf method, and leaf-cutting method were used to evaluate the safety of strain Yb-1 on peppers. The results indicated that Yb-1 did not affect the normal growth of peppers. The results of further field trials showed that the control effect of strain Yb-1 on pepper anthracnose was 59.45%. Thus, the strain B. velezensis Yb-1 has great biocontrol potential for pepper anthracnose and promotes plant growth.

Evaluation of a Molecular Mosprie Assay for Detection of Helicobacter pylori and Resistance to Clarithromycin and Levofloxacin

Helicobacter pylori eradication regimens should be guided by antimicrobial susceptibility testing. The objective of this study was to evaluate the molecular-based Mosprie assay for detecting H. pylori resistance to clarithromycin and levofloxacin using gastric biopsies. A total of 185 culture-positive frozen gastric biopsies were included for Mosprie assay and also for 23S rRNA and gyrA gene sequencing. The susceptibility results by the Mosprie assay were compared with the E-test results retrospectively retrieved. The discordant results were analyzed by sequencing of the 23S rRNA and gyrA genes. Susceptibility concordance between the Mosprie assay and E-test for clarithromycin and levofloxacin was 97.30% (180/185) and 88.11% (163/185), respectively. The full agreement between clarithromycin genotypes by Mosprie assay and the 23S rRNA sequencing results was observed in the 5 samples with discordant Mosprie assay and E-test results. However, for levofloxacin, of the 16 discordant samples with resistant phenotype but a susceptible genotype by Mosprie assay, 6 were found to have levofloxacin resistance-related gyrA gene mutations. The rapid and reliable Mosprie assay can be recommended for H. pylori susceptibility testing of clarithromycin and levofloxacin on gastric biopsies. Future technical improvements are needed in detecting levofloxacin resistance-associated gene mutations.

Biocontrol potential of Bacillus subtilis CTXW 7-6-2 against kiwifruit soft rot pathogens revealed by whole-genome sequencing and biochemical characterisation.

Soft rot causes significant economic losses in the kiwifruit industry. This study isolated strain CTXW 7-6-2 from healthy kiwifruit tissue; this was a gram-positive bacterium that produced the red pigment pulcherrimin. The phylogenetic tree based on 16S ribosomal RNA, gyrA, rpoB, and purH gene sequences identified CTXW 7-6-2 as a strain of Bacillus subtilis. CTXW 7-6-2 inhibited hyphal growth of pathogenic fungi that cause kiwifruit soft rot, namely, Botryosphaeria dothidea, Phomopsis sp., and Alternaria alternata, by 81.76, 69.80, and 32.03%, respectively. CTXW 7-6-2 caused the hyphal surface to become swollen and deformed. Volatile compounds (VOC) produced by the strain inhibited the growth of A. alternata and Phomopsis sp. by 65.74 and 54.78%, respectively. Whole-genome sequencing revealed that CTXW 7-6-2 possessed a single circular chromosome of 4,221,676 bp that contained 4,428 protein-coding genes, with a guanine and cytosine (GC) content of 43.41%. Gene functions were annotated using the National Center for Biotechnology Information (NCBI) non-redundant protein, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes, Clusters of Orthologous Groups of proteins, Gene Ontology, Pathogen-Host Interactions, Carbohydrate-Active enZYmes, and Rapid Annotations using Subsystem Technology databases, revealing non-ribosomal pathways associated with antifungal mechanisms, biofilm formation, chemotactic motility, VOC 3-hydroxy-2-butanone, cell wall-associated enzymes, and synthesis of various secondary metabolites. antiSMASH analysis predicted that CTXW 7-6-2 can produce the active substances bacillaene, bacillibactin, subtilosin A, bacilysin, and luminmide and has four gene clusters of unknown function. Quantitative real-time PCR (qRT-PCR) analysis verified that yvmC and cypX, key genes involved in the production of pulcherrimin, were highly expressed in CTXW 7-6-2. This study elucidates the mechanism by which B. subtilis strain CTXW 7-6-2 inhibits pathogenic fungi that cause kiwifruit soft rot, suggesting the benefit of further studying its antifungal active substances.

Isolation and Identification of a Bacillus sp. from Freshwater Sediment Displaying Potent Activity Against Bacteria and Phytopathogen Fungi.

A bacterium strain isolated from freshwater sediment of San Pablo river of Santiago de Cuba, Cuba was identified as a Bacillus sp. by Matrix-Assisted Laser Desorption/Ionization Time Of Flight Mass Spectrometry. A 16S rRNA gene analysis showed that the isolate A3 belongs to the operational group Bacillus amyloliquefaciens, while the phylogenetic analysis of the gyrA gene sequence grouped it within B. amyloliquefaciens subsp. plantarum cluster, referred now as Bacillus velezensis. In vitro antibacterial studies demonstrated the capacity of the isolate A3 to produce bioactive metabolites against Bacillus subtilis ATCC 11,778, Bacillus cereus ATCC 6633, and Staphylococcus aureus ATCC 25,923 by cross-streak, overlay, and microdilution methods. The strain also showed a high potential against the multidrug-resistant Staphylococcus aureus ATCC 700,699, ATCC 29,213, and ATCC 6538. At pH 8 and 96h in the medium 2 of A3 culture conditions, the produced metabolites with antibacterial potential were enhanced. Some alterations in the morphology of the phytopathogens Aspergillus niger ATCC 9642, Alternaria alternata CECT 2662, and Fusarium solani CCEBI 3094 were induced by the cell-free supernatant of B. velezensis A3. A preliminary study of the nature of the bioactive compounds produced by the strain A3 showed the presence of both lipids and peptides in the culture. Those results highlight B. velezensis A3 as a promissory bacterium capable to produce bioactive metabolites with antibacterial and antifungal properties against pathogens.

Identification of Bacillus velezensis SBB and Its Antifungal Effects against Verticillium dahliae.

Traditional control methods have drawbacks in controlling Verticillium wilt diseases caused by Verticillium dahliae Kleb.; therefore, an efficient and environmentally friendly strategy for disease control must be identified and the mechanisms determined. In this study, a soil-isolated strain SBB was identified as Bacillus velezensis based on 16S rRNA, gyrA, and gyrB gene sequences. In vitro, strain SBB had excellent inhibitory effects on V. dahliae, with the highest inhibition rate of 70.94%. Moreover, strain SBB inhibited production of the conidia of V. dahliae and suppressed the production of microsclerotia and melanin. Through gas chromatograph–mass spectrometer analysis, nine compounds were detected from the volatile organic compounds produced by SBB, among which 2-nonanol, 2-heptanone, 6-methyl-2-heptanone, and 2-nonanone could completely inhibit V. dahliae growth. Strain SBB produced cellulase, amylase, protease, and siderophore. During inhibitory action on V. dahliae, strain SBB showed upregulated expression of genes encoding non-volatile inhibitory metabolites, including difficidin, bacilysin, and bacillaene, at 1.923-, 1.848-, and 1.448-fold higher, respectively. Thus, our study proved that strain SBB had an efficient antagonistic effect on V. dahliae, suggesting strain SBB can be used as a potential biological control agent against Verticillium wilt.

Isolation and characterization of Bacillus amyloliquefaciens YL-1 with ochratoxin A degradation ability and biocontrol activity against Aspergillus westerdijkiae

Ochratoxin A (OTA), a toxic secondary metabolite mainly produced by Aspergillus and Penicillium species, is often found in food and agricultural products. Microbiological degradation appears to be a promising method for mycotoxins detoxification. In the present work, a bacterial strain YL-1 with OTA removal ability was isolated and identified to be Bacillus amyloliquefaciens based on 16S rRNA and gyrA gene sequences analyses. Up to 79.7% of OTA with an initial concentration of 2.5 μg/mL could be removed in liquid medium by this isolated bacterium in 48 h. Extracellular enzymatic degradation is suggested to be mainly responsible for the OTA removal ability of B. amyloliquefaciens YL-1, with lactone-opened ochratoxin α as the possible degradation product. Moreover, B. amyloliquefaciens YL-1 could inhibit the fungal growth of Aspergillus westerdijkiae both on agar plate and rice grains. These findings indicated that B. amyloliquefaciens YL-1 could be considered as a promising biological agent against OTA contamination and A. westerdijkiae growth in cereal grains.

Induction of Systemic Resistance to Tobacco mosaic virus in Tomato through Foliar Application of Bacillus amyloliquefaciens Strain TBorg1 Culture Filtrate.

The application of microbe-derived products as natural biocontrol agents to boost systemic disease resistance to virus infections in plants is a prospective strategy to make agriculture more sustainable and environmentally friendly. In the current study, the rhizobacterium Bacillus amyloliquefaciens strain TBorg1 was identified based on 16S rRNA, rpoB, and gyrA gene sequences, and evaluated for its efficiency in conferring protection of tomato from infection by Tobacco mosaic virus (TMV). Under greenhouse circumstances, foliar sprays of TBorg1 culture filtrate (TBorg1-CF) promoted tomato growth, lowered disease severity, and significantly decreased TMV accumulation in systemically infected leaves of treated plants relative to untreated controls. TMV accumulation was reduced by 90% following the dual treatment, applied 24 h before and after TMV infection. Significant increases in levels of total soluble carbohydrates, proteins, and ascorbic acid were also found. In addition, a significant rise in activities of enzymes capable of scavenging reactive oxygen species (PPO and POX), as well as decreased levels of non-enzymatic oxidative stress markers (H2O2 and MDA) were observed, compared to untreated plants. Enhanced systemic resistance to TMV was indicated by significantly increased transcript accumulation of polyphenolic pathway (C4H, HCT, and CHI) and pathogenesis-related (PR-1 and PR-5) genes. Out of the 15 compounds identified in the GC-MS analysis, 1,2-benzenedicarboxylic acid mono(2-ethylhexyl) ester and phenol, 2,4-bis(1,1-dimethylethyl), as well as L-proline, N-valeryl-, and heptadecyl ester were present in the highest concentrations in the ethyl acetate extract of TBorg1-CF. In addition, significant amounts of n-hexadecanoic acid, pyrrolo [1,2-a] pyrazine-1,4-dione hexahydro-3-(2-methylpropyl)-, nonane, 5-butyl-, and eicosane were also detected. These compounds may act as inducers of systemic resistance to viral infection. Our findings indicate that the newly isolated B. amyloliquefaciens strain TBorg1 could be a potentially useful rhizobacterium for promoting plant growth and a possible source of biocontrol agents for combating plant virus infections.

Identification of Two Bacillus Strains with Antimicrobial Activity and Preliminary Evaluation of Their Biocontrol Efficiency

Cabbage Fusarium Wilt (CFW) is a serious disease caused by Fusarium oxysporum f. sp. conglutinans in many parts of the world. The use of chemical fungicides has placed a heavy burden on the environment and is prone to drug resistance in plant pathogens. As a method with great potential, biological control has attracted the attention of many academics both at home and abroad. In this study, we have found that strains B5 and B6 had a strong inhibitory effect on various pathogens and significantly inhibited mycelium growth. They were both identified as Bacillus velezensis by morphological features, biochemical determinations, 16S rRNA gene and gyrA gene sequence analysis. When different concentrations of bacterial suspension were applied to cabbage seeds, hypocotyl and taproot length increased to varying degrees. The in vivo results showed that B5 and B6 decreased the incidence of cabbage seedling wilt disease, with B6 performing significantly better. Furthermore, B. velezensis B6 had the ability to colonize cabbage plants and rhizosphere soil. Thus, strain B6 has great potential for biocontrol development and this research could lead to the development of a promising biological agent for CFW.

Helicobacter turcicus sp. nov., a catalase-negative new member of the Helicobacter genus, isolated from Anatolian Ground Squirrel (Spermophilus xanthoprymnus) in Turkey.

Eleven Gram-negative, curved and S-shaped, oxidase activity positive, catalase activity negative bacterial isolates recovered from faeces of Anatolian ground squirrel (Spermophilus xanthoprymnus) in the city of Kayseri, Turkey, were subjected to a polyphasic taxonomic study. Results of a genus-specific PCR revealed that these isolates belonged to the genus Helicobacter. The 16S rRNA gene sequence analysis revealed that the 11 isolates had over 99 % sequence identity with each other and were most closely related to Helicobacter ganmani CMRI H02T with 97.0-97.1 % identity levels and they formed a novel phylogenetic line within the genus Helicobacter. Faydin-H64 and Faydin-H70T strains were subjected to gyrA and atpA gene and whole genome sequence analyses. These two Helicobacter strains formed separate phylogenetic clades, divergent from other known Helicobacter species. The DNA G+C content and genome size of the strain Faydin-H70T were 35.3 mol% and 1.7 Mb, respectively. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain Faydin-H70T and its close phylogenetic neighbour H. winghamensis ATCC BAA-430T were determined as 81.7 and 34.9 %, respectively. Pairwise sequence comparison showed that it was closely related to H. ganmani CMRI H02T however it shared the highest ANI and dDDH values with H. winghamensis ATCC BAA-430T. The data obtained from the polyphasic taxonomy approach, including phenotypic characterization and whole-genome sequences, revealed that these strains represent a novel species within the genus Helicobacter, for which the name Helicobacter turcicus sp. nov., is proposed with Faydin-H70T as the type strain (=DSM 112556T=LMG 32335T).

Description of the two novel species of the genus Helicobacter: Helicobacter anatolicus sp. nov., and Helicobacter kayseriensis sp. nov., isolated from feces of urban wild birds

A total of 26 Gram-negative, motile, gently curved, and rod-shaped isolates were recovered, during a study to determine the faeco-prevalence of Helicobacter spp. in urban wild birds. Pairwise comparisons of the 16S rRNA gene sequences indicated that these isolates belonged to the genus Helicobacter and phylogenetic analysis based on the 16S rRNA gene sequences showed that the isolates were separated into two divergent groups. The first group consisted of 20 urease-positive isolates sharing the highest 16S rRNA gene sequence identity levels of 98.5–98.6% to H. mustelae ATCC 43772T, while the second group contained six urease-negative isolates with the sequence identity level of 98.5% to the type strain of H. pametensis ATCC 51478T. Five isolates were chosen and subjected to comparative whole-genome analysis. The phylogenetic analysis of the 16S rRNA, gyrA and atpA gene sequences showed that Helicobacter isolates formed two separate phylogenetic clades, differentiating the isolates from the other Helicobacter species. Digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) analyses between strains faydin-H8T, faydin-H23T and their close neighbors H. anseris MIT 04-9362T and H. pametensis ATCC 51478T, respectively, confirmed that both strains represent novel species in the genus Helicobacter. The DNA G+C contents of the strains faydin-H8T and faydin-H23T are 32.0% and 37.6%, respectively. The results obtained for the characterization of the wild bird isolates indicate that they represent two novel species, for which the names Helicobacter anatolicus sp. nov., and Helicobacter kayseriensis sp. nov., are proposed, with faydin-H8T(=LMG 32237T = DSM 112312T) and faydin-H23T(=LMG 32236T = CECT 30508T) as respective type strains.

First report of Klebsiella aerogenes Inciting Stem Rot of Pearl Millet in Haryana, India.

First report of Klebsiella aerogenes Inciting Stem Rot of Pearl Millet in Haryana, India.

Identification and Genomic Characterization of Pathogenic Bacillus altitudinis from Common Pear Trees in Morocco

Bacterial burn is one of the major diseases affecting pear trees worldwide, with serious impacts on producers and economy. In Morocco, several pear trees (Pyrus communis) have shown leaf burns since 2015. To characterize the causal agent of this disease, we isolated fourteen bacterial strains from different parts of symptomatic pear trees (leaves, shoots, fruits and flowers) that were tested in planta for their pathogenicity on Louise bonne and Williams cultivars. The results showed necrotic lesions with a significant severity range from 47.63 to 57.77% on leaves of the Louise bonne cultivar inoculated with isolate B10, while the other bacterial isolates did not induce any disease symptom. 16S rRNA gene sequencing did not allow robust taxonomic discrimination of the incriminated isolate. Thus, we conducted whole-genome sequencing (WGS) and phylogenetic analyzes based on gyrA, gyrB and cdaA gene sequences, indicating that this isolate belongs to the Bacillus altitudinis species. This taxonomic classification was further confirmed by the Average Nucleotide Identity (ANI) and the in silico DNA-DNA hybridization (isDDH) analyzes compared to sixty-five Bacillus spp. type strains. The genome was mined for genes encoding carbohydrate-active enzymes (CAZymes) known to play a role in the vegetal tissue degradation. 177 candidates with functions that may support the in planta phytopathogenicity results were identified. To the best of our knowledge, this is the first data reporting B. altitudinis as agent of leaf burn in P. communis in Morocco. Our dataset will improve our knowledge on spread and pathogenicity of B. altitudinis genotypes that appears as emergent phytopathogenic agent, unveiling virulence factors and their genomic location (i.e., within genomic islands or the accessory genome) to induce trees disease.

Antisense peptide nucleic acid inhibits the growth of KPC-producing Klebsiella pneumoniae strain

Klebsiella pneumoniae causes common and severe hospital- and community-acquired infections with a high incidence of multidrug resistance (MDR) and mortality. In this study, we investigated the ability of the antisense peptide nucleic acids (PNA) conjugated to the (KFF)3K cell-penetrating peptide (CPP) to target the gyrA KPC-producing K. pneumoniae and inhibit bacterial growth in vitro. The inhibitory effect on gyrA gene was evaluated by measuring 16s gene amplification in KPC-producing K. pneumoniae treated with the antisense PNA conjugate. The hemolytic property of the antisense PNA conjugate was accessed toward mice red blood cells. Finally, molecular modeling and dynamics simulations analyses in aqueous solutions were performed to predict the PNA conformation alone in contact with DNA (gyrA gene sequence). PNA was capable of inhibiting bacterial growth at 50 μM, also reducing 16S gene amplification in 96.7%. Besides, PNA presented low hemolytic activity (21.1% hemolysis) at this same concentration. Bioinformatics analysis demonstrated that the structure of the PNA is stable in water without major changes in its secondary structure. The ability of PNA and its conjugated CPP ((KFF)3K) to inhibit bacterial growth demonstrates the potential of this new class of antibacterial agents, encouraging further in vivo studies to confirm its therapeutic efficacy.